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Sample records for capillary basement membrane

  1. Basement membrane changes in capillaries of the ageing human retina

    PubMed Central

    Powner, Michael B; Scott, Andrew; Zhu, Meidong; Munro, Peter M G; Foss, Alexander J E; Hageman, Gregory S; Gillies, Mark C; Fruttiger, Marcus

    2014-01-01

    Objectives The ultrastructural appearance of retinal capillaries can yield important information about disease mechanisms, but is not well characterised in human post mortem samples. We therefore aimed to create a baseline for the appearance of capillaries and establish how this is influenced by post mortem fixation delays and donor age. Methods Electron microscopy was used to characterise retinal capillaries in 20 anonymous donors (with no known eye diseases) of various ages and with various post mortem fixation delays. In addition, samples from six patients with conditions that are known to affect the retinal vasculature (four cases of type 2 diabetes without diabetic retinopathy, one case of diabetic retinopathy and one case of macular telangiectasia type 2) were analysed. Results Vacuoles were found in capillary basement membranes at the vessel—glia interface in all samples, from both the normal and disease cases. Vacuole frequency increased with donor age but was not influenced by post mortem fixation delays. Conclusion Vacuoles in the basement membrane are a normal feature of adult human retinal capillaries and do not indicate disease. Their incidence increases with age and might be a contributing factor to late-onset pathologies of the retinal vasculature. PMID:21606466

  2. Ultrastructural morphometry of capillary basement membrane thickness in normal and transgenic diabetic mice.

    PubMed

    Carlson, Edward C; Audette, Janice L; Veitenheimer, Nicole J; Risan, Jessica A; Laturnus, Donna I; Epstein, Paul N

    2003-04-01

    Capillary basement membrane (CBM) thickening is an ultrastructural hallmark in diabetic patients and in animal models of diabetes. However, the wide variety of tissues sampled and diverse methods employed have made the interpretation of thickness data difficult. We showed previously that acellular glomerular BMs in OVE26 transgenic diabetic mice were thickened beyond normal age-related thickening, and in the current study we hypothesized that other microvascular BMs likewise would show increased widths relative to age-matched controls. Accordingly, a series of tissues, including skeletal and cardiac muscle, ocular retina and choriod, peripheral nerve, lung, pancreas, and renal glomerulus was collected from 300-350-day-old normal and transgenic mice. Transmission electron micrographs of cross sections through capillary walls were prepared, and CBM thickness (CBMT) was determined by the "orthogonal intercept" method. Morphometric analyses showed highly variable transgene-related BMT increases in the sampled tissues, with glomerular BM showing by far the greatest increase (+87%). Significant thickness increases were also seen in the retina, pulmonary alveolus, and thoracoabdominal diaphragm. BMT increases were not universal; however, most were modestly widened, and those that were thickest in controls generally showed the greatest increase. Although the pathogenesis of diabetes-related increases in CBM is poorly understood, data in the current study showed that in OVE26 transgenic mice increased BMT was a frequent concomitant of hyperglycemia. Accordingly, it seems likely that hyperglycemia-induced microvascular damage may be a contributing factor in diabetic BM disease, and that microvessel cellular and extracellular heterogeneity may limit the extent of CBM thickening in diverse tissues. PMID:12629676

  3. Anti-glomerular basement membrane blood test

    MedlinePlus

    GBM antibody test; Antibody to human glomerular basement membrane; Anti-GBM antibodies ... Normally, there are none of these antibodies in the blood. Normal ... labs use different measurements or test different samples. Talk ...

  4. Basement Membranes: Cell Scaffoldings and Signaling Platforms

    PubMed Central

    Yurchenco, Peter D.

    2011-01-01

    Basement membranes are widely distributed extracellular matrices that coat the basal aspect of epithelial and endothelial cells and surround muscle, fat, and Schwann cells. These extracellular matrices, first expressed in early embryogenesis, are self-assembled on competent cell surfaces through binding interactions among laminins, type IV collagens, nidogens, and proteoglycans. They form stabilizing extensions of the plasma membrane that provide cell adhesion and that act as solid-phase agonists. Basement membranes play a role in tissue and organ morphogenesis and help maintain function in the adult. Mutations adversely affecting expression of the different structural components are associated with developmental arrest at different stages as well as postnatal diseases of muscle, nerve, brain, eye, skin, vasculature, and kidney. PMID:21421915

  5. Vascular basement membranes as pathways for the passage of fluid into and out of the brain.

    PubMed

    Morris, Alan W J; Sharp, Matthew MacGregor; Albargothy, Nazira J; Fernandes, Rute; Hawkes, Cheryl A; Verma, Ajay; Weller, Roy O; Carare, Roxana O

    2016-05-01

    In the absence of conventional lymphatics, drainage of interstitial fluid and solutes from the brain parenchyma to cervical lymph nodes is along basement membranes in the walls of cerebral capillaries and tunica media of arteries. Perivascular pathways are also involved in the entry of CSF into the brain by the convective influx/glymphatic system. The objective of this study is to differentiate the cerebral vascular basement membrane pathways by which fluid passes out of the brain from the pathway by which CSF enters the brain. Experiment 1: 0.5 µl of soluble biotinylated or fluorescent Aβ, or 1 µl 15 nm gold nanoparticles was injected into the mouse hippocampus and their distributions determined at 5 min by transmission electron microscopy. Aβ was distributed within the extracellular spaces of the hippocampus and within basement membranes of capillaries and tunica media of arteries. Nanoparticles did not enter capillary basement membranes from the extracellular spaces. Experiment 2: 2 µl of 15 nm nanoparticles were injected into mouse CSF. Within 5 min, groups of nanoparticles were present in the pial-glial basement membrane on the outer aspect of cortical arteries between the investing layer of pia mater and the glia limitans. The results of this study and previous research suggest that cerebral vascular basement membranes form the pathways by which fluid passes into and out of the brain but that different basement membrane layers are involved. The significance of these findings for neuroimmunology, Alzheimer's disease, drug delivery to the brain and the concept of the Virchow-Robin space are discussed. PMID:26975356

  6. Laminin isoforms in endothelial and perivascular basement membranes

    PubMed Central

    Yousif, Lema F.; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis. PMID:23263631

  7. Decreased glomerular basement membrane heparan sulfate proteoglycan in essential hypertension.

    PubMed

    Heintz, B; Stöcker, G; Mrowka, C; Rentz, U; Melzer, H; Stickeler, E; Sieberth, H G; Greiling, H; Haubeck, H D

    1995-03-01

    Heparan sulfate proteoglycans are major components of the glomerular basement membrane and play a key role in the molecular organization and function of the basement membrane. Moreover, their presence is essential for maintenance of the selective permeability of the glomerular basement membrane. Recently, we isolated and characterized a novel small basement membrane-associated heparan sulfate proteoglycan from human aorta and kidney. Partial amino acid sequence data clearly show that this heparan sulfate proteoglycan is distinct from the large basement membrane-associated heparan sulfate proteoglycan (perlecan). Using specific monoclonal antibodies, we have shown that the novel heparan sulfate proteoglycan is located predominantly in the glomerular basement membrane and, to a lesser extent, in the basement membrane of tubuli. Turnover or, in the course of kidney diseases, degradation of heparan sulfate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulfate proteoglycan, which can be measured by a sensitive enzyme immunoassay. The aim of the present study was to analyze whether changes in the structure and function of glomerular basement membranes can be directly detected by measurement of the excretion of a component of this basement membrane, eg, heparan sulfate proteoglycan into urine. The excretion of this small heparan sulfate proteoglycan was compared after physical exercise in normotensive and hypertensive subjects. Normotensive subjects and treated, essential hypertensive patients underwent a standardized workload on a bicycle ergometer. Biochemical characterization of the urinary proteins and heparan sulfate proteoglycan was performed before and 15 and 45 minutes after exercises.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7875766

  8. Basement membrane stiffening promotes retinal endothelial activation associated with diabetes.

    PubMed

    Yang, Xiao; Scott, Harry A; Monickaraj, Finny; Xu, Jun; Ardekani, Soroush; Nitta, Carolina F; Cabrera, Andrea; McGuire, Paul G; Mohideen, Umar; Das, Arup; Ghosh, Kaustabh

    2016-02-01

    Endothelial activation is a hallmark of the high-glucose (HG)-induced retinal inflammation associated with diabetic retinopathy (DR). However, precisely how HG induces retinal endothelial activation is not fully understood. We hypothesized that HG-induced up-regulation of lysyl oxidase (LOX), a collagen-cross-linking enzyme, in retinal capillary endothelial cells (ECs) enhances subendothelial basement membrane (BM) stiffness, which, in turn, promotes retinal EC activation. Diabetic C57BL/6 mice exhibiting a 70 and 50% increase in retinal intercellular adhesion molecule (ICAM)-1 expression and leukocyte accumulation, respectively, demonstrated a 2-fold increase in the levels of BM collagen IV and LOX, key determinants of capillary BM stiffness. Using atomic force microscopy, we confirmed that HG significantly enhances LOX-dependent subendothelial matrix stiffness in vitro, which correlated with an ∼2.5-fold increase in endothelial ICAM-1 expression, a 4-fold greater monocyte-EC adhesion, and an ∼2-fold alteration in endothelial NO (decrease) and NF-κB activation (increase). Inhibition of LOX-dependent subendothelial matrix stiffening alone suppressed HG-induced retinal EC activation. Finally, using synthetic matrices of tunable stiffness, we demonstrated that subendothelial matrix stiffening is necessary and sufficient to promote EC activation. These findings implicate BM stiffening as a critical determinant of HG-induced retinal EC activation and provide a rationale for examining BM stiffness and underlying mechanotransduction pathways as therapeutic targets for diabetic retinopathy. PMID:26443820

  9. Epidermal Basement Membrane in Health and Disease.

    PubMed

    Has, Cristina; Nyström, Alexander

    2015-01-01

    Skin, as the organ protecting the individual from environmental aggressions, constantly meets external insults and is dependent on mechanical toughness for its preserved function. Accordingly, the epidermal basement membrane (BM) zone has adapted to enforce tissue integrity. It harbors anchoring structures created through unique organization of common BM components and expression of proteins exclusive to the epidermal BM zone. Evidence for the importance of its correct assembly and the nonredundancy of its components for skin integrity is apparent from the multiple skin blistering disorders caused by mutations in genes coding for proteins associated with the epidermal BM and from autoimmune disorders in which autoantibodies target these molecules. However, it has become clear that these proteins not only provide mechanical support but are also critically involved in tissue homeostasis, repair, and regeneration. In this chapter, we provide an overview of the unique organization and components of the epidermal BM. A special focus will be given to its function during regeneration, and in inherited and acquired diseases. PMID:26610913

  10. Immunology of anti-glomerular basement membrane disease.

    PubMed

    Salama, Alan D; Pusey, Charles D

    2002-05-01

    Anti-glomerular basement membrane disease is a form of autoimmune glomerulonephritis often accompanied by lung haemorrhage. It is characterized by circulating and deposited antibodies that bind basement membrane components in the glomerulus and lung alveolus. Since early descriptions of the deposition of immunoglobulin on the glomerular basement membrane, work has focused on the binding properties of the autoantibodies, and this has led to the identification of the autoantigen as the non-collagenous region of the alpha 3 chain of type IV collagen. Despite being thought of as a prototypic antibody mediated autoimmune disease, it is becoming apparent that both humoral and cellular immune mechanisms act in concert to initiate and perpetuate disease. Recent data have shed light on the molecular pathogenesis of anti-glomerular basement membrane disease and provided a more complete framework on which to build our understanding of autoimmune renal disease. This should lead to novel approaches to immunotherapy for patients with glomerulonephritis. PMID:11981257

  11. The macula densa tubular basement membrane: a unique plaque of basement membrane specialization.

    PubMed

    Bonsib, S M

    1986-01-01

    The inner (luminal) surface of the macula densa (MD) basement membrane (BM) was exposed by solubilization of the overlying epithelium permitting examination of its structural features by scanning electron microscopy (SEM). Nineteen macula densa BM were identified in five autopsy kidneys and seven renal biopsies by this SEM technique. The MD BM consists of an oval plaque of specialized BM, restricted to the glomerular hilum and arteriolar aspect of the thick ascending limb of Henle. It contains haphazardly oriented shallow tunnels, slender pleats, and bridges of BM which do not resemble any other nephron BM examined by this technique. The MD BM, critically located between MD and lacis cells appears to firmly anchor the MD to the juxtaglomerular apparatus and may amplify the extent of interaction between lacis cell processes or lacis cell interstitial space and MD cell processes. PMID:3453363

  12. Heterotypic Control of Basement Membrane Dynamics During Branching Morphogenesis

    PubMed Central

    Nelson, Deirdre A.; Larsen, Melinda

    2015-01-01

    Many mammalian organs undergo branching morphogenesis to create highly arborized structures with maximized surface area for specialized organ function. Cooperative cell-cell and cell-matrix adhesions that sculpt the emerging tissue architecture are guided by dynamic basement membranes. Properties of the basement membrane are reciprocally controlled by the interacting epithelial and mesenchymal cell populations. Here we discuss how basement membrane remodeling is required for branching morphogenesis to regulate cell-matrix and cell-cell adhesions that are required for cell patterning during morphogenesis and how basement membrane impacts morphogenesis by stimulation of cell patterning, force generation, and mechanotransduction. We suggest that in addition to creating mature epithelial architecture, remodeling of the epithelial basement membrane during branching morphogenesis is also essential to promote maturation of the stromal mesenchyme to create mature organ structure. Recapitulation of developmental cell-matrix and cell-cell interactions are of critical importance in tissue engineering and regeneration strategies that seek to restore organ function. PMID:25527075

  13. Experimental orchitis induced in rats by passive transfer of an antiserum to seminiferous tubule basement membrane.

    PubMed

    Lustig, L; Denduchis, B; González, N N; Puig, R P

    1978-09-01

    A multifocal damage of the testis was obtained when rats were injected intravenously or under the tunica albuginea of the testis with a rabbit antiseminiferous tubule basement membrane serum. The damage was characterized by foci of perivascular and peritubular infiltrates of mononuclear round cells, infolding, thickening, and rupture of the seminiferous tubular wall and different degrees of injury of the germinal epithelium such as, cell disorganization, cell sloughing, and atrophy. Delamination and thickening of seminiferous tubule basement membrane and vacuolization of the Sertoli cell cytoplasm was often observed by electron microscopy. A linear deposit of rabbit gamma-globulin was detected by immunohistochemical techniques along the basement membranes of the seminiferous tubules and vessels. Testicular damage was not detected in rats injected with normal rabbit serum, used as control. In the kidneys of rats injected intravenously with the immune serum, a deposit of rabbit gamma-globulin was detected along glomerular basement membrane. Focal areas of mononuclear cell infiltrates, hypercellularity of glomeruli and thickening of glomerular capillary walls and Bowman's capsule were also observed. PMID:367304

  14. Glomerular basement membrane composition and the filtration barrier

    PubMed Central

    2011-01-01

    The glomerular basement membrane (GBM) is an especially thick basement membrane that contributes importantly to the kidney’s filtration barrier. The GBM derives from the fusion of separate podocyte and endothelial cell basement membranes during glomerulogenesis and consists primarily of laminin-521 (α5β2γ1), collagen α3α4α5(IV), nidogens-1 and -2, and agrin. Of these nine proteins, mutations in the genes encoding four of them (LAMB2, COL4A3, COL4A4, and COL4A5) cause glomerular disease in humans as well as in mice. Furthermore, mutation of a fifth (Lama5) gene in podocytes in mice causes proteinuria, nephrotic syndrome, and progression to renal failure. These results highlight the importance of the GBM for establishing and maintaining a properly functioning glomerular filtration barrier. PMID:21327778

  15. Developmental and Pathogenic Mechanisms of Basement Membrane Assembly

    PubMed Central

    Yurchenco, Peter D.; Patton, Bruce L.

    2010-01-01

    Basement membranes are sheet-like cell-adherent extracellular matrices that serve as cell substrata and solid-phase agonists, contributing to tissue organization, stability and differentiation. These matrices are assembled as polymers of laminins and type IV collagens that are tethered to nidogens and proteoglycans. They bind to cell surface molecules that include signal-transducing receptors such as the integrins and dystroglycan and form attachments to adjacent connective tissues. The cell receptors, in turn, provide links between the matrix and underlying cytoskeleton. Genetic diseases of basement membrane and associated components, collectively the basement membrane zone, disrupt the extracellular matrix and/or its linkages to affect nerve, muscle, skin, kidney and other tissues. These diseases can arise due to a loss of matrix integrity, adhesion strength and/or receptor-mediated signaling. An understanding of the mechanisms of basement membrane zone assembly and resulting structure can provide insights into the development of normal tissues and the pathogenic mechanisms that underlie diverse disorders. PMID:19355968

  16. Beta-amyloid fibrils of Alzheimer's disease: pathologically altered, basement membrane-associated microfibrils?

    PubMed

    Inoue, S; Kisilevsky, R

    2001-01-01

    Beta amyloid fibrils were examined in situ in the cerebral cortex of brains from patients with Alzheimer's disease using high resolution ultrastructural and immunohistochemical techniques. The main body of the fibril was identical with that of microfibrils and was made up of a core containing amyloid P component (AP), and a surface layer. Beta amyloid protein (Abeta) in the form of 1 nm wide flexible filaments was associated with the external surface of the microfibril. In cerebrovascular amyloid angiopathy the fibrils were formed at the outer surface of the vascular basement membrane. Overproduction of microfibrils has been reported at the basement membrane of "leaky" capillaries including the glomerular capillary in disease or leaky alveolar-capillary walls of normal lungs. Similarly, in Alzheimer's disease overproduction of microfibril-like beta amyloid fibrils in amyloid angiopathy coincided with breakdown of the blood-brain barrier of the cerebromicrovasculature. Thus, in the above three locations, the presence of abundant microfibrils, or microfibril-like structures, may be related to plasma which leaks out of the circulation into the adjoining vascular basement membrane. AP is an essential constituent of microfibrils and since the only site where AP is available in the cerebral cortex is in leaky microvasculature, a chronic, steady supply of AP into perivascular areas may be the cause of overproduction of microfibrils. Brain "microfibrils" may further be altered pathologically into beta amyloid fibrils by the addition of Abeta. The origin of the fibrils in senile plaques may also be the microvasculature since in the area of the plaques no source of AP is apparent. PMID:11730002

  17. The clinical features of thin basement membrane nephropathy.

    PubMed

    Gregory, Martin C

    2005-05-01

    Thin basement membrane nephropathy (TBMN) is a common, lifelong condition affecting the kidneys that is characterized by microscopic glomerular hematuria, minimal or no proteinuria, and normal renal function. It often is discovered incidentally, and usually has an excellent prognosis. Many cases are familial and show autosomal-dominant inheritance. The defining characteristic is a glomerular basement membrane (GBM) that is thinned to about half its normal thickness on ultrastructural examination of the renal biopsy specimen. However, occasionally patients with TBMN develop marked proteinuria or renal impairment. It is unclear whether individuals with TBMN and impaired renal function represent part of the spectrum of TBMN associated with heterozygous COL4A3 or COL4A4 mutations, or if their disease is caused by mutations of other genes, or whether it is caused by a second coexistent renal lesion or is misdiagnosed Alport syndrome. PMID:15880323

  18. Regulation of the basement membrane by epithelia generated forces

    PubMed Central

    Tanner, Kandice

    2012-01-01

    Tumor metastasis involves a progressive loss of tissue architecture and dissolution of structural boundaries between the epithelium and connective tissue. The basement membrane (BM), a specialized network of extracellular matrix proteins forms a barrier that physically restricts pre- invasive lesions such that they remain as local insults. The BM is not a static structure, but one that is constantly regenerated and remodeled in the adult organism. Matrix organization also regulates cell function. Thus alterations in the balance of synthesis, remodeling and proteolytic degradation of the extracellular matrix proteins may contribute to a loss of structural integrity. However, the de novo assembly and maintenance of the complex structural properties of in vivo basement membranes remain elusive. Here, this paper highlights the current understanding on the structural properties and the establishment of the BM, and discusses the potential role of self-generated forces in adult tissue remodeling and the maintenance of the BM as a malignancy suppressor. PMID:23196920

  19. Granzyme B promotes cytotoxic lymphocyte transmigration via basement membrane remodeling.

    PubMed

    Prakash, Monica D; Munoz, Marcia A; Jain, Rohit; Tong, Philip L; Koskinen, Aulikki; Regner, Matthias; Kleifeld, Oded; Ho, Bosco; Olson, Matthew; Turner, Stephen J; Mrass, Paulus; Weninger, Wolfgang; Bird, Phillip I

    2014-12-18

    Granzyme B (GzmB) is a protease with a well-characterized intracellular role in targeted destruction of compromised cells by cytotoxic lymphocytes. However, GzmB also cleaves extracellular matrix components, suggesting that it influences the interplay between cytotoxic lymphocytes and their environment. Here, we show that GzmB-null effector T cells and natural killer (NK) cells exhibited a cell-autonomous homing deficit in mouse models of inflammation and Ectromelia virus infection. Intravital imaging of effector T cells in inflamed cremaster muscle venules revealed that GzmB-null cells adhered normally to the vessel wall and could extend lamellipodia through it but did not cross it efficiently. In vitro migration assays showed that active GzmB was released from migrating cytotoxic lymphocytes and enabled chemokine-driven movement through basement membranes. Finally, proteomic analysis demonstrated that GzmB cleaved basement membrane constituents. Our results highlight an important role for GzmB in expediting cytotoxic lymphocyte diapedesis via basement membrane remodeling. PMID:25526309

  20. The role of laminins in basement membrane function

    PubMed Central

    AUMAILLEY, MONIQUE; SMYTH, NEIL

    1998-01-01

    Laminins are a family of multifunctional macromolecules, ubiquitous in basement membranes, and represent the most abundant structural noncollagenous glycoproteins of these highly specialised extracellular matrices. Their discovery started with the difficult task of isolating molecules produced by cultivated cells or extracted from tissues. The development of molecular biology techniques has facilitated and accelerated the identification and the characterisation of new laminin variants making it feasible to identify full-length polypeptides which have not been purified. Further, genetically engineered laminin fragments can be generated for studies of their structure-function relationship, permitting the demonstration that laminins are involved in multiple interactions with themselves, with other components of the basal lamina, and with cells. It endows laminins with a central role in the formation, the architecture, and the stability of basement membranes. In addition, laminins may both separate and connect different tissues, i.e. the parenchymal and the interstitial connective tissues. Laminins also provide adjacent cells with a mechanical scaffold and biological information either directly by interacting with cell surface components, or indirectly by trapping growth factors. In doing so they trigger and control cellular functions. Recently, the structural and biological diversity of the laminins has started to be elucidated by gene targeting and by the identification of laminin defects in acquired or inherited human diseases. The consequent phenotypes highlight the pivotal role of laminins in determining heterogeneity in basement membrane functions. PMID:9758133

  1. Capillary origami and superhydrophobic membrane surfaces

    NASA Astrophysics Data System (ADS)

    Geraldi, N. R.; Ouali, F. F.; Morris, R. H.; McHale, G.; Newton, M. I.

    2013-05-01

    Capillary origami uses surface tension to fold and shape solid films and membranes into three-dimensional structures. It uses the fact that solid surfaces, no matter how hydrophobic, will tend to adhere to and wrap around the surface of a liquid. In this work, we report that a superhydrophobic coating can be created, which can completely suppress wrapping as a contacting water droplet evaporates. We also show that using a wetting azeotropic solution of allyl alcohol, which penetrates the surface features, can enhance liquid adhesion and create more powerful Capillary Origami. These findings create the possibility of selectively shaping membrane substrates.

  2. Effects of radiation on the permeability of human basement membranes

    NASA Astrophysics Data System (ADS)

    Fan, B.-T.; Achour, S.; Simmonet, F.; Guerin, D.

    1999-02-01

    The influence of radiation on the permeability properties of human basement membrane was investigated by measuring the diffusion rate of several organic compounds (glycine, proline, glucose, urea and insulin) through human anterior lens capsules. The basement membranes borne an γ-irradiation treatment change significantly their permeability vis-a-vis studied organic substances. This modification in physico-chemical properties is probably due to the radiation, which alters or degrades the complex structure (or architecture) of basement membranes. Moreover the change in permeability is dependent upon the diffusing compounds. An increase in diffusion has been observed for glucose, glycine and urea. However for insulin and proline, a decrease in diffusion rate was observed. L'influence de radiation sur la perméabilité de la membrane basale a été étudiée par la mesure de la vitesse de diffusion de plusieurs composés organiques d'intérêt biologique (glycine, proline, glucose, urée et insuline) à travers la lame basale antérieure du cristallin de l'oil humain. Les membranes basales qui sont traitées avec l'irradiation γ changent significativement leur perméabilité vis-à-vis des substances organiques. Ce changement de propriétés physico-chimiques est probablement dû à l'altération ou la dégradation de la structure (ou de l'architecture) de la membrane basale entraînée par l'irradiation. De plus, la modification de la perméabilité de la membrane basale est dépendante des composés diffusants. Une augmentation de la vitesse de diffusion a été observée pour le glucose, le glycine et l'urée. Par contre, dans les cas de l'insuline et de la proline, on a observé une diminution de la vitesse de diffusion.

  3. Basement membranes in the worm: a dynamic scaffolding that instructs cellular behaviors and shapes tissues

    PubMed Central

    Clay, Matthew R.; Sherwood, David R.

    2015-01-01

    The nematode worm Caenorhabditis elegans has all the major basement membrane proteins found in vertebrates, usually with a smaller gene family encoding each component. With its powerful forward genetics, optical clarity, simple tissue organization, and the capability to functionally tag most basement membrane components with fluorescent proteins, C. elegans has facilitated novel insights into the assembly and function of basement membranes. Although basement membranes are generally thought of as static structures, studies in C. elegans have revealed their active properties and essential functions in tissue formation and maintenance. Here we review discoveries from C. elegans development that highlight dynamic aspects of basement membrane assembly, function, and regulation during organ growth, tissue polarity, cell migration, cell invasion, and tissue attachment. These studies have helped transform our view of basement membranes from static support structures to dynamic scaffoldings that play broad roles in regulating tissue organization and cellular behavior that are essential for development and have important implications in human diseases. PMID:26610919

  4. An active role for basement membrane assembly and modification in tissue sculpting

    PubMed Central

    Morrissey, Meghan A.; Sherwood, David R.

    2015-01-01

    Basement membranes are a dense, sheet-like form of extracellular matrix (ECM) that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stress. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instructs coordinated cellular behaviors and acts mechanically to sculpt tissues. In this Commentary, we highlight recent studies that support the idea that far from being a passive matrix, basement membranes play formative roles in shaping tissues. PMID:25717004

  5. [Basement membranes as the regulatory system between epithelial cell connections and connective tissue].

    PubMed

    Heine, H

    1986-01-01

    Basement membranes develop by an interaction between epithelium and underlying mesenchymal. Basement membranes are to an great extend involved in the metabolic induced informational exchange between these 2 compartments. Informational selectivity is not only given with the construction of basement membranes. By redox systems like ascorbic acid/dehydroascorbic acid, preferentially located in basement membranes, the capability exists of scavenging free oxygen radicals on the border line between epithelia and connective tissue. This seems to be a very important factor in coupling the biorhythms between epithelia and connective tissue. PMID:3743995

  6. IgA-mediated anti-glomerular basement membrane disease: an uncommon mechanism of Goodpasture's syndrome

    PubMed Central

    Moulis, Guillaume; Huart, Antoine; Guitard, Joëlle; Fortenfant, Françoise; Chauveau, Dominique

    2012-01-01

    Goodpasture's (GP) disease is usually mediated by IgG autoantibodies. We describe a case of IgA-mediated GP, in a patient presenting with isolated rapidly progressive glomerulonephritis. The diagnosis was established on kidney biopsy, since routine enzyme-linked immunosorbent assay (ELISA) targeted at IgG circulating autoantibodies failed to detect the nephritogenic antibodies. Immunofluorescence microscopy showed intense linear deposition of IgA along the glomerular capillary walls. An elevated titre (1:80) of circulating IgA anti-glomerular basement membrane (GBM) antibodies was retrospectively demonstrated by indirect fluorescence. Despite immunosuppressive regimen, the disease progressed to end-stage renal failure (ESRF). Transplantation was not associated with recurrence in the kidney graft. We reviewed the 11 previously reported cases of IgA-mediated GP. PMID:26069798

  7. Building from the ground up: basement membranes in Drosophila development

    PubMed Central

    Isabella, Adam J.; Horne-Badovinac, Sally

    2016-01-01

    Basement Membranes (BMs) are sheet-like extracellular matrices found at the basal surfaces of epithelial tissues. The structural and functional diversity of these matrices within the body endows them with the ability to affect multiple aspects of cell behavior and communication; for this reason, BMs are integral to many developmental processes. The power of Drosophila genetics, as applied to the BM, has yielded substantial insight into how these matrices influence development. Here, we explore three facets of BM biology to which Drosophila research has made particularly important contributions. First we discuss how newly synthesized BM proteins are secreted to and assembled exclusively on basal epithelial surfaces. Next, we examine how regulation of the structural properties of the BM mechanically supports and guides tissue morphogenesis. Finally, we explore how BMs influence development through the modulation of several major signaling pathways. PMID:26610918

  8. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini

    PubMed Central

    Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

    2015-01-01

    The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function. PMID:26674091

  9. Basement membrane components are potent promoters of rat intestinal epithelial cell differentiation in vitro.

    PubMed

    Hahn, U; Stallmach, A; Hahn, E G; Riecken, E O

    1990-02-01

    Basement membranes have been implicated in morphogenesis and cell differentiation. In this study, the effect of basement membrane components on intestinal epithelial cell maturation in a mesenchyme-free environment was investigated. Fetal rat small intestinal epithelial cells (from the 14th-17th day of gestation) were exposed to basement membrane-derived proteins (laminin, collagen type IV, and a complex basement membrane-enriched extract from the Engelbreth-Holm-Swarm sarcoma) and other extracellular matrix proteins (collagen type I and fibronectin) coated onto Petri dishes. The cells attached readily only to fibronectin and basement membrane proteins. For 5 days the developing epithelial colonies were monitored in vitro, assessing morphological and functional parameters of cell maturation. Colonies grown on laminin and the basement membrane extract were larger and of greater cell density. An increase in alkaline phosphatase and lactase activity was observed after 3-4 days in these colonies which could be enhanced to yield 90%-100% positive cells by the addition of dexamethasone to the medium while no sucrase-isomaltase activity was elicited. Electron microscopy confirmed a high degree of cellular polarization illustrated by tight junctions and apical microvilli in epithelial cells grown on a basement membrane-like support. In contrast, none of the other proteins stimulated the cells to mature in vitro. The authors conclude that certain basement membrane components actively promote fetal intestinal epithelial cell differentiation. PMID:2295387

  10. Fluid Mechanics of the Vascular Basement Membrane in the Brain

    NASA Astrophysics Data System (ADS)

    Coloma, Mikhail; Hui, Jonathan; Chiarot, Paul; Huang, Peter; Carare, Roxana; McLeod, Kenneth; Schaffer, David

    2013-11-01

    Beta-amyloid is a normal product of brain metabolic function and is found within the interstitial fluid of the brain. Failure of the clearance of beta-amyloid from the aging brain leads to its accumulation within the walls of arteries and to Alzheimer's disease. The vascular basement membrane (VBM) within the walls of cerebral arteries surrounds the spirally arranged smooth muscle cells and represents an essential pathway for removal of beta-amyloid from the brain. This process fails with the stiffening of arterial walls associated with aging. In this study we hypothesize that the deformation of the VBM associated with arterial pulsations drives the interstitial fluid to drain in the direction opposite of the arterial blood flow. This hypothesis is theoretically investigated by modeling the VBM as a thin, coaxial, fluid-filled porous medium surrounding a periodically deforming cylindrical tube. Flow and boundary conditions required to achieve such a backward clearance are derived through a control volume analysis of mass, momentum, and energy.

  11. Unaltered anionic sites of glomerular basement membrane in aminonucleoside nephrosis

    SciTech Connect

    Kanwar, Y.S.; Jakubowski, M.L.

    1984-04-01

    Quantitative electron microscopic autoradiography was employed to determine the changes in the binding of /sup 125/I-cationic ferritin (CFI, pI approximately 7.2 to 7.4) to the anionic sites of the glomerular basement membrane (GBM) of rats following the induction of nephrosis. Animals were rendered nephrotic by a single intravenous injection of puromycin aminonucleoside (PAN) and sacrificed 0, 7, 14, and 21 days after its administration. CFI (10 mg/mCi/100 g body weight) was given intravenously. The kidneys were subsequently fixed by perfusion and processed for electron microscopic autoradiography. The mean grain densities over the GBMs on 0, 7, 14, and 21 days of PAN nephrosis were 1.50 +/- 0.04, 1.49 +/- 0.05, 1.50 +/- 0.05, and 1.51 +/- 0.04, respectively. These results indicate that there are no significant alterations in the anionic sites rich in heparan sulfate proteoglycan during the entire course of PAN nephrosis.

  12. Nanoscale protein architecture of the kidney glomerular basement membrane

    PubMed Central

    Suleiman, Hani; Zhang, Lei; Roth, Robyn; Heuser, John E; Miner, Jeffrey H; Shaw, Andrey S; Dani, Adish

    2013-01-01

    In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM. DOI: http://dx.doi.org/10.7554/eLife.01149.001 PMID:24137544

  13. The vascular basement membrane as "soil" in brain metastasis.

    PubMed

    Carbonell, W Shawn; Ansorge, Olaf; Sibson, Nicola; Muschel, Ruth

    2009-01-01

    Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Paget's "seed and soil" concept. However, there is little direct evidence for this "neurotropic" growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the "soil" for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM) as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesion and invasion of malignant cells and was sufficient for tumor growth prior to any evidence of angiogenesis. Blockade or loss of the beta1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies. PMID:19516901

  14. High-resolution ultrastructural study of the rat glomerular basement membrane in aminonucleoside nephrosis.

    PubMed

    Inoue, S; Bendayan, M

    1996-01-01

    In the initial stages of aminonucleoside nephrosis, functional alterations in the glomerular basement membrane occur, as evidenced by the development of proteinuria. However, it has not been possible to observe important ultrastructural modifications at the level of the basement membrane, probably because the changes are taking place at the molecular level. In this study, by the use of high-resolution electron microscopy, an attempt was made to evaluate such changes in rat glomerular basement membrane during acute aminonucleoside nephrosis. As previously reported, in control animals the glomerular basement membrane is composed of a network of 4-nm-wide irregular anastomosing strands, referred to as "cords," which are known to contain a core filament of type IV collagen surrounded by a "sheath" of other components, such as laminin and heparan sulfate proteoglycan (HSPG). The most conspicuous ultrastructural alteration of the nephrotic glomerular basement membrane, recognizable only at high magnification, is that the cords were denuded leaving only the core filament through the loss of the sheath material. Thus, the cord network was transformed, with the progress of pathological conditions, into a network of fine filaments. On the other hand, abundance and distribution of HSPG molecules known to be present in the form of 4.5- to 5-nm-wide ribbon-like "double tracks," were found to be similar in control and nephrotic tissues. Since HSPG is one of the charge proteins of the basement membrane, the little changes observed for HSPG are difficult to interpret in view of reported decreases in basement membrane anionic sites in nephrosis. In conclusion, the glomerular basement membrane in aminonucleoside nephrosis loses its cord network components and replaces them with a more perforated network, which could be a cause for the increased permeability of this basement membrane. PMID:8883324

  15. Cellular and circulating immunity to diabetic basement membrane, a negative finding.

    PubMed Central

    De Bats, A; Park, J R; Rhodes, E L

    1975-01-01

    A linear deposition of IgG was seen by immunofluorescence on the glomerular basement membrane of a diabetic kidney. However, when the remaining kidney was subjected to elution with acidic buffer the eluate had no affinity for glomerular basement membrane. The leucocytes from normal and diabetic subjects were also tested for an in vitro cell-mediated response to diabetic and normal basement membrane. No difference was found between the reaction of leucocytes from diabetics and those from normal age- and sex-matched controls. Images Fig. 1 Fig. 2 Fig. 3 PMID:765023

  16. Possible continuity of subplasmalemmal cytoplasmic network with basement membrane cord network: ultrastructural study.

    PubMed

    Inoue, S

    1995-05-01

    The ultrastructure of the subplasmalemmal cytoplasm of the cell and the associated basement membrane as well as the area of the cell-basement membrane border were observed with high resolution electron microscopy after preparation of the tissues with cryofixation or glutaraldehyde fixation followed by freeze substitution. The subplasmalemmal cytoplasm of the smooth muscle cells of rat epididymal tubules and the podocyte processes of the mouse glomerular visceral epithelium were found to be composed of a fine network of irregular anastomosing strands. This network closely resembled the previously characterized cord network of the basement membrane. The cords are known to be composed of a 1.5 to 3 nm thick core filament made up of type IV collagen which is surrounded by an irregular 'sheath' of other components. The strands in the subplasmalemmal network showed ultrastructural features similar to those of the cord network. Ribbon-like, 4.5 nm wide heparan sulfate proteoglycan 'double tracks' were previously reported to be associated with the cord network. Structures similar in size and appearance to the double tracks were also found in the subplasmalemmal network. At the cell-basement membrane border, the lamina densa of the basement membrane was in contact with the cell without the intervening space of a lamina lucida which was recently found to be an artefact caused by conventional tissue processing. Furthermore, the subplasmalemmal network appeared to be continuous through the plasma membrane, with the cord network of the basement membrane.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7657717

  17. Fabricating PFPE Membranes for Capillary Electrophoresis

    NASA Technical Reports Server (NTRS)

    Lee, Michael C.; Willis, Peter A.; Greer, Frank; Rolland, Jason

    2009-01-01

    A process has been developed for fabricating perfluoropolyether (PFPE) membranes that contain microscopic holes of precise sizes at precise locations. The membranes are to be incorporated into laboratory-on-a-chip microfluidic devices to be used in performing capillary electrophoresis. The present process is a modified version of part of the process, described in the immediately preceding article, that includes a step in which a liquid PFPE layer is cured into solid (membrane) form by use of ultraviolet light. In the present process, one exploits the fact that by masking some locations to prevent exposure to ultraviolet light, one can prevent curing of the PFPE in those locations. The uncured PFPE can be washed away from those locations in the subsequent release and cleaning steps. Thus, holes are formed in the membrane in those locations. The most straightforward way to implement the modification is to use, during the ultraviolet-curing step, an ultraviolet photomask similar to the photomasks used in fabricating microelectronic devices. In lieu of such a photomask, one could use a mask made of any patternable ultraviolet-absorbing material (for example, an ink or a photoresist).

  18. Remodeling of basement membrane in patients with asthma.

    PubMed

    Grigoraş, Adriana; Grigoraş, Constantin Cristian; Giuşcă, Simona Eliza; Căruntu, Irina Draga; Amălinei, Cornelia

    2016-01-01

    The "bronchial remodeling" specific for the asthmatic disease consists in irreversible changes of the bronchial wall, including glandular and smooth muscle fibers hyperplasia and÷or hypertrophy, goblet cells hyperplasia, and thickening of basement membrane (BM). We aimed to analyze the BM thickness in asthma patients, in order to validate the relationship between its changes and the disease severity defined in agreement with the Global Initiative for Asthma (GINA) criteria. The study group has been formed of 38 patients with different degrees of severity of asthma established by spirometry using Forced Expiratory Volume in one second (FEV1), and two patients without asthma symptoms as controls. The specimens harvested by fibrobronchoscopy have been processed by paraffin embedding followed by Hematoxylin-Eosin (HE) and Periodic Acid-Schiff (PAS) staining. For each case, the BM measurement has been realized by a "point-by-point" method. Statistical analysis has been performed using SPSS 17 software, by applying non-parametric correlation tests. The quantitative assessment revealed a progressive increase in BM thickness during the course of the disease, from a mean value of 11.2 μm in stage 1 to that of 15.6 μm in stage 4. Even if this process has been noticed starting with the first stage of asthma, the differences in the BM size were statistically significant only for stages 1 and 3 (p=0.047), stages 1 and 4 (p=0.000), stages 2 and 3 (p=0.000), and stages 3 and 4 (p=0.000). Spearman's test has shown an opposite correlation between the BM thickness and asthma severity defined by FEV1 values (r=-0.86, p<0.01, 95% CI). Our study demonstrates that the collagen deposition at the epithelium-connective interface is initiated in early stages of asthma and continues in a progressive modality, the BM thickening being correlated with the disease severity. Thus, we support the concept of biological consequences of BM thickening in asthma pathogenesis, a mechanism still

  19. Binding of Streptococcus mutans antigens to heart and kidney basement membranes.

    PubMed Central

    Stinson, M W; Barua, P K; Bergey, E J; Nisengard, R J; Neiders, M E; Albini, B

    1984-01-01

    Using indirect immunofluorescence, alkali-extracted components of Streptococcus mutans were found to bind in vitro to capillary walls and sarcolemmal sheaths of monkey cardiac muscle and to glomerular and tubular basement membranes of monkey kidney. Adsorption of S. mutans components to tissue fragments was also detected by indirect radioimmunoassay and immunoblotting on nitrocellulose paper. Antibodies did not bind to untreated, control tissues in these experiments, proving that antigens shared by S. mutans and tissue components were not involved. Rabbit and monkey heart and kidney components bound S. mutans antigens of 24,000, 35,000, and 65,000 Mr. Monkey heart also bound molecules of 90,000 and 120,000 Mr. Rabbits immunized by intravenous injection of disrupted S. mutans cells developed severe nephritis that was characterized by the deposition of immunoglobulins, complement component C3, and S. mutans antigens in the glomeruli. Immunoglobulin G eluted from nephritic kidneys reacted in immunoblots with the 24,000, 35,000, and 65,000 Mr components of S. mutans extract, indicating that the antigens that bound to tissue in vitro also bound in vivo and reacted with antibodies in situ. Antibodies to other S. mutans antigens were not detected in the kidney eluate, although they were present in the serum of the same rabbit. Images PMID:6384042

  20. Basement membrane proteins promote progression of intraepithelial neoplasia in 3-dimensional models of human stratified epithelium.

    PubMed

    Andriani, Frank; Garfield, Jackie; Fusenig, Norbert E; Garlick, Jonathan A

    2004-01-20

    We have developed novel 3-dimensional in vitro and in vivo tissue models that mimic premalignant disease of human stratified epithelium in order to analyze the stromal contribution of extracellular matrix and basement membrane proteins to the progression of intraepithelial neoplasia. Three-dimensional, organotypic cultures were grown either on a de-epidermalized human dermis with pre-existing basement membrane components on its surface (AlloDerm), on a Type I collagen gel that lacked basement membrane proteins or on polycarbonate membranes coated with purified extracellular matrix proteins. When tumor cells (HaCaT-II4) were mixed with normal keratinocytes (4:1/normals:HaCaT-II4), tumor cells selectively attached, persisted and proliferated at the dermal-epidermal interface in vitro and generated dysplastic tissues when transplanted to nude mice only when grown in the presence of the AlloDerm substrate. This stromal interface was permissive for tumor cell attachment due to the rapid assembly of structured basement membrane. When tumor cells were mixed with normal keratinocytes and grown on polycarbonate membranes coated with individual extracellular matrix or basement membrane components, selective attachment and significant intraepithelial expansion occurred only on laminin 1 and Type IV collagen-coated membranes. This preferential adhesion of tumor cells restricted the synthesis of laminin 5 to basal cells where it was deposited in a polarized distribution. Western blot analysis revealed that tumor cell attachment was not due to differences in the synthesis or processing of laminin 5. Thus, intraepithelial progression towards premalignant disease is dependent on the selective adhesion of cells with malignant potential to basement membrane proteins that provide a permissive template for their persistence and expansion. PMID:14648700

  1. Visualization of basement membranes in normal breast and breast cancer tissues using multiphoton microscopy

    PubMed Central

    WU, XIUFENG; CHEN, GANG; QIU, JINGTING; LU, JIANPING; ZHU, WEIFENG; CHEN, JIANXIN; ZHUO, SHUANGMU; YAN, JUN

    2016-01-01

    Since basement membranes represent a critical barrier during breast cancer progression, timely imaging of these signposts is essential for early diagnosis of breast cancer. A label-free method using multiphoton microscopy (MPM) based on two-photon excited fluorescence signals and second harmonic generation signals for analyzing the morphology of basement membrane in normal and cancerous breast tissues is likely to enable a better understanding of the pathophysiology of breast cancer and facilitate improved clinical management and treatment of this disease. The aim of this study was to determine whether MPM has the potential for label-free assessment of the morphology of basement membrane in normal and cancerous breast tissues. A total of 60 tissue section samples (comprising 30 fresh breast cancer specimens and 30 normal breast tissues) were first imaged (fresh, unfixed and unstained) with MPM and are then processed for routine hematoxylin and eosin (H&E) histopathology. Comparisons were made between MPM imaging and gold standard sections for each specimen stained with H&E. Simply by visualizing morphological features appearing on multiphoton images, cancerous lesions may be readily identified by the loss of basement membrane and tumor cells characterized by irregular size and shape, enlarged nuclei and increased nuclear-cytoplasmic ratio. These results suggest that MPM has potential as a label-free method of imaging the morphology of basement membranes and cell features to effectively distinguish between normal and cancerous breast tissues. PMID:27313695

  2. Acute effects of antiglomerular basement membrane antibody on the process of glomerular filtration in the rat.

    PubMed Central

    Blantz, R C; Wilson, C B

    1976-01-01

    Nehron filtration rate (sngfr) and the factors controlling filtration were examined before and with 60 min of the intravenous infusion of 225-450 mug of antiglomerular basement membrane antibody (AGBM Ab) (greater than 50% antigenic saturation) in plasma-expanded (2.5% body wt) Munich-Wistar rats. Pressures in glomerular capillaries (PG) and bowman's space (Pt) were measured with a servo-nulling device, systemic (piA) and efferent arteriolar oncotic pressures (piE) were measured by microprotein methods, and nephron plasma flow (rpf) and sngfr were measured by micropuncture techniques in both control and post-AGBM Ab conditions in each rat. The sngfr fell from 52.7+/-2.9 to 24.1+/-1.9 nl/min per g kidney wt (n = 7, P less than 0.001). Both afferent and efferent arteriolar resistances increased and rpf fell from 221+/-25 to 90+/-9 nl/min per g kidney wt (P less than 0.001) but the hydrostatic pressure gradient across the glomerular membrane deltaP = PG - Pt) increased from 37+/- 1 to 50+/-2 mm Hg (P less than 0.001). The increase in deltaP and a numerical decrease in piA both acted to maintain sngfr after AGBM Ab and effectively nullified the influence of decreased rpf upon sngfr. The mean effective filtration pressure (EFP = deltaP - pi) increased from 14+/-2 to 30+/-3 mm Hg (P less than 0.001) while sngfr decreased. The major and critical reason for this reduction in sngfr was a decrease in the glomerular permeability coefficient from 0.077+/-0.017 to 0.014+/-0.001 nl/s per g kidney wt per mm Hg P less than 0.001) where sngfr=EFP-LpA. Images PMID:61207

  3. Dystroglycan protein distribution coincides with basement membranes and muscle differentiation during mouse embryogenesis.

    PubMed

    Anderson, Claire; Winder, Steven J; Borycki, Anne-Gaëlle

    2007-09-01

    Using immunohistochemistry, we have examined beta-Dystroglycan protein distribution in the mouse embryo at embryonic stages E9.5 to E11.5. Our data show that Dystroglycan expression correlates with basement membranes in many tissues, such as the notochord, neural tube, promesonephros, and myotome. In the myotome, we describe the timing of Dystroglycan protein re-distribution at the surface of myogenic precursor cells as basement membrane assembles into a continuous sheet. We also report on non-basement-membrane-associated Dystroglycan expression in the floor plate and the myocardium. This distribution often corresponds to sites of expression previously reported in adults, suggesting that Dystroglycan is continuously produced during development. PMID:17676646

  4. Thy-1 antigen: selective association in lymphoid organs with the vascular basement membrane involved in lymphocyte recirculation.

    PubMed Central

    Ritter, M A; Morris, R J

    1980-01-01

    The cell surface differentiation antigen, Thy-1, was demonstrated by immunofluorescence to be associated with collagen-based connective tissue (mainly basement membrane) around some blood vessels in rat lymphoid organs. This association is highly selective: only certain types of blood vessel within a given lymphoid organ were found to be Thy-1+; and different lymphoid organs (thymus, bone marrow, lymph node and spleen) had characteristic differences in the types of blood vessel that bear Thy-1. In lymph node and spleen the vessels that were Thy-1+ were those involved in lymphocyte recirculation and homing (post-capillary venules and arterioles of white pulp); the possibility that Thy-1 may function in mediating selective adhesion of small lymphocytes to extracellular substrates during recirculation is discussed. Images Figure 2 Figure 3 Figure 1 Figure 4 PMID:6991398

  5. Extracellular chloride signals collagen IV network assembly during basement membrane formation.

    PubMed

    Cummings, Christopher F; Pedchenko, Vadim; Brown, Kyle L; Colon, Selene; Rafi, Mohamed; Jones-Paris, Celestial; Pokydeshava, Elena; Liu, Min; Pastor-Pareja, Jose C; Stothers, Cody; Ero-Tolliver, Isi A; McCall, A Scott; Vanacore, Roberto; Bhave, Gautam; Santoro, Samuel; Blackwell, Timothy S; Zent, Roy; Pozzi, Ambra; Hudson, Billy G

    2016-05-23

    Basement membranes are defining features of the cellular microenvironment; however, little is known regarding their assembly outside cells. We report that extracellular Cl(-) ions signal the assembly of collagen IV networks outside cells by triggering a conformational switch within collagen IV noncollagenous 1 (NC1) domains. Depletion of Cl(-) in cell culture perturbed collagen IV networks, disrupted matrix architecture, and repositioned basement membrane proteins. Phylogenetic evidence indicates this conformational switch is a fundamental mechanism of collagen IV network assembly throughout Metazoa. Using recombinant triple helical protomers, we prove that NC1 domains direct both protomer and network assembly and show in Drosophila that NC1 architecture is critical for incorporation into basement membranes. These discoveries provide an atomic-level understanding of the dynamic interactions between extracellular Cl(-) and collagen IV assembly outside cells, a critical step in the assembly and organization of basement membranes that enable tissue architecture and function. Moreover, this provides a mechanistic framework for understanding the molecular pathobiology of NC1 domains. PMID:27216258

  6. Tissue specificity of a baculovirus expressed, basement membrane-degrading protease in larvae of Heliothis virescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ScathL is a cathepsin L-like cysteine protease from flesh fly Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm, Heliothis virescens, significantly faste...

  7. Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes

    SciTech Connect

    Verrando, P.; Pisani, A.; Serieys, N.; Ortonne, J.P. ); Hsi, Baeli; Yeh, Changjing )

    1987-05-01

    A monoclonal antibody, GB3, has been raised against human amnion. Not only does GB3 bind to amniotic basement membrane, but it also recognizes an antigenic structure expressed by epidermal as well as by some other human basement membranes. This antigen is synthesized (and excreted) by cultured normal human epidermal keratinocytes. It is expressed to a lesser extent by the A431 epidermoid carcinoma cell line, but is not expressed by the SV40 virus-transformed SVK14 keratinocyte cell line. In ultrastructural studies, this antigen was located in the epidermal basement membrane, both in the lamina densa and in the lamina lucida, associated with hemidesmosomes. It was identified as a protein by in vitro proteolytic cleavage studies. The radio-immunoprecipitates from cultured human keratinocytes, analyzed by SDS-PAGE, showed that GB3 recognized five polypeptides of 93.5, 125, 130, 146 and 150 kD under reducing conditions. The tissue distribution of the antigen and the molecular weights (MWs) of its constitutive polypeptides suggest that it is different from other known components of basement membranes. It may provide a biochemical marker for hemidesmosomes. Furthermore, GB3 represents an interesting and original clinical probe, since the antigenic structure recognized by GB3 is lacking in Junctional Epidermolysis Bullosa, a lethal genodermatosis in which a dermo-epidermal splitting occurs at the level of lamina lucida.

  8. Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly.

    PubMed

    Villacorte, Mylah; Delmarcelle, Anne-Sophie; Lernoux, Manon; Bouquet, Mahé; Lemoine, Pascale; Bolsée, Jennifer; Umans, Lieve; de Sousa Lopes, Susana Chuva; Van Der Smissen, Patrick; Sasaki, Takako; Bommer, Guido; Henriet, Patrick; Refetoff, Samuel; Lemaigre, Frédéric P; Zwijsen, An; Courtoy, Pierre J; Pierreux, Christophe E

    2016-06-01

    Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5(dKO)) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO) embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa(KO)) thyroids, Smad1/5(dKO) thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO) and Smad1/5(dKO) thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5(dKO) and Vegfa(KO) thyroids. Laminin α1, β1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane. PMID:27068110

  9. Heparan sulfate proteoglycan is present in basement membrane as a double-tracked structure.

    PubMed

    Inoue, S; Grant, D; Leblond, C P

    1989-05-01

    Basement membranes contain 4.5-nm wide sets of two parallel lines, along which short prongs called "spikes" occur at regular intervals. The nature of this structure, referred to as "double tracks," was investigated in Lowicryl sections of mouse kidney and rat Reichert's membrane immunolabeled for basement membrane components using secondary antibodies conjugated to 5-nm gold particles. When the mouse glomerular basement membrane and rat Reichert's membrane were exposed to antibodies directed to the core protein of heparan sulfate proteoglycan, 95% or more of the gold particles were over double tracks, whereas after exposure of Reichert's membrane to antisera against laminin, collagen IV, or entactin, labeling of the double tracks remained at the random level. When heparan sulfate proteoglycan was incubated in Tris buffer, pH 7.4, at 35 degrees C for 1 hr, a precipitate resulted which, on electron microscopic examination, was found to consist of 5- to 6-nm wide sets of two parallel lines along which densities were observed. Immunolabeling confirmed the presence of the proteoglycan's core protein in the sets. Since double tracks were closely similar to this structure and were labeled with the same antibodies, they were likely to be also composed of heparan sulfate proteoglycan. PMID:2522961

  10. Isotropic Versus Bipolar Functionalized Biomimetic Artificial Basement Membranes and Their Evaluation in Long-Term Human Cell Co-Culture.

    PubMed

    Rossi, Angela; Wistlich, Laura; Heffels, Karl-Heinz; Walles, Heike; Groll, Jürgen

    2016-08-01

    In addition to dividing tissues into compartments, basement membranes are crucial as cell substrates and to regulate cellular behavior. The development of artificial basement membranes is indispensable for the ultimate formation of functional engineered tissues; however, pose a challenge due to their complex structure. Herein, biodegradable electrospun polyester meshes are presented, exhibiting isotropic or bipolar bioactivation as a biomimetic and biofunctional model of the natural basement membrane. In a one-step preparation process, reactive star-shaped prepolymer additives, which generate a hydrophilic fiber surface, are electrospun with cell-adhesion-mediating peptides, derived from major components of the basement membrane. Human skin cells adhere to the functionalized meshes, and long-term co-culture experiments confirm that the artificial basement membranes recapitulate and preserve tissue specific functions. Several layers of immortalized human keratinocytes grow on the membranes, differentiating toward the surface and expressing typical epithelial markers. Fibroblasts migrate into the reticular lamina mimicking part of the mesh. Both cells types begin to produce extracellular matrix proteins and to remodel the initial membrane. It is shown at the example of skin that the artificial basement membrane design provokes biomimetic responses of different cell types and can thus be used as basis for the future development of basement membrane containing tissues. PMID:27283510

  11. Dibutyryl cyclic AMP does not influence glomerular collagen or basement membrane production in vitro.

    PubMed

    Uw, V Y; Cohen, M P

    1980-02-01

    Glomeruli isolated from normal rat renal cortex were incubated for 3 hr with radiolabeled proline in the presence or absence of dibutyryl cyclic AMP. Following incubation, glomerular basement membranes were purified with osmotic lysis followed by selective solubilization of the cell membranes and intracellular proteins with detergents. This technique permitted quantitative recovery of radiolabeled membranes synthesized under different incubational conditions. Dibutyryl cyclic AMP did not affect the incorporation of radioactive precursor glomerular basement membrane (control = 14.72 +/- 1.08 cpm/microgram of membrane protein; cyclic AMP = 14.43 +/- 1.13). Nondialyzable [14C]protein and hydroxy[14C]proline were also measured in the media and in the various glomerular cell fractions obtained during isolation of the basement membranes. Protein ([14C]proline) and collagen (OH[14C]proline) secretion into the media in incubations with cyclic AMP did not differ from that in control incubations. OH[14C]proline content was greatest (congruent to 23% in the water-soluble fraction recovered after osmotic lysis, but significant amounts of OH[14C]proline were also associated with the detergent-solubilized cell fractions. Dibutyryl cyclic AMP had no effect on either glomerular protein or collagen synthesis in these experiments. The results suggest that total glomerular basement membrane production in mixed cell populations is not modulated via a cyclic AMP--coordinated mechanism but do not exclude the possibility that cyclic AMP modulates the amount or kind of collagen synthesis by individual glomerular cell types. PMID:6243687

  12. Numerical Simulation of Particle Distribution in Capillary Membrane during Backwash

    PubMed Central

    Mansour, Hussam; Keller, Anik; Gimbel, Rolf; Kowalczyk, Wojciech

    2013-01-01

    The membrane filtration with inside-out dead-end driven UF-/MF- capillary membranes is an effective process for particle removal in water treatment. Its industrial application increased in the last decade exponentially. To date, the research activities in this field were aimed first of all at the analysis of filtration phenomena disregarding the influence of backwash on the operation parameters of filtration plants. However, following the main hypothesis of this paper, backwash has great potential to increase the efficiency of filtration. In this paper, a numerical approach for a detailed study of fluid dynamic processes in capillary membranes during backwash is presented. The effect of particle size and inlet flux on the backwash process are investigated. The evaluation of these data concentrates on the analysis of particle behavior in the cross sectional plane and the appearance of eventually formed particle plugs inside the membrane capillary. Simulations are conducted in dead-end filtration mode and with two configurations. The first configuration includes a particle concentration of 10% homogeneously distributed within the capillary and the second configuration demonstrates a cake layer on the membrane surface with a packing density of 0.6. Analyzing the hydrodynamic forces acting on the particles shows that the lift force plays the main role in defining the particle enrichment areas. The operation parameters contribute in enhancing the lift force and the heterogeneity to anticipate the clogging of the membrane. PMID:24957056

  13. Numerical Simulation of Particle Distribution in Capillary Membrane during Backwash.

    PubMed

    Mansour, Hussam; Keller, Anik; Gimbel, Rolf; Kowalczyk, Wojciech

    2013-01-01

    The membrane filtration with inside-out dead-end driven UF-/MF- capillary membranes is an effective process for particle removal in water treatment. Its industrial application increased in the last decade exponentially. To date, the research activities in this field were aimed first of all at the analysis of filtration phenomena disregarding the influence of backwash on the operation parameters of filtration plants. However, following the main hypothesis of this paper, backwash has great potential to increase the efficiency of filtration. In this paper, a numerical approach for a detailed study of fluid dynamic processes in capillary membranes during backwash is presented. The effect of particle size and inlet flux on the backwash process are investigated. The evaluation of these data concentrates on the analysis of particle behavior in the cross sectional plane and the appearance of eventually formed particle plugs inside the membrane capillary. Simulations are conducted in dead-end filtration mode and with two configurations. The first configuration includes a particle concentration of 10% homogeneously distributed within the capillary and the second configuration demonstrates a cake layer on the membrane surface with a packing density of 0:6. Analyzing the hydrodynamic forces acting on the particles shows that the lift force plays the main role in defining the particle enrichment areas. The operation parameters contribute in enhancing the lift force and the heterogeneity to anticipate the clogging of the membrane. PMID:24957056

  14. Ultrastructural appearance of renal and other basement membranes in the Bull terrier model of autosomal dominant hereditary nephritis.

    PubMed

    Hood, J C; Savige, J; Seymour, A E; Dowling, J; Martinello, P; Colville, D; Sinclair, R; Naito, I; Jennings, G; Huxtable, C

    2000-08-01

    Bull terrier hereditary nephritis may represent a model for autosomal dominant Alport's syndrome because affected dogs have the typically lamellated glomerular basement membrane (GBM) and father-to-son disease transmission occurs. This study examined the ultrastructural appearance of the renal and extrarenal basement membranes and their composition in affected Bull terriers. Affected stillborn animals and puppies had subepithelial frilling and vacuolation of the GBM. In adult dogs, lamellation was common, and subepithelial frilling and vacuolation were less prominent. Foot-process effacement and mesangial matrix expansion occurred frequently. Basement membranes in the glomeruli, tubules, and Bowman's capsule were significantly thickened and often mineralized. Immunohistochemical examination showed alpha 1(IV) and alpha 2(IV) collagen chains in all renal basement membranes; alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains in the GBM, distal tubular basement membrane, and Bowman's capsule; and the alpha 6(IV) chain in Bowman's capsule. Conversely, the basement membranes from the affected Bull terrier cornea, lens capsule, retina, skin, lung, and muscle had a normal ultrastructural appearance and were not thickened compared with membranes in normal age-matched dogs. The distribution of basement membrane abnormalities in Bull terrier hereditary nephritis may occur because the defective protein is present exclusively or more abundantly in the kidney and is structurally more important in the kidney or because of local intrarenal stresses. PMID:10922317

  15. Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis.

    PubMed Central

    Kalluri, R; Shield, C F; Todd, P; Hudson, B G; Neilson, E G

    1997-01-01

    Normal glomerular capillaries filter plasma through a basement membrane (GBM) rich in alpha3(IV), alpha4(IV), and alpha5(IV) chains of type IV collagen. We now show that these latter isoforms are absent biochemically from the glomeruli in patients with X-linked Alport syndrome (XAS). Their GBM instead retain a fetal distribution of alpha1(IV) and alpha2(IV) isoforms because they fail to developmentally switch their alpha-chain use. The anomalous persistence of these fetal isoforms of type IV collagen in the GBM in XAS also confers an unexpected increase in susceptibility to proteolytic attack by collagenases and cathepsins. The incorporation of cysteine-rich alpha3(IV), alpha4(IV), and alpha5(IV) chains into specialized basement membranes like the GBM may have normally evolved to protectively enhance their resistance to proteolytic degradation at the site of glomerular filtration. The relative absence of these potentially protective collagen IV isoforms in GBM from XAS may explain the progressive basement membrane splitting and increased damage as these kidneys deteriorate. PMID:9153291

  16. The γ3 Chain of Laminin is Widely But Differentially Expressed in Murine Basement Membranes: Expression and Functional Studies

    PubMed Central

    Li, Yong N.; Radner, Stephanie; French, Margaret M.; Pinzón-Duarte, Germán; Daly, Gerard H.; Burgeson, Robert E.; Koch, Manuel; Brunken, William J.

    2012-01-01

    Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an α, a β and a γ chain. Previous studies have shown that the γ3 chain is partnered with either the β1 chain (in placenta) or β2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the γ3 chain is expressed in both apical and basal compartments (Gersdorff et al., 2005; Koch et al., 1999; Yan and Cheng, 2006). This study investigates the expression pattern of the γ3 chain in mouse. We developed three new γ3-reactive antibodies, and we show that the γ3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the γ1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where γ3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the γ3 chain in mice; unlike other laminin null mice (α5, β2, γ1 nulls) (Miner et al., 1998; Noakes et al., 1995; Smyth et al., 1999), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the γ3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system. PMID:22222602

  17. Histones have high affinity for the glomerular basement membrane. Relevance for immune complex formation in lupus nephritis

    SciTech Connect

    Schmiedeke, T.M.; Stoeckl, F.W.W.; Weber, R.; Sugisaki, Y.; Batsford, S.R.; Vogt, A.

    1989-06-01

    An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns, have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of /sup 125/I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary.

  18. Delayed reepithelialization and basement membrane regeneration after wounding in mice lacking CXCR3

    PubMed Central

    Yates, Cecelia C.; Whaley, Diana; Hooda, Shveta; Hebda, Patricia A.; Bodnar, Richard J.; Wells, Alan

    2010-01-01

    Wound healing is a complex, orchestrated series of biological events that is controlled by extracellular components that communicate between cell types to re-establish lost tissue. We have found that signaling by ELR-negative CXC chemokines through their common CXCR3 receptor is critical for dermal maturation during the resolving phase. In addition there needs to be complete maturation of the epidermis and regeneration of a delineating basement membrane for proper functioning. The role of this ligand–receptor system appears confounding as one ligand, CXCL4/(PF4), is present during the initial dissolution and two others, CXCL10/(IP-10) and CXCL11/(IP-9/I-TAC), are expressed by keratinocytes in the later regenerative and resolving phases during which the basement membrane is re-established. We examined CXCR3 signaling role in healing using a mouse lacking this receptor, as all three ligands act solely via the common receptor. Reepithelialization was delayed in CXCR3-deficient mice in both full and partial-thickness excisional wounds. Even at 90 days postwounding, the epidermis of these mice appeared less mature with lower levels of E-cadherin and cytokeratin 18. The underlying basement membrane, a product of both dermal fibroblasts and epidermal keratinocytes, was not fully established with persistent diffuse expression of the matrix components laminin 5, collagen IV, and collagen VII throughout the wound bed. These results suggest that CXCR3 and its ligands play an important role in the re-establishment of the basement membrane and epidermis. These studies further establish the emerging signaling network that involves the CXCR3 chemokine receptor and its ligands as a key regulator of wound repair. PMID:19152649

  19. Glomerular basement membrane expansion in passive Heymann nephritis. Absence of increased synthesis of type IV collagen, laminin, or fibronectin.

    PubMed Central

    Fogel, M. A.; Boyd, C. D.; Leardkamolkarn, V.; Abrahamson, D. R.; Minto, A. W.; Salant, D. J.

    1991-01-01

    The distribution and synthetic rate of glomerular basement membrane components was examined in the Passive Heymann Nephritis model of experimental membranous nephropathy. The extensive tissue injury that developed included subepithelial electron-dense deposits, podocyte foot process effacement, and expansion of the glomerular basement membrane. Levels of mRNA for type IV collagen, laminin, and fibronectin from isolated glomeruli was quantitated by slot-blot analysis and showed no change in experimental animals as compared to controls at either 1 week, 3 weeks, or 3 months after disease induction. Immunoelectron microscopy with gold-labeled anti-laminin IgG revealed no difference in the number of particles bound to the glomerular basement membrane of experimental animals and controls. Immunofluorescence with both type IV collagen antisera and anti-laminin antibody showed no difference in the intensity or pattern of staining. Despite extensive glomerular damage and glomerular basement membrane thickening, no evidence was found for either an increase in the synthetic rate of type IV collagen, laminin, or fibronectin or for an accumulation of basement membrane laminin within the damaged glomeruli. Alternate processes, such as diminished density of matrix components or accumulation of other unmeasured matrix constituents, presumably account for the expansion of the glomerular basement membrane seen in experimental membranous nephropathy. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1992771

  20. Corneal nerve architecture in a donor with unilateral epithelial basement membrane dystrophy

    PubMed Central

    He, Jiucheng; Bazan, Haydee E.P.

    2014-01-01

    Background: Epithelial basement membrane dystrophy (EBMD) is by far the most common corneal dystrophy. In this study, we used a newly developed method of immunofluorescence staining and imaging to study the entire corneal nerve architecture of a donor with unilateral EBMD. Method: Two fresh eyes from a 56-year-old male donor were obtained; the right eye of the donor was diagnosed with EBMD and the left was normal. After slit lamp examination, the corneas were immunostained with anti-β-tubulin III antibody. Images were recorded by a fluorescent microscope equipped with a Photometrics digital camera using MetaVue imaginig software. Results: The left cornea appeared normal as observed by slit lamp and stereomicroscope, but the right eye had numerous irregular geographic patches in the basement membrane. Immunofluorescence showed no difference in the stromal nerve distribution between the two eyes, but there were areas without innervations in the EBMD cornea. Subbasal nerve fibers also showed tortuous courses and fewer divisions. There was a significant decrease in the density of subbasal nerve fibers and the number of terminals in the right eye. Conclusion: We show for the first time detailed nerve architecture in an EBMD cornea. Our results suggest that EBMD-induced abnormalities of basement membrane altered epithelial nerve architecture and decreased nerve density, contributing to the pathology of the disease. PMID:23306594

  1. Antigens of the basement membranes of the seminiferous tubules induce autoimmunity in Wistar rats.

    PubMed

    Lustig, L; Satz, M L; Sztein, M B; Denduchis, B

    1982-05-01

    A preparation enriched in basement membranes from seminiferous tubules was isolated from rat testes (STBM) and injected with complete Freund's adjuvant into Wistar rats. In 60% of animals a mild multifocal orchitis was observed. In damaged areas, perivascular and peritubular mononuclear cell infiltrates and different degrees of cell sloughing of some seminiferous tubules were observed. Electron microscopy revealed focal thickenings and delamination of the basement membrane of the seminiferous tubules as well as vacuolization of Sertoli cell cytoplasm. Using immunofluorescence discontinuous linear deposits of IgG were detected along the seminiferous tubular wall. Moreover, the same pattern of immunofluorescence was observed when the IgG eluted from the testes of the immunized rats was layered on sections of normal rat testis. Circulating antibodies to STBM were detected using passive haemagglutination in approximately 45% of the immunized rats, with titers ranging from 1:20 to 1:80. Leukocyte migration was inhibited when the spleen cells of the immunized rats were incubated with antigens from the basement membrane of seminiferous tubules, whilst a negative reaction was obtained when the soluble fraction of testis homogenate was used. PMID:7050376

  2. The basement membranes of cryofixed or aldehyde-fixed, freeze-substituted tissues are composed of a lamina densa and do not contain a lamina lucida.

    PubMed

    Chan, F L; Inoue, S; Leblond, C P

    1993-07-01

    When tissues are processed for electron microscopy by conventional methods, such as glutaraldehyde fixation followed by rapid dehydration in acetone, basement membranes show two main layers: the electron-lucent "lamina lucida". (or rara) and the electron-dense "lamina densa". In an attempt to determine whether this subdivision is real or artefactual, two approaches have been used. Firstly, rat and mouse seminiferous tubules, mouse epididymis and associated tissues, and anterior parts of mouse eyes were subjected to cryofixation by instant freezing followed by freeze substitution in a -80 degrees C solution of osmium tetroxide in dry acetone, which was gradually warmed to room temperature over a 3-day period. The results indicate that, in areas devoid of ice crystals, basement membranes consist of a lamina densa in direct contact with the plasmalemma of the associated cells without an intervening lamina lucida. Secondly, a series of tissues from mice perfused with 3% glutaraldehyde were cryoprotected in 30% glycerol, frozen in Freon 22 and subjected to a 3-day freeze substitution in osmium tetroxide-acetone as above. Under these conditions, no lamina lucida accompanies the lamina densa in the basement membranes of the majority of tissues, including kidney, thyroid gland, smooth and skeletal muscle, ciliary body, seminiferous tubules, epididymis and capillary endothelium. Thus, even though these tissues have been fixed in glutaraldehyde, no lamina lucida appears when they are slowly dehydrated by freeze substitution. It is concluded that the occurrence of this lamina in conventionally processed tissues is not due to fixation but to the rapid dehydration. However, in this series of experiments, the basement membranes of trachea and plantar epidermis include a lamina lucida along their entire length, while those of esophagus and vas deferens may or may not include a lamina lucida. To find out if the lamina lucida appearing under these conditions is a real structure or

  3. Normal mammary epithelial cells promote carcinoma basement membrane invasion by inducing microtubule-rich protrusions

    PubMed Central

    Lee, Meng-Horng; Wu, Pei-Hsun; Gilkes, Daniele; Aifuwa, Ivie; Wirtz, Denis

    2015-01-01

    Recent work suggests that the dissemination of tumor cells may occur in parallel with, and even preceed, tumor growth. The mechanism for this early invasion is largely unknown. Here, we find that mammary epithelial cells (MECs) induce neighboring breast carcinoma cells (BCCs) to cross the basement membrane by secreting soluble laminin. Laminin continuously produced by MECs induce long membrane cellular protrusions in BCCs that promote their contractility and invasion into the surrounding matrix. These protrusions depend on microtubule bundles assembled de novo through laminin-integrin β1 signaling. These results describe how non-cancerous MECs can actively participate in the invasive process of BCCs. PMID:26334095

  4. Basement membrane of mouse bone marrow sinusoids shows distinctive structure and proteoglycan composition: a high resolution ultrastructural study.

    PubMed

    Inoue, S; Osmond, D G

    2001-11-01

    Venous sinusoids in bone marrow are the site of a large-scale traffic of cells between the extravascular hemopoietic compartment and the blood stream. The wall of the sinusoids consists solely of a basement membrane interposed between a layer of endothelial cells and an incomplete covering of adventitial cells. To examine its possible structural specialization, the basement membrane of bone marrow sinusoids has now been examined by high resolution electron microscopy of perfusion-fixed mouse bone marrow. The basement membrane layer was discontinuous, consisting of irregular masses of amorphous material within a uniform 60-nm-wide space between apposing endothelial cells and adventitial cell processes. At maximal magnifications, the material was resolved as a random arrangement of components lacking the "cord network" formation seen in basement membranes elsewhere. Individual components exhibited distinctive ultrastructural features whose molecular identity has previously been established. By these morphological criteria, the basement membrane contained unusually abundant chondroitin sulfate proteoglycan (CSPG) revealed by 3-nm-wide "double tracks," and moderate amounts of both laminin as dense irregular coils and type IV collagen as 1-1.5-nm-wide filaments, together with less conspicuous amounts of amyloid P forming pentagonal frames. In contrast, 4.5-5-nm-wide "double tracks" characteristic of heparan sulfate proteoglycan (HSPG) were absent. The findings demonstrate that, in comparison with "typical" basement membranes in other tissues, the bone marrow sinusoidal basement membrane is uniquely specialized in several respects. Its discontinuous nature, lack of network organization, and absence of HSPG, a molecule that normally helps to maintain membrane integrity, may facilitate disassembly and reassembly of basement membrane material in concert with movements of adventitial cell processes as maturing hemopoietic cells pass through the sinusoidal wall: the

  5. Basement membrane protein nidogen-1 is a target of meprin β in cisplatin nephrotoxicity

    PubMed Central

    Herzog, Christian; Marisiddaiah, Raju; Haun, Randy S.; Kaushal, Gur P.

    2015-01-01

    Meprins are oligomeric metalloproteinases that are abundantly expressed in the brush-border membranes of renal proximal tubules. During acute kidney injury (AKI) induced by cisplatin or ischemia-reperfusion, membrane-bound meprins are shed and their localization is altered from the apical membranes toward the basolateral surface of the proximal tubules. Meprins are capable of cleaving basement membrane proteins in vitro, however, it is not known whether meprins are able of degrade extracellular matrix proteins under pathophysiological conditions in vivo. The present study demonstrates that a basement membrane protein, nidogen-1, is cleaved and excreted in the urine of mice subjected to cisplatin-induced nephrotoxicity, a model of AKI. Cleaved nidogen-1 was not detected in the urine of untreated mice, but during the progression of cisplatin nephrotoxicity, the excretion of cleaved nidogen-1 increased in a time-dependent manner. The meprin inhibitor actinonin markedly prevented urinary excretion of the cleaved nidogen-1. In addition, meprin β-deficient mice, but not meprin α-deficient mice, subjected to cisplatin nephrotoxicity significantly suppressed excretion of cleaved nidogen-1, further suggesting that meprin β is involved in the cleavage of nidogen-1. These studies provide strong evidence for a pathophysiological link between meprin β and urinary excretion of cleaved nidogen-1 during cisplatin-induced AKI. PMID:25957482

  6. Intraepidermal expression of basement membrane components in the lesional skin of a patient with dystrophic epidermolysis bullosa.

    PubMed

    Muramatsu, T; Ko, T; Honoki, K; Hatoko, M; Shirai, T; Vnittanakom, P

    1999-02-01

    The patient was a 15-year-old male. Since birth, he had developed blistering and erosion of the skin. Biopsy skin specimen of the bullous lesions showed subepidermal blister formation. Electron microscopic examination revealed that tissue separation had occurred at the sublamina densa level. By indirect immunofluorescence using antibodies specific for alpha 6 integrin, laminin 5, type IV collagen, and type VII collagen, all of these basement membrane components were detected as coarse granular intracytoplasmic deposits only in the basal and suprabasal cells of the blister roof. In the non-blistered regions, these basement membrane components showed a linear pattern similar to that seen in normal skin. These findings suggest that intraepidermal expression of basement membrane components was closely related to the blister formation. The biological meaning of intraepidermal expression of basement membrane components were also discussed. PMID:10091480

  7. Lectin-binding sites and silver affinity of the macula densa basement membranes in the rabbit kidney.

    PubMed Central

    Ojeda, J L; Piedra, S

    1994-01-01

    Fluorochrome-labelled lectins and the Jones method of silver impregnation preceded by different oxidation and enzymatic digestion procedures were used to study the patterns of glycosylation and silver affinity of the macula densa (MD) and thick ascending limb (TAL) basement membranes of the rabbit kidney. The goal of this study was to analyse the morphological basis of MD basement membrane permeability and its possible role in modulation of the signal involved in tubuloglomerular feedback control of the juxtaglomerular apparatus. The lectin-binding pattern and silver affinity of basement membrane differed clearly from those of the TAL basement membrane. The former had greater WGA and Con A affinity than the latter. Furthermore, the MD basement membrane lost argyrophilia in permanganate oxidized sections whereas that of the TAL did not. The cell coat of MD cells differed from that of the TAL cells in that it had N-acetyl neuraminic acid and Con A binding sites. Our results suggest that the MD basement membrane has a distinctive macromolecular composition which may be related to its permeability to high molecular weight molecules. Images Fig. 1 Fig. 2 Fig. 3 PMID:7544331

  8. Anionic charge concentration of rat kidney glomeruli and glomerular basement membrane.

    PubMed Central

    Comper, W D; Lee, A S; Tay, M; Adal, Y

    1993-01-01

    Estimates of levels of glomerular and glomerular-basement-membrane anion charge should serve as useful quantitative markers for the integrity of the tissues in health and disease. We have developed a simple, rapid, technique to measure this charge through the use of ion exchange with radioisotopes 22Na+ and 36Cl- at low ionic strengths in phosphate buffer. When this technique is used, normal glomeruli isolated from rat have a measured net anion charge concentration of 17.4 +/- 3.7 p-equiv. per glomerulus (n = 20). Perfused rat kidneys that lose approximately half of their glomerular heparan [35S]sulphate content (owing to oxygen-radical damage) exhibited a lower anion charge, of 7.5 +/- 1.6 p-equiv. per glomerulus (n = 5). Glomerular basement membranes prepared from rat glomeruli by a sonication-centrifugation procedure in the presence of enzyme inhibitors had a charge concentration of 6.3 +/- 0.7 mu-equiv./g wet wt. of tissue (n = 4), whereas membranes prepared by sonication, centrifugation, DNAse and detergent treatment had a charge concentration of 7.1 +/- 1.6 mu-equiv./g wet wt. (n = 4). Isotope-dilution experiments with 3H2O on these detergent-prepared glomerular basement membranes demonstrated that they had a water content of approx. 93%, which would then give a net anion charge concentration of 7.6 +/- 1.7 m-equiv./l (n = 4). These values are in good agreement with those obtained by others using titration techniques [Bray and Robinson (1984) Kidney Int. 25, 527-533]. The relatively low magnitude of glomerular anion charge in normal kidneys is consistent with other recent findings that glomerular anion charge is too low to affect the glomerular transport of charged molecules in a direct, passive, biophysical manner through electrostatic interactions. PMID:8435064

  9. Permselectivity Replication of Artificial Glomerular Basement Membranes in Nanoporous Collagen Multilayers

    PubMed Central

    Pullela, Srinivasa R.; Andres, Christine; Chen, Wei; Xu, Chuanlai; Wang, Libing; Kotov, Nicholas A.

    2011-01-01

    Basement membranes (BMs) play important roles in many biological functions such as tissue regeneration, cancer proliferation, nutrient/drug delivery, breathing, and many others. While there are many theoretical models, adequate experimental analogs of BMs describing basic physicochemical properties of BM, such as diffusion and permselectivity are not available. Taking BMs found in glomerulus of kidneys as an example, adequate reproduction of their permselectivity requires biomimetic membranes with submicron thickness, high uniformity, nanoscale porosity, and size-selective permeability. Artificial kidney BMs were assembled from poly(acrylic acid) and collagen using layer-by-layer (LBL) assembly technology and display multiple structural similarities with glomerular BMs. Diffusional transport through the artificial BMs faithfully replicate cut-off parameters of kidney membranes. Their utilization in understanding of unique diffusion processes in kidneys, in vitro studies of blood clearance time of small drugs/nanoscale drug carriers and design of more complex organoids including live cells for cancer proliferation studies is anticipated. PMID:22200004

  10. Laminin and Type IV Collagen Isoform Substitutions Occur in Temporally and Spatially Distinct Patterns in Developing Kidney Glomerular Basement Membranes

    PubMed Central

    St. John, Patricia L.; Stroganova, Larysa; Zelenchuk, Adrian; Steenhard, Brooke M.

    2013-01-01

    Kidney glomerular basement membranes (GBMs) undergo laminin and type IV collagen isoform substitutions during glomerular development, which are believed to be required for maturation of the filtration barrier. Specifically, GBMs of earliest glomeruli contain laminin α1β1γ1 and collagen α1α2α1(IV), whereas mature glomeruli contain laminin α5β2γ1 and collagen α3α4α5(IV). Here, we used confocal microscopy to simultaneously evaluate expression of different laminin and collagen IV isoforms in newborn mouse GBMs. Our results show loss of laminin α1 from GBMs in early capillary loop stages and continuous linear deposition of laminin bearing the α5 chain thereafter. In contrast, collagen α1α2α1(IV) persisted in linear patterns into late capillary loop stages, when collagen α3α4α5(IV) first appeared in discontinuous, non-linear patterns. This patchy pattern for collagen α3α4α5(IV) continued into maturing glomeruli where there were lengths of linear, laminin α5-positive GBM entirely lacking either isoform of collagen IV. Relative abundance of laminin and collagen IV mRNAs in newborn and 5-week-old mouse kidneys also differed, with those encoding laminin α1, α5, β1, β2, and γ1, and collagen α1(IV) and α2(IV) chains all significantly declining at 5 weeks, but α3(IV) and α4(IV) were significantly upregulated. We conclude that different biosynthetic mechanisms control laminin and type IV collagen expression in developing glomeruli. PMID:23896970

  11. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    PubMed

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states. PMID:19189961

  12. Nephrotoxic potency of antisera to three rat glomerular basement membrane glycoproteins.

    PubMed Central

    Devulder, B; Bardos, P; Plouvier, B; Martin, J C; Muh, J P; Tacquet, A

    1978-01-01

    In a previous article, we cited studies which have allowed us to isolate diverse glycoproteins of the rat glomerular basement membrane (GMB) and to study their biochemical structures and antigenicity. This present study attempts to examine, using the heterologous nephrotoxic nephritis model (Masugi's nephritis) the nephrotoxicity of immune sera prepared from three of these glycoproteins: one fairly rich in collagen-like structures (A3), another lacking collagen-like structures (A1), and a third of intermediate composition (A2). The results obtained are discussed in relation to those already published concerning the nature of the GBM antigen(s) responsible for the nephrotoxicity of the sera. PMID:357054

  13. Immunolocalization of entactin, a sulfated basement membrane component, in rodent tissues, and comparison with GP-2 (laminin).

    PubMed Central

    Bender, B. L.; Jaffe, R.; Carlin, B.; Chung, A. E.

    1981-01-01

    Entactin is a sulfated glycoprotein in the extracellular basement membrane like matrix produced by M1536-B3 cells, a mouse endodermal line derived from an embryonal carcinoma. It has a molecular weight of 158,000 and is chemically and immunologically distinguishable from GP-2 (laminin) and fibronectin. Antibodies produced against entactin and GP-2 react with subepithelial and vascular basement membranes in rat lung, liver, spleen, and kidney and mouse placenta and kidney when examined by light microscopy. Both antibodies yield staining around the marginal sinus of the white pulp of the spleen. Antientactin reacts with basement membrane and mesangium in rat glomeruli, and anti-GP2 does not. Ultrastructurally, staining in kidneys is strongest at epithelial or endothelial cell membranes bordering basement membranes, with only moderate staining of the basement membrane proper. Intracellular staining is not present. The location of entactin suggests that it has a role in the interaction of cells with extracellular matrix, possibly in adhesion. Lack of intracellular staining suggests that the tissues studied are not actively producing entactin or GP-2 and that these substances may be fairly stable in adult organisms. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:6165248

  14. Distribution and origin of the basement membrane component perlecan in rat liver and primary hepatocyte culture.

    PubMed Central

    Rescan, P. Y.; Loréal, O.; Hassell, J. R.; Yamada, Y.; Guillouzo, A.; Clément, B.

    1993-01-01

    Basement membranes contain three major components (ie collagen IV, laminin, and the heparan sulfate proteoglycan termed perlecan). Although the distribution and origin of both collagen IV and laminin have been well documented in the liver, perlecan has been poorly investigated, so far. We have studied the distribution and cellular origin of perlecan in rat livers in various conditions as well as in hepatocyte primary culture. By immunolocalization in both adult and 18-day-old fetal liver, perlecan was found in portal spaces, around central veins, and throughout the lobule. Immunoelectron microscopy revealed its presence at the level of basement membranes surrounding bile ducts and blood vessels, and in the space of Disse discontinuously interacting with hepatocyte microvilli. Precursors of perlecan were detected in the rough endoplasmic reticulum of bile duct cells and both vascular and sinusoidal endothelial cells. Both hepatocytes and Ito cells were negative. Northern-blot analysis confirmed the lack of appreciable expression of perlecan in hepatocytes isolated from either fetal or adult livers. In 18-month-diethylnitrosamine-treated rat liver, perlecan was abundant in neoplastic nodules. Electron microscopic investigation revealed an almost continuous layer of perlecan in the space of Disse and intracellular staining in sinusoidal endothelial cells, only. Perlecan mRNAs were detectable in malignant nodules, and absent in hepatocytes from nontumorous areas. Hepatocytes expressed high levels of perlecan mRNAs only when put in culture. This expression was reduced in conditions that allow improvement of hepatocyte survival and function (ie addition of corticoids, dimethylsulfoxide or nicotinamide to the medium, or in coculture with liver epithelial cells from biliary origin). Immunolocalization by light and electron microscopy showed that deposition of the proteoglycan occurred in coculture, in basement membranelike structures located around hepatocyte cords. In

  15. Matriglycan: a novel polysaccharide that links dystroglycan to the basement membrane

    PubMed Central

    Yoshida-Moriguchi, Takako; Campbell, Kevin P

    2015-01-01

    Associations between cells and the basement membrane are critical for a variety of biological events including cell proliferation, cell migration, cell differentiation and the maintenance of tissue integrity. Dystroglycan is a highly glycosylated basement membrane receptor, and is involved in physiological processes that maintain integrity of the skeletal muscle, as well as development and function of the central nervous system. Aberrant O-glycosylation of the α subunit of this protein, and a concomitant loss of dystroglycan's ability to function as a receptor for extracellular matrix (ECM) ligands that bear laminin globular (LG) domains, occurs in several congenital/limb-girdle muscular dystrophies (also referred to as dystroglycanopathies). Recent genetic studies revealed that mutations in DAG1 (which encodes dystroglycan) and at least 17 other genes disrupt the ECM receptor function of dystroglycan and cause disease. Here, we summarize recent advances in our understanding of the enzymatic functions of two of these disease genes: the like-glycosyltransferase (LARGE) and protein O-mannose kinase (POMK, previously referred to as SGK196). In addition, we discuss the structure of the glycan that directly binds the ECM ligands and the mechanisms by which this functional motif is linked to dystroglycan. In light of the fact that dystroglycan functions as a matrix receptor and the polysaccharide synthesized by LARGE is the binding motif for matrix proteins, we propose to name this novel polysaccharide structure matriglycan. PMID:25882296

  16. Pericytes Regulate Vascular Basement Membrane Remodeling and Govern Neutrophil Extravasation during Inflammation

    PubMed Central

    Wang, Shijun; Cao, Canhong; Chen, Zhongming; Bankaitis, Vytas; Tzima, Eleni; Sheibani, Nader; Burridge, Keith

    2012-01-01

    During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation. PMID:23029055

  17. Laminin- and basement membrane-polycaprolactone blend nanofibers as a scaffold for regenerative medicine

    PubMed Central

    Neal, Rebekah A.; Lenz, Steven M.; Wang, Tiffany; Abebayehu, Daniel; Brooks, Benjamin P.C.; Ogle, Roy C.; Botchwey, Edward A.

    2016-01-01

    Mimicking one or more components of the basement membrane (BM) holds great promise for overcoming insufficiencies in tissue engineering therapies. We have electrospun laminin nanofibers (NFs) isolated from the murine Engelbreth-Holm Swarm (EHS) tumor and evaluated them as a scaffold for embryonic stem cell culture. Seeded human embryonic stem cells were found to better maintain their undifferentiated, colony environment when cultured on laminin NFs compared to laminin mats, with 75% remaining undifferentiated on NFs. Mouse embryonic stem cells cultured on 10% laminin-polycaprolactone (PCL) NFs maintained their colony formation for twice as long without passage compared to those on PCL or gelatin substrates. In addition, we have established a protocol for electrospinning reconstituted basement membrane aligned (RBM)-PCL NFs within 10° of angular deviation. Neuron-like PC12 cells show significantly greater attachment (p < 0.001) and percentage of neurite-extending cells in vitro on 10% RBM-PCL NFs when compared to 1% and 0% RBM-PCL NFs (p < 0.015 and p < 0.001, respectively). Together, these results implicate laminin- and RBM-PCL scaffolds as a promising biomimetic substrate for regenerative medicine applications.

  18. Binding of human plasminogen to basement-membrane (type IV) collagen.

    PubMed

    Stack, M S; Moser, T L; Pizzo, S V

    1992-05-15

    Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix. PMID:1599390

  19. Experimental study on nerve regeneration through the basement membrane tubes of the nerve, muscle, and artery.

    PubMed

    Itoh, S; Shinomiya, K; Samejima, H; Ohta, T; Ishizuki, M; Ichinose, S

    1996-01-01

    We evaluated neurotization after transplantation with lyophilized nerves, muscles, and arteries, and examined the possibility of practical application of long bridging grafts. Grafts of 10 mm and 25 mm of lyophilized nerves, muscles, and arteries harvested from Fisher rats were transplanted to the sciatic nerves of recipient Lewis rats. The histological changes undergone by short grafts were observed at weekly intervals. The sham-operated and isograft groups were used to compare the results of long grafts. In both the nerve and muscle-graft group, regenerated axons grew out through the residual basement membrane tube. But in the muscle graft group, phagocytosis of myofibril debris took longer than that of degenerated axons. No statistical differences were found between results of TSI, induced EMG, and quantitative analysis of myelinated axons in the nerve and muscle graft groups. No neurotization was noted in the long artery graft. In long grafts, laminin found on the basement membrane may not be sufficient to accelerate neurotization, and arteries should not be used for tubulization. PMID:9431514

  20. Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G.

    PubMed Central

    Heck, L. W.; Blackburn, W. D.; Irwin, M. H.; Abrahamson, D. R.

    1990-01-01

    To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2356859

  1. Mechanical Stretch on Human Skin Equivalents Increases the Epidermal Thickness and Develops the Basement Membrane

    PubMed Central

    Tokuyama, Eijiro; Nagai, Yusuke; Takahashi, Ken; Kimata, Yoshihiro; Naruse, Keiji

    2015-01-01

    All previous reports concerning the effect of stretch on cultured skin cells dealt with experiments on epidermal keratinocytes or dermal fibroblasts alone. The aim of the present study was to develop a system that allows application of stretch stimuli to human skin equivalents (HSEs), prepared by coculturing of these two types of cells. In addition, this study aimed to analyze the effect of a stretch on keratinization of the epidermis and on the basement membrane. HSEs were prepared in a gutter-like structure created with a porous silicone sheet in a silicone chamber. After 5-day stimulation with stretching, HSEs were analyzed histologically and immunohistologically. Stretch-stimulated HSEs had a thicker epidermal layer and expressed significantly greater levels of laminin 5 and collagen IV/VII in the basal layer compared with HSEs not subjected to stretch stimulation. Transmission electron microscopy revealed that the structure of the basement membrane was more developed in HSEs subjected to stretching. Our model may be relevant for extrapolating the effect of a stretch on the skin in a state similar to an in vivo system. This experimental system may be useful for analysis of the effects of stretch stimuli on skin properties and wound healing and is also expected to be applicable to an in vitro model of a hypertrophic scar in the future. PMID:26528823

  2. Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis.

    PubMed Central

    Kashtan, C; Fish, A J; Kleppel, M; Yoshioka, K; Michael, A F

    1986-01-01

    We probed epidermal basement membranes (EBM) of acid-urea denatured skin from members of kindreds with Alport-type familial nephritis (FN) for the presence of antigens reactive with Goodpasture sera (GPS) and serum (FNS) from an Alport patient who developed anti-glomerular basement membrane (GBM) nephritis in a renal allograft. By immunoblotting, GPS reacted primarily with the 28,000 molecular weight (mol wt) monomer but also the 24,000 mol wt and 26,000 mol wt monomers of the noncollagenous globular domain (NC1) of type IV collagen from normal human GBM, while FNS identified only the 26,000-mol wt monomer. FNS reacted with EBM of 12 controls and nine unaffected male kindred members but not EBM of eight affected males. Five affected females exhibited interrupted reactivity of FNS with EBM. GPS showed variable reactivity with EBM and was not discriminating with respect to Alport-type FN. FNS did not stain renal basement members of five affected males. However, the EBM, tubular basement membrane, and Bowman's capsules of affected males contained antigens reactive with GPS. These immunochemical studies suggest that the FNS antigen is distinct from Goodpasture antigen(s). The expression of FNS antigen located on the NC1 domain of type IV collagen is altered in basement membranes of patients with Alport-type FN, and the distribution of this antigenic anomaly within kindreds suggests X-linked dominant transmission of a defective gene. Images PMID:2428839

  3. In vitro irradiation of basement membrane enhances the invasiveness of breast cancer cells

    PubMed Central

    Paquette, B; Baptiste, C; Therriault, H; Arguin, G; Plouffe, B; Lemay, R

    2007-01-01

    Following removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast. Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation. PMID:17987037

  4. Development of an enzyme immunoassay specific for a core protein epitope of a novel small basement membrane associated heparan sulphate proteoglycan from human kidney.

    PubMed

    Stöcker, G; Stickeler, E; Switalla, S; Fischer, D C; Greiling, H; Haubeck, H D

    1997-02-01

    Heparan sulphate proteoglycans are major components of the glomerular basement membrane and play a key role in their molecular organization and function. Moreover, their presence is essential for the maintenance of the selective permeability of the glomerular basement membrane. Recently, we have isolated and characterized a novel, small basement membrane associated heparan sulphate proteoglycan from human aorta and kidney. Using specific monoclonal antibodies we have shown that the novel heparan sulphate proteoglycan is predominantly located in the glomerular basement membrane, to a lesser extent in the basement membrane of tubuli, and also in the mesangium. Turnover or, in the course of kidney diseases, degradation of heparan sulphate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulphate proteoglycan. Therefore, changes in the structure and function of glomerular basement membranes may be directly detected by measuring the excretion of a component of this basement menbrane, e. g. heparan sulphate proteoglycan into urine. Here we describe the establishment of an enzyme immunoassay for the sensitive detection of the novel, small heparan sulphate proteoglycan in urine. In this assay the specific monoclonal antibody 1F10/B8, which recognizes a core protein epitope, was used to detect the polyanionic heparan sulphate proteoglycan bound to the surface of a cationic charge modified microtitre plate. This assay allows the sensitive and specific detection of the small heparan sulphate proteoglycan, which is released from the glomerular basement membrane into urine during normal turnover and also in the course of kidney diseases. PMID:9056750

  5. Urinary excretion of glomerular basement membrane antigens in Alport's syndrome. A new diagnostic approach.

    PubMed

    Lubec, G; Balzar, E; Weissenbacher, G; Syré, G

    1978-05-01

    Alport's syndrome is defined by the combination of hereditary nephropathy and neurosensory deafness, and is diagnosed from the family history combined with renal electron microscopy. Immunoelectrophoresis of the urine of 8 of 12 children suspected of Alport's syndrome showed a precipitation line moving into the beta-zone, applying an antiglomerular basement membrane antibody derived from an immunised rabbit. All patients who showed the typical pattern of Alport's syndrome on renal electron microscopy were among the 8 cases whose urine gave this immunoelectrophoresis pattern. Additionally, 5 of the mothers of the 8 children excreted the same antigen in their urine. The urine of 30 healthy children and of 10 patients with the idiopathic nephrotic syndrome did not show the presence of this antigen. This characteristic sign of Alport's syndrome may therefore be useful for its detection. PMID:666354

  6. Fibrillary glomerulonephritis masquerading as rapidly progressive glomerulonephritis with pseudo-linear glomerular basement membrane staining.

    PubMed

    El-Husseini, Amr; Aycinena, Juan-Carlos; George, Bennet; Jennings, Stuart; Cornea, Virgilius; Sawaya, B Peter

    2015-10-01

    Fibrillary glomerulonephritis (FGN) is a rare disorder with poor renal prognosis. It is a heterogeneous disease associated with significant risk of end-stage renal disease (ESRD). Its etiology and pathogenesis have not been clearly identified. We report a case of a patient presenting with hypertensive crisis, nephrotic range proteinuria, and rapidly progressive glomerulonephritis (RPGN). The kidney biopsy demonstrates crescentic GN on light microscopy (LM) and strong pseudo-linear/globular glomerular basement membrane (GBM) staining for immunoglobulin G on immunofluorescence (IF), suggestive of anti-GBM disease. However, circulating anti-GBM antibodies were negative. Electron microscopy (EM) revealed fibrillary deposits in the GBM, confirming the diagnosis of FGN. Review of the literature revealed very few reported similar cases. It appears that severe hypertension and heavy proteinuria, while uncommon in anti-GBM disease, are consistent findings in RPGN form of FGN. PMID:26249548

  7. Rapidly progressive glomerulonephritis due to coexistent anti-glomerular basement membrane disease and fibrillary glomerulonephritis

    PubMed Central

    Cheungpasitporn, Wisit; Zacharek, Claudia C.; Fervenza, Fernando C.; Cornell, Lynn D.; Sethi, Sanjeev; Herrera Hernandez, Loren P.; Nasr, Samih H.; Alexander, Mariam P.

    2016-01-01

    Anti-glomerular basement membrane (anti-GBM) disease is a major cause of rapidly progressive glomerulonephritis (RPGN). On the other hand, fibrillary glomerulonephritis (GN) typically presents as proteinuria, hematuria and renal insufficiency, but rarely as RPGN. Without electron microscopy, the diagnosis of fibrillary GN can be missed. We report a 68-year-old white woman who presented with RPGN with kidney biopsy demonstrating diffuse crescentic GN on light microscopy. By immunofluorescence, there was bright linear staining of the GBMs and smudgy mesangial staining for immunoglobulin G, C3, and kappa and lambda light chain. Electron microscopy revealed fibrillary deposits in the GBM and mesangium. A serum test for anti-GBM antibody was positive. To our knowledge, this is the first report of coexistence of fibrillary GN in a patient with anti-GBM disease. Electron microscopy is critical to identify the coexistence of other GN in patients presenting with crescentic GN. PMID:26798468

  8. Alveolar haemorrhage in anti‐glomerular basement membrane disease without detectable antibodies by conventional assays

    PubMed Central

    Serisier, D J; Wong, R C W; Armstrong, J G

    2006-01-01

    Anti‐glomerular basement membrane (anti‐GBM) disease represents the spectrum of disease attributable to circulating anti‐GBM antibodies. While active anti‐GBM disease in the absence of circulating anti‐GBM antibodies has been described, it is considered rare with the use of current routinely available assays. We report four subjects with features consistent with active anti‐GBM antibody disease without detectable antibodies by routinely available enzyme linked immunosorbent assay (ELISA) and immunoblot techniques. All were smokers who presented with diffuse alveolar haemorrhage, minimal renal involvement, and undetectable anti‐GBM antibodies. Seronegative anti‐GBM disease with predominant pulmonary involvement may be more common than previously appreciated and should be part of the differential diagnosis for otherwise unexplained diffuse alveolar haemorrhage. Renal biopsy with immunofluorescent studies should be considered in the diagnostic evaluation of such subjects, including those with idiopathic pulmonary haemosiderosis. PMID:16807392

  9. Cell Receptor-Basement Membrane Interactions in Health and Disease: A Kidney-Centric View.

    PubMed

    Borza, Corina M; Chen, Xiwu; Zent, Roy; Pozzi, Ambra

    2015-01-01

    Cell-extracellular matrix (ECM) interactions are essential for tissue development, homeostasis, and response to injury. Basement membranes (BMs) are specialized ECMs that separate epithelial or endothelial cells from stromal components and interact with cells via cellular receptors, including integrins and discoidin domain receptors. Disruption of cell-BM interactions due to either injury or genetic defects in either the ECM components or cellular receptors often lead to irreversible tissue injury and loss of organ function. Animal models that lack specific BM components or receptors either globally or in selective tissues have been used to help with our understanding of the molecular mechanisms whereby cell-BM interactions regulate organ function in physiological and pathological conditions. We review recently published works on animal models that explore how cell-BM interactions regulate kidney homeostasis in both health and disease. PMID:26610916

  10. The Cerebrovascular Basement Membrane: Role in the Clearance of β-amyloid and Cerebral Amyloid Angiopathy

    PubMed Central

    Morris, Alan W. J.; Carare, Roxana O.; Schreiber, Stefanie; Hawkes, Cheryl A.

    2014-01-01

    Cerebral amyloid angiopathy (CAA), the accumulation of β-amyloid (Aβ) peptides in the walls of cerebral blood vessels, is observed in the majority of Alzheimer’s disease (AD) brains and is thought to be due to a failure of the aging brain to clear Aβ. Perivascular drainage of Aβ along cerebrovascular basement membranes (CVBMs) is one of the mechanisms by which Aβ is removed from the brain. CVBMs are specialized sheets of extracellular matrix that provide structural and functional support for cerebral blood vessels. Changes in CVBM composition and structure are observed in the aged and AD brain and may contribute to the development and progression of CAA. This review summarizes the properties of the CVBM, its role in mediating clearance of interstitial fluids and solutes from the brain, and evidence supporting a role for CVBM in the etiology of CAA. PMID:25285078

  11. A Novel Human Adipocyte-derived Basement Membrane for Tissue Engineering Applications

    NASA Astrophysics Data System (ADS)

    Damm, Aaron

    Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.

  12. Cell Division and Targeted Cell Cycle Arrest Opens and Stabilizes Basement Membrane Gaps

    PubMed Central

    Matus, David Q.; Chang, Emily; Makohon-Moore, Sasha C.; Hagedorn, Mary A.; Chi, Qiuyi; Sherwood, David R.

    2014-01-01

    Large gaps in basement membrane (BM) occur during organ remodeling and cancer cell invasion. Whether dividing cells, which temporarily reduce their attachment to BM, influence these breaches is unknown. Here we analyse uterine-vulval attachment during development across 21 species of rhabditid nematodes and find that the BM gap that forms between these organs is always bounded by a non-dividing vulval cell. Through cell cycle manipulation and live cell imaging in Caenorhabditis elegans, we show that actively dividing vulval cells facilitate enlargement of this breach by promoting BM movement. In contrast, targeted cell-cycle arrest halts BM movement and limits gap opening. Further, we demonstrate that the BM component laminin accumulates at the BM gap edge and promotes increased integrin levels in non-dividing vulval cells, stabilizing gap position. Together, these studies reveal that cell division can be used as a mechanism to regulate BM breaches, thus controlling the exchange of cells between tissues. PMID:24924309

  13. Cell Receptor-Basement Membrane Interactions in Health and Disease: a Kidney-Centric View

    PubMed Central

    Borza, Corina M.; Chen, Xiwu; Zent, Roy; Pozzi, Ambra

    2016-01-01

    Cell-extracellular matrix (ECM) interactions are essential for tissue development, homeostasis, and response to injury. Basement membranes (BMs) are specialized ECMs that separate epithelial or endothelial cells from stromal components and interact with cells via cellular receptors, including integrins and discoidin domain receptors. Disruption of cell-BM interactions due to either injury or genetic defects in either the ECM components or cellular receptors often lead to irreversible tissue injury and loss of organ function. Animal models that lack specific BM components or receptors either globally or in selective tissues have been used to help with our understanding of the molecular mechanisms whereby cell-BM interactions regulate organ function in physiological and pathological conditions. We review recently published work on animal models that explore how cell-BM interactions regulate kidney homeostasis in both health and disease. PMID:26610916

  14. A unique covalent bond in basement membrane is a primordial innovation for tissue evolution.

    PubMed

    Fidler, Aaron L; Vanacore, Roberto M; Chetyrkin, Sergei V; Pedchenko, Vadim K; Bhave, Gautam; Yin, Viravuth P; Stothers, Cody L; Rose, Kristie Lindsey; McDonald, W Hayes; Clark, Travis A; Borza, Dorin-Bogdan; Steele, Robert E; Ivy, Michael T; Hudson, Julie K; Hudson, Billy G

    2014-01-01

    Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad-a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids-is a primordial innovation of the ECM essential for organogenesis and tissue evolution. PMID:24344311

  15. A unique covalent bond in basement membrane is a primordial innovation for tissue evolution

    PubMed Central

    Fidler, Aaron L.; Vanacore, Roberto M.; Chetyrkin, Sergei V.; Pedchenko, Vadim K.; Bhave, Gautam; Yin, Viravuth P.; Stothers, Cody L.; Rose, Kristie Lindsey; McDonald, W. Hayes; Clark, Travis A.; Borza, Dorin-Bogdan; Steele, Robert E.; Ivy, Michael T.; Hudson, Julie K.; Hudson, Billy G.

    2014-01-01

    Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad—a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids—is a primordial innovation of the ECM essential for organogenesis and tissue evolution. PMID:24344311

  16. FoxC1 is essential for vascular basement membrane integrity and hyaloid vessel morphogenesis

    PubMed Central

    Skarie, Jonathan M.; Link, Brian A.

    2013-01-01

    Purpose Alterations in FOXC1 dosage lead to a spectrum of highly penetrant, ocular anterior segment dysgenesis phenotypes. The most serious outcome is development of glaucoma, but this only occurs in 50–75% of patients. Therefore, the need to identify specific pathways and genes that interact with FOXC1 to promote glaucoma is great. In this study, we investigated loss of foxC1 in the zebrafish to characterize phenotypes and gene interactions that may impact glaucoma pathogenesis. Methods Morpholino knockdown in zebrafish, RNA and protein marker analyses, transgenic reporter lines, and angiography, along with histology and transmission electron microscopy were used to study foxC1 function and gene interactions. Results Zebrafish foxC1 genes were expressed dynamically in the developing vasculature and periocular mesenchyme during development. Multiple ocular and vascular defects were found after knockdown of foxC1. Defects in the hyaloid vasculature, arterial-venous malformations, and coarctation of the aorta were observed with maximal depletion of foxC1. Partial loss of foxC1 resulted in CNS and ocular hemorrhages, defects in intersegmental vessel patterning, and increased vascular permeability. To investigate the basis for these disruptions, ultrastructure of foxC1-depleted hyaloid vascular cells was studied. These experiments, along with Laminin-111 immunoreactivity, revealed disruptions in basement membrane integrity. Finally, co-depletion of laminin alpha-1 and foxC1 uncovered a genetic interaction between these genes during development. Conclusions Genetic interactions between FOXC1 and basement membrane components influence vascular stability and may impact glaucoma development and increase stroke risk in FOXC1 patients. PMID:19458328

  17. An Overlapping Case of Alport Syndrome and Thin Basement Membrane Disease

    PubMed Central

    Alganabi, Mashriq; Eter, Ahmad

    2016-01-01

    We report a case of a 48-year-old male who presented with hematuria of at least 10 years, and has a daughter with hematuria as well. The patient has a history of degenerative hearing loss, decreased vision and cataract formation, but no diabetes, hypertension or proteinuria. A full serology and urology workup was negative for any abnormality. A kidney biopsy for the patient revealed a diagnosis of Alport syndrome but was unable to rule out thin basement membrane disease. The biopsy was inconclusive in making the diagnosis but the patient’s clinical presentation led to the diagnosis of Alport syndrome. The patient’s 10-year-old daughter also has hematuria with no clear etiology but now can subsequently be anticipatorily managed for Alport syndrome progression. Due to the rarity of the disease, diagnosis is often missed or delayed by primary care providers especially when no associated proteinuria has yet developed. This can lead to confusion and misdiagnosis with thin basement membrane disease, a generally benign hematuria without kidney failure progression. Additionally, biopsy can be inconclusive in these patients, relying on the physician’s history and physical examination findings to diagnose. It is important to appropriately diagnose Alport syndrome not only to manage the patient’s rate of kidney failure progression but also allow for a higher degree of suspicion, screening and intervention in the patient’s family members. Both the inconclusive nature of kidney biopsies and the usefulness of diagnosis for family member screening are often overlooked in medical literature but are explored in this case.

  18. Precise, biomimetic replication of the multiscale structure of intestinal basement membrane using chemical vapor deposition.

    PubMed

    Pfluger, Courtney A; McMahon, Brian J; Carrier, Rebecca L; Burkey, Daniel D

    2013-03-01

    While it has been shown that cells respond to topographical cues, most studies of the influence of topography have been restricted to culture substrates with regular, single-scale features, such as grooves. In contrast, in vivo topography is highly complex, irregular, and multiscale. In this work, we demonstrate the use of chemical vapor deposition (CVD) on native tissue to fabricate a precise nonbiological replica of irregular macro-to-microscale biological topography. Specifically, the porcine intestinal basement membrane was decellularized and used as a template to create a silica replica from which tissue was removed to produce a free-standing topographically biomimetic silica film. Preservation of the crypt-villus structure (tens to hundreds of micrometers in scale), which is theorized to influence intestinal cell development and behavior, as well as the porosity of the native tissue membrane (1-5 μM in scale), was demonstrated; however, submicrometer topography appeared to be masked by ball-like structures believed to be a result of the CVD process. CVD process parameters, including reactor pressure and deposition temperature, were explored in efforts to enhance structural and mechanical integrity of the silica replica. A rigid inorganic replica can be used as a template for casting of biocompatible polymeric membranes; thus, this is the first step in fabricating cell culture substrates that precisely mimic their in vivo counterparts in terms of irregular, multiscale topography. PMID:23013380

  19. Insecticidal Activity of a Basement Membrane-Degrading Protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, signific...

  20. Association of malachite green-positive material with heparan sulfate proteoglycan double tracks in basement membrane of mouse kidney tubules.

    PubMed

    Inoue, S

    1995-03-01

    The presence of lipids in the basement membrane of the mouse kidney tubules was examined by histochemical staining with malachite green. Pieces of mouse kidney cortex were immersed in a fixative containing 3% glutaraldehyde and 0.1% malachite green in 0.067 M sodium cacodylate buffer, pH 6.8, for 18 hr at 4 degrees C. Control tissue was fixed in the same way except that no malachite green was added to the fixative. The tissue pieces were cryoprotected, frozen in Freon 22, and subjected to freeze-substitution in dry acetone containing 1% OsO4. Thin sections of Epon-embedded specimens were observed by electron microscopy at first without uranyl-lead counterstaining. The basement membrane of mouse kidney tubules was positively stained in a pattern composed of an irregular assembly of 5-8-nm wide strands. The nature of these malachite green-positive strands was further examined by counterstaining thin sections with uranyl-lead, and they were identified as 4.5-5-nm wide ribbon-like "double tracks" previously characterized as the form taken by heparan sulfate proteoglycan in basement membranes. It is concluded that lipids are present in the basement membrane of mouse kidney tubules in association with heparan sulfate proteoglycan. PMID:7868858

  1. Vitamin K1-induced localized scleroderma (morphea) with linear deposition of IgA in the basement membrane zone.

    PubMed

    Alonso-Llamazares, J; Ahmed, I

    1998-02-01

    We describe a 45-year-old white man in whom distinctive clinical and histologic features of localized scleroderma developed at sites of injection of vitamin K1 (phytonadione). A direct immunofluorescence test demonstrated prominent linear deposition of IgA along the basement membrane zone. No circulating antibasement membrane zone IgA antibodies were identified on indirect immunofluorescence testing. We believe that the unusual immunofluorescence finding in our patient is nonspecific and represents an epiphenomenon caused by cutaneous injury. PMID:9486707

  2. MT1-MMP-mediated basement membrane remodeling modulates renal development

    SciTech Connect

    Riggins, Karen S.; Mernaugh, Glenda; Su, Yan; Quaranta, Vito; Koshikawa, Naohiko; Seiki, Motoharu; Pozzi, Ambra; Zent, Roy

    2010-10-15

    Extracellular matrix (ECM) remodeling regulates multiple cellular functions required for normal development and tissue repair. Matrix metalloproteinases (MMPs) are key mediators of this process and membrane targeted MMPs (MT-MMPs) in particular have been shown to be important in normal development of specific organs. In this study we investigated the role of MT1-MMP in kidney development. We demonstrate that loss of MT1-MMP leads to a renal phenotype characterized by a moderate decrease in ureteric bud branching morphogenesis and a severe proliferation defect. The kidneys of MT1-MMP-null mice have increased deposition of collagen IV, laminins, perlecan, and nidogen and the phenotype is independent of the MT-1MMP target, MMP-2. Utilizing in vitro systems we demonstrated that MTI-MMP proteolytic activity is required for renal tubule cells to proliferate in three dimensional matrices and to migrate on collagen IV and laminins. Together these data suggest an important role for MT1-MMP in kidney development, which is mediated by its ability to regulate cell proliferation and migration by proteolytically cleaving kidney basement membrane components.

  3. Diabetes-induced morphological, biomechanical, and compositional changes in ocular basement membranes.

    PubMed

    To, Margaret; Goz, Alexandra; Camenzind, Leon; Oertle, Philipp; Candiello, Joseph; Sullivan, Mara; Henrich, Paul Bernhard; Loparic, Marko; Safi, Farhad; Eller, Andrew; Halfter, Willi

    2013-11-01

    The current study investigates the structural and compositional changes of ocular basement membranes (BMs) during long-term diabetes. By comparing retinal vascular BMs and the inner limiting membrane (ILM) from diabetic and non-diabetic human eyes by light and transmission electron microscopy (TEM), a massive, diabetes-related increase in the thickness of these BMs was detected. The increase in ILM thickness was confirmed by atomic force microscopy (AFM) on native ILM flat-mount preparations. AFM also detected a diabetes-induced increase in ILM stiffness. The changes in BM morphology and biophysical properties were accompanied by partial changes in the biochemical composition as shown by immunocytochemistry and western blots: agrin, fibronectin and tenascin underwent relative increases in concentration in diabetic BMs as compared to non-diabetic BMs. Fibronectin and tenascin were particularly high in the BMs of outlining microvascular aneurisms. The present data showed that retinal vascular BMs and the ILM undergo morphological, biomechanical and compositional changes during long-term diabetes. The increase in BM thickness not only resulted from an up-regulation of the standard BM proteins, but also from the expression of diabetes-specific extracellular matrix proteins that are not normally found in retinal BMs. PMID:24095823

  4. Co-existence of thin basement membrane nephropathy with other glomerular pathologies; a single center experience

    PubMed Central

    Qazi, Rizwan A.; Bastani, Bahar

    2015-01-01

    Background: The co-existence of thin basement membrane nephropathy (TBMN) and another glomerular pathology portends a worse prognosis than TBMN alone. Objectives: The purpose of our study was to investigate the prevalence of TBMN and associated glomerular pathologies at our institution. Patients and Methods: We reviewed all renal biopsies performed at Saint Louis University hospital over a 7-year period. We excluded all post transplant biopsies, and biopsies showing diabetic glomerulopathy, membranoproliferative glomerulopathy, membranous glomerulopathy, and biopsies where no electron microscopy or immunofluorescent studies were done. All other biopsies were included. Results: A total of 634 biopsies were included in the study. The prevalence of TBMN was 47 (7.4%), of whom 17 (36.2%) had TBMN alone. In the remaining 30 (63.8%) patients TBMN was associated with other glomerular pathologies: IgAN 9 (19.1%) and FSGS 9 (19.1%). We found significantly higher prevalence of IgAN in patients with TBMN versus all biopsies (19.1% vs. 7.7%, respectively, P = 0.002). We found significant similarities in biopsy indications for TBMN and IgAN group. Conclusions: Around two thirds of the cases of TBMN were associated with other glomerular pathologies. The prevalence of IgAN, but not focal segmental glomerulosclerosis, was significantly higher in patients with TBMN as compared to the general renal biopsy specimens. PMID:25964888

  5. Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles

    NASA Technical Reports Server (NTRS)

    Costes, S.; Streuli, C. H.; Barcellos-Hoff, M. H.

    2000-01-01

    We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.

  6. Capillaries demonstrate changes in membrane potential in response to pharmacological stimuli.

    PubMed

    McGahren, E D; Beach, J M; Duling, B R

    1998-01-01

    It has been proposed that capillaries can detect changes in tissue metabolites and generate signals that are communicated upstream to resistance vessels. The mechanism for this communication may involve changes in capillary endothelial cell membrane potentials which are then conducted to upstream arterioles. We have tested the capacity of capillary endothelial cells in vivo to respond to pharmacological stimuli. In a hamster cheek pouch preparation, capillary endothelial cells were labeled with the voltage-sensitive dye di-8-ANEPPS. Fluorescence from capillary segments (75-150 microns long) was excited at 475 nm and recorded at 560 and 620 nm with a dual-wavelength photomultiplier system. KCl was applied using pressure injection, and acetylcholine (ACh) and phenylephrine (PE) were applied iontophoretically to these capillaries. Changes in the ratio of the fluorescence emission at two emission wavelengths were used to estimate changes in the capillary endothelial membrane potential. Application of KCl resulted in depolarization, whereas application of the vehicle did not. Application of ACh and PE resulted in hyperpolarization and depolarization, respectively. The capillary responses could be blocked by including a receptor antagonist (atropine or prazosin, respectively) in the superfusate. We conclude that the capillary membrane potential is capable of responding to pharmacological stimuli. We hypothesize that capillaries can respond to changes in the milieu of surrounding tissue via changes in endothelial membrane potential. PMID:9458852

  7. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

    SciTech Connect

    BARCELLOS-HOFF, M. H; AGGELER, J.; RAM, T. G; BISSELL, M. J

    1989-02-01

    An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrixensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar

  8. [Effect of dithranol therapy on membrane, basement membrane and nuclear markers in psoriasis lesions].

    PubMed

    Wollina, U; Schlesier, F; Schaarschmidt, H; Knopf, B; Hipler, C; Henkel, U; Roth, H; Bartá, U

    1987-02-15

    We investigated the effects of anti-psoriatic therapy with dithranol (1/20-1%) in salicylic acid (0.5%) in white petrolatum on lesional skin. FITC-labeled lectins and pemphigus vulgaris antibodies (PV) served as analytical means to study the glycocalyx. Antibodies of bullous pemphigoid (BP) were used as basal membrane markers. Nuclear antigens were recorded according to the binding of speckled, anti-nuclear antibodies (ANA) as well as antibodies to dsDNA. With some lectins, dithranol therapy resulted in pronounced fluorescence of the lower parts of the basal cells. ConA was fixed by the basal cell layer. To a lesser degree, ANA were fixed by nuclei of keratinocytes. PV antibodies were not fixed at all. PMID:3554801

  9. Role for Macrophage Metalloelastase in Glomerular Basement Membrane Damage Associated with Alport Syndrome

    PubMed Central

    Rao, Velidi H.; Meehan, Daniel T.; Delimont, Duane; Nakajima, Motowo; Wada, Takashi; Ann Gratton, Michael; Cosgrove, Dominic

    2006-01-01

    Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic strategy for Alport glomerular disease. PMID:16816359

  10. Degradation of endothelial basement membrane by human breast cancer cell lines

    SciTech Connect

    Yee, C.; Shiu, R.P.

    1986-04-01

    During metastasis, it is believed that tumor cells destroy the basement membrane (BM) of blood vessels in order to disseminate through the circulatory system. By radioactively labeling the extracellular matrix produced by primary endothelial cells in vitro, the ability of human breast cancer cells to degrade BM components was studied. We found that T-47D, a human breast cancer line, was able to degrade significant amounts of (35S)methionine-labeled and (3H)proline-labeled BM, but not 35SO4-labeled BM. Six other tumor cell lines of human breast origin were assayed in the same manner and were found to degrade BM to varying degrees. Several non-tumor cell lines tested showed relatively little degrading activity. The use of serum-free medium greatly enhanced degradation of the BM by tumor cells, suggesting a role for naturally occurring enzyme inhibitors in the serum. Direct cell contact with the BM was required for BM degradation, suggesting that the active enzymes are cell associated. The addition of hormones implicated in the etiology of breast cancer did not significantly alter the ability of T-47D cells to degrade the BM. The use of this assay affords future studies on the mechanism of invasion and metastasis of human breast cancer.

  11. SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane

    PubMed Central

    Morrissey, Meghan A.; Jayadev, Ranjay; Miley, Ginger R.; Blebea, Catherine A.; Chi, Qiuyi; Ihara, Shinji; Sherwood, David R.

    2016-01-01

    Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells. PMID:26926673

  12. SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane.

    PubMed

    Morrissey, Meghan A; Jayadev, Ranjay; Miley, Ginger R; Blebea, Catherine A; Chi, Qiuyi; Ihara, Shinji; Sherwood, David R

    2016-02-01

    Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells. PMID:26926673

  13. Cleavage of Nidogen-1 by Cathepsin S Impairs Its Binding to Basement Membrane Partners

    PubMed Central

    Sage, Juliette; Leblanc-Noblesse, Emmanuelle; Nizard, Carine; Sasaki, Takako; Schnebert, Sylvianne; Perrier, Eric; Kurfurst, Robin; Brömme, Dieter; Lalmanach, Gilles; Lecaille, Fabien

    2012-01-01

    Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine ctss−/− spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases. PMID:22952693

  14. Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa.

    PubMed

    Arduino, Paolo G; Carrozzo, Marco; Pagano, Marco; Broccoletti, Roberto; Scully, Crispian; Gandolfo, Sergio

    2010-06-01

    Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone. PMID:19506920

  15. Does Tensile Rupture of Tumor Basement Membrane Mark the Onset of Cancer Metastasis?

    NASA Astrophysics Data System (ADS)

    Prakash, Sai

    2015-03-01

    Recognizing a conceptual analogy from polymer physics and reasoning via induction, we infer the plausibility that a malignant tumor (carcinoma) grows in size until a threshold determined by its mechanochemical state in relation to its microenvironment whence, peripheral cells undergo epithelial-to-mesenchymal transitions (EMT) facilitating metastasis. This state is equated to the tensile yielding/rupture of the proteolytically-weakened basement membrane (BM) that encapsulates the growing neoplasm. BMs are typically constituted of tri-continuous hydrogel networks of collagen-IV, laminin, and interstitial fluid, with connector proteins such as nidogens, and perlecans. We test this postulate by formulating a theoretical model based on continuum fluid-solid mechanics, diffusion, and biochemical kinetics of energy metabolism. Herein, a prototypical, viscous tumor spheroid grows radially, consuming metabolic nutrients while being constrained by an elastic BM ca. 0.5-2 microns-thick, and cell adhesion molecules (CAMs), chiefly cadherins and integrins. The model is computationally analyzed via Comsol®. Results validate the a priori conjecture, and predict subsequent crack-tip stresses shifting strains on the CAMs from compressive to tensile, that might also indicate mechanotransduced switches in their conformations, such as from non-invasive, adhesive E-cadherins to invasive, non-adhesive N-cadherin phenotypes. Grant from Brady Urological Institute, JHMI.

  16. The role of the basement membrane as a modulator of intestinal epithelial-mesenchymal interactions.

    PubMed

    Simon-Assmann, P; Spenle, C; Lefebvre, O; Kedinger, M

    2010-01-01

    Intestinal development is a process of continuous dynamic bidirectional crosstalk between epithelial and underlying mesenchymal cells. This crosstalk is mediated by well-dissected signaling pathways. Another crucial actor in the epithelio-mesenchymal interactions is the stromal microenvironment, which is composed of extracellular matrix molecules. Among them, the basement membrane (BM) molecules are secreted by the epithelium and mesenchyme in a complementary manner. These molecules signal back to the cells via the integrins or other specific receptors. In this review, we mainly focus on the BM molecules, particularly laminins. The major BM molecules are organized in a complex molecular network, which is highly variable among organs. Cell culture, coculture, and grafting models have been of great interest in understanding the importance of these molecules. Mouse gene ablation of laminin chains are interesting models, which often lead to embryonic death and are frequently accompanied by compensatory processes. Overall, the BM molecules have a crucial role in the careful maintenance of intestinal homeostasis. PMID:21075345

  17. Cadherin 11 Involved in Basement Membrane Damage and Dermal Changes in Melasma.

    PubMed

    Kim, Nan-Hyung; Choi, Soo-Hyun; Lee, Tae Ryong; Lee, Chang-Hoon; Lee, Ai-Young

    2016-06-15

    Basement membrane (BM) disruption and dermal changes (elastosis, collagenolysis, vascular ectasia) have been reported in melasma. Although ultraviolet (UV) irradiation can induce these changes, UV is not always necessary for melasma development. Cadherin 11 (CDH11), which is upregulated in some melasma patients, has previously been shown to stimulate melanogenesis. Because CDH11 action requires cell-cell adhesion between fibroblasts and melanocytes, BM disruption in vivo should facilitate this. The aim of this study was to examine whether CDH11 overexpression leads to BM disruption and dermal changes, independent of UV irradiation. Immunohistochemistry/immunofluorescence, real-time PCR, Western blotting, and zymography suggested that BM disruption/dermal changes and related factors were present in the hyperpigmented skin of CDH11-upregulated melasma patients and in CDH11-overexpressing fibroblasts/keratinocytes. The opposite was seen in CDH11-knockdown cells. UV irradiation of the cultured cells did not increase CDH11 expression. Collectively, these data demonstrate that CDH11 overexpression could induce BM disruption and dermal changes in melasma, regardless of UV exposure. PMID:26671310

  18. Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila.

    PubMed

    Isabella, Adam J; Horne-Badovinac, Sally

    2015-10-15

    Basement membranes (BMs) are sheet-like extracellular matrices that provide essential support to epithelial tissues. Recent evidence suggests that regulated changes in BM architecture can direct tissue morphogenesis, but the mechanisms by which cells remodel BMs are largely unknown. The Drosophila egg chamber is an organ-like structure that transforms from a spherical to an ellipsoidal shape as it matures. This elongation coincides with a stage-specific increase in Type IV Collagen (Col IV) levels in the BM surrounding the egg chamber; however, the mechanisms and morphogenetic relevance of this remodeling event have not been established. Here, we identify the Collagen-binding protein SPARC as a negative regulator of egg chamber elongation, and show that SPARC down-regulation is necessary for the increase in Col IV levels to occur. We find that SPARC interacts with Col IV prior to secretion and propose that, through this interaction, SPARC blocks the incorporation of newly synthesized Col IV into the BM. We additionally observe a decrease in Perlecan levels during elongation, and show that Perlecan is a negative regulator of this process. These data provide mechanistic insight into SPARC's conserved role in matrix dynamics and demonstrate that regulated changes in BM composition influence organ morphogenesis. PMID:26348027

  19. D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen maturation and basement membrane function

    PubMed Central

    Sasaki, Masato; Knobbe, Christiane B.; Itsumi, Momoe; Elia, Andrew J.; Harris, Isaac S.; Chio, Iok In Christine; Cairns, Rob A.; McCracken, Susan; Wakeham, Andrew; Haight, Jillian; Ten, Annick You; Snow, Bryan; Ueda, Takeshi; Inoue, Satoshi; Yamamoto, Kazuo; Ko, Myunggon; Rao, Anjana; Yen, Katharine E.; Su, Shinsan M.; Mak, Tak Wah

    2012-01-01

    Isocitrate dehydrogenase-1 (IDH1) R132 mutations occur in glioma, but their physiological significance is unknown. Here we describe the generation and characterization of brain-specific Idh1 R132H conditional knock-in (KI) mice. Idh1 mutation results in hemorrhage and perinatal lethality. Surprisingly, intracellular reactive oxygen species (ROS) are attenuated in Idh1-KI brain cells despite an apparent increase in the NADP+/NADPH ratio. Idh1-KI cells also show high levels of D-2-hydroxyglutarate (D2HG) that are associated with inhibited prolyl-hydroxylation of hypoxia-inducible transcription factor-1α (Hif1α) and up-regulated Hif1α target gene transcription. Intriguingly, D2HG also blocks prolyl-hydroxylation of collagen, causing a defect in collagen protein maturation. An endoplasmic reticulum (ER) stress response induced by the accumulation of immature collagens may account for the embryonic lethality of these mutants. Importantly, D2HG-mediated impairment of collagen maturation also led to basement membrane (BM) aberrations that could play a part in glioma progression. Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects. PMID:22925884

  20. Relationship between blood and bronchial submucosal eosinophilia and reticular basement membrane thickening in chronic obstructive pulmonary disease.

    PubMed

    Eltboli, Osama; Mistry, Vijay; Barker, Bethan; Brightling, Chris E

    2015-05-01

    A sputum eosinophilia is observed in 10-40% of COPD subjects. The blood eosinophil count is a biomarker of sputum eosinophilia, but whether it is associated with bronchial submucosal eosinophils is unclear. In 20 COPD subjects and 21 controls we assessed the number of bronchial submucosal eosinophils and reticular basement membrane thickening and found these were positively correlated with the blood eosinophil percentage. In COPD, blood eosinophils are a good biomarker of bronchial eosinophilia and remodelling. PMID:25645275

  1. Relationship between blood and bronchial submucosal eosinophilia and reticular basement membrane thickening in chronic obstructive pulmonary disease

    PubMed Central

    Eltboli, Osama; Mistry, Vijay; Barker, Bethan

    2015-01-01

    A sputum eosinophilia is observed in 10–40% of COPD subjects. The blood eosinophil count is a biomarker of sputum eosinophilia, but whether it is associated with bronchial submucosal eosinophils is unclear. In 20 COPD subjects and 21 controls we assessed the number of bronchial submucosal eosinophils and reticular basement membrane thickening and found these were positively correlated with the blood eosinophil percentage. In COPD, blood eosinophils are a good biomarker of bronchial eosinophilia and remodelling. PMID:25645275

  2. Organogenesis of mild ocular coloboma in FLS mice: failure of basement membrane disintegration at optic fissure margins.

    PubMed

    Tsuji, Naho; Kita, Katsutoshi; Ozaki, Kiyokazu; Narama, Isao; Matsuura, Tetsuro

    2012-01-01

    Fatty Liver Shionogi (FLS) mice have been shown to develop a hereditary disorder characterized by localized retinochoroidal defects of the ventral fundus very similar to human typical ocular coloboma without microphthalmia. The objective of this study was to determine when and how the failure of the optic fissure closure occurs, and to clarify the disturbed mechanism of basement membrane disintegration during embryonal stage in FLS mice. Fetuses at day 11.5-15.5 of gestation were obtained from dams of FLS and BALB/c strain of mice. Coronal serial sections through the eye were examined by light and electron microscopy. The sections were followed by observation of the basement membrane using reaction with periodic acid-Schiff (PAS) reagent and immunohistochemical staining with anti-Laminin and anti-Type IV collagen antibodies. Both optic fissure margins closely approached each other up to GD 11.5 in all FLS and BALB/c embryos. The inner and outer layers of the optic cup did not normally fuse at midlenticular levels of the optic fissure in almost 70% of FLS fetuses by GD 15.5, whereas both margins were completely fused in all BALB/c fetuses of the same gestational day. In the FLS fetuses at GD 12.5, rolling on one side of fissure margins and consequent asymmetry were observed at the ventral optic fissure. The basement membrane persisted after the close contact of both sides of the fissure margins during GD 11.5 and 15.5. Ultrastructurally, the basal lamina was not disintegrated and mesenchymal cells intervened between the two neuroepithelial layers, resulting in complete separation of both fissure margins at GD 13.0. It is highly probable that the disturbed basement membrane disintegration right before optic fissure closure causes mild ocular coloboma without microphthalmia in FLS mice. PMID:22182670

  3. Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa.

    PubMed

    Watt, Stephen A; Dayal, Jasbani H S; Wright, Sheila; Riddle, Megan; Pourreyron, Celine; McMillan, James R; Kimble, Roy M; Prisco, Marco; Gartner, Ulrike; Warbrick, Emma; McLean, W H Irwin; Leigh, Irene M; McGrath, John A; Salas-Alanis, Julio C; Tolar, Jakub; South, Andrew P

    2015-01-01

    Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention. PMID:26380979

  4. Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

    PubMed Central

    Watt, Stephen A.; Dayal, Jasbani H. S.; Wright, Sheila; Riddle, Megan; Pourreyron, Celine; McMillan, James R.; Kimble, Roy M.; Prisco, Marco; Gartner, Ulrike; Warbrick, Emma; McLean, W. H. Irwin; Leigh, Irene M.; McGrath, John A.; Salas-Alanis, Julio C.; Tolar, Jakub; South, Andrew P.

    2015-01-01

    Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention. PMID:26380979

  5. Laminin Production and Basement Membrane Deposition by Mesenchymal Stem Cells upon Adipogenic Differentiation

    PubMed Central

    Sillat, Tarvo; Virtanen, Ismo; Ingerpuu, Sulev; Bäck, Nils; Konttinen, Yrjö T.; Korhonen, Matti

    2013-01-01

    The aim was to study laminin (LM) synthesis, integration, and deposition into the basement membrane (BM) during adipogenesis. Human bone marrow-derived mesenchymal stromal cells (MSCs) were induced along the adipogenic lineage. LM chain mRNA and protein levels were followed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, transmission electron microscopy (TEM), and immunoprecipitation. MSCs produced low levels of LM mRNAs but were not surrounded by BM in IF and TEM imaging. LM-α4, LM-β1, and LM-γ1 mRNAs increased during adipogenesis 3.9-, 5.8-, and 2.8-fold by day 28. LM-411 was immunoprecipitated from the ECM of the differentiated cells. Immunostaining suggested deposition of LM-411 and some LM-421. BM build-up was probably organized in part by integrin (Int) α6β1. At day 28, TEM images revealed BM-like structures around fat droplet-containing cells. The first signs of BM formation and Int α6β1 were seen using IF imaging at day 14. Laminin-411 and Int α6β1 were expressed in vivo in mature human subcutaneous fat tissue. Undifferentiated human MSCs did not organize LM subunits into BM, whereas LM-411 and some LM-421 are precipitated in the BM around adipocytes. This is the first demonstration of LM-411 precipitation during hMSC adipogenesis around adipocytes as a structural scaffold and Int-regulated signaling element. PMID:23900596

  6. Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid arteries.

    PubMed

    Southgate, K M; Fisher, M; Banning, A P; Thurston, V J; Baker, A H; Fabunmi, R P; Groves, P H; Davies, M; Newby, A C

    1996-12-01

    Basement membrane-degrading metalloproteinases (gelatinases) appear necessary for vascular smooth muscle cell migration and proliferation in culture and for intimal migration of cells after balloon injury to the rat carotid artery. We investigated in the present study the secretion of gelatinases from pig carotid artery tissue after balloon injury. Segments of injured artery and segments proximal and distal to the area of injury were removed 3, 7, and 21 days after balloon dilatation. Medial explants from these segments were then cultured for 3 days, and the serum-free conditioned media were subjected to gelatin zymography. Production of 72- and 95-kD gelatinases was quantified by densitometry. Balloon-injured segments secreted significantly more 72- and 95-kD gelatinase than did paired distal segments at all time points. Release of both gelatinase activities was increased at 3 and 7 days relative to segments from uninjured arteries but declined again by 21 days after balloon injury. Similar results were found for gelatinase levels in extracts of arterial tissue. Consistent with the protein secretion data, in situ hybridization demonstrated that the mRNAs for both gelatinases were upregulated after balloon injury. Expression was prominent in medial smooth muscle cells, particularly around foci of necrosis, and in neointimal cells 3 and 7 days after balloon injury; 72-kD gelatinase mRNA persisted after 21 days and was prominent in regrown endothelial cells. The upregulation of gelatinase activity paralleled the time course of smooth muscle cell migration and proliferation in this model. We conclude that increased gelatinase production occurs in response to balloon injury and may play a role in permitting migration and proliferation of vascular smooth muscle cells. PMID:8943956

  7. Effects of dietary protein on glomerular mesangial area and basement membrane thickness in aged uninephrectomized dogs.

    PubMed Central

    McCarthy, R A; Steffens, W L; Brown, C A; Brown, S A; Ard, M; Finco, D R

    2001-01-01

    The primary objective of this study was to determine the effects of diets containing 18% or 34% protein on glomerular mesangial area (GMA) and basement membrane thickness (GBMT) in uninephrectomized aged dogs. A secondary objective was to determine the combined effects of aging and uninephrectomy on GMA and GBMT in dogs. Ten clinically healthy, pure-bred dogs were unilaterally nephrectomized at about 8 y of age. After 2 mo, 5 dogs were fed an 18% protein diet and 5 dogs were fed a 34% protein diet for 48 mo. At month 48, the dogs were euthanized and the remaining kidney was collected. Samples of kidney from both times of collection were used to measure GMA and GBMT using electron microscopy. The effects of diet on GMA and GBMT were analyzed (student's t-test) using necropsy/nephrectomy score ratios. The effects of time-nephrectomy were determined by comparing nephrectomy values for GMA and GBMT with necropsy values (paired t-test). Dogs fed 34% dietary protein did not have a significant increase in GMA and GBM thickness when compared to dogs fed the 18% protein diet. A significant increase in GMA and GBMT occurred with time-nephrectomy (P = 0.011 and 0.018, respectively). Although dietary protein intake was not a significant factor in causing structural changes to glomeruli in uninephrectomized aged dogs, the power to detect a difference was low. However, significant effects of aging and nephrectomy were detected despite the low power of the study. These results suggest that the increases in GMA and GBMT that occur over time are not markedly influenced by dietary protein intake. However, subtle protein effects cannot be eliminated as a possibility based on this study. Images Figure 2. Figure 3. PMID:11346257

  8. A Case of Fibrillary Glomerulonephritis Associated with Thrombotic Microangiopathy and Anti-Glomerular Basement Membrane Antibody

    PubMed Central

    Momose, Akishi; Nakajima, Taku; Chiba, Shigetoshi; Kumakawa, Kenjirou; Shiraiwa, Yasuo; Sasaki, Nobuhiro; Watanabe, Kazuo; Kitano, Etsuko; Hatanaka, Mitiyo; Kitamura, Hajime

    2015-01-01

    We present the first report of a case of fibrillary glomerulonephritis (FGN) associated with thrombotic microangiopathy (TMA) and anti-glomerular basement membrane antibody (anti-GBM antibody). A 54-year-old man was admitted to our hospital for high fever and anuria. On the first hospital day, we initiated hemodialysis for renal dysfunction. Laboratory data revealed normocytic-normochromic anemia with schistocytes in the peripheral smear, thrombocytopenia, increased serum lactate dehydrogenase, decreased serum haptoglobin, and negative results for both direct and indirect Coombs tests. Based on these results, we diagnosed TMA. Assays conducted several days later indicated a disintegrin-like and metalloprotease with a thrombospondin motif 13 (ADAMTS13) activity of 31.6%, and ADAMTS13 inhibitors were negative. We started plasma exchange using fresh frozen plasma and steroid pulse therapy. Anti-GBM antibody was found to be positive. Renal biopsy showed FGN. Blood pressure rose on the 46th hospital day, and mild convulsions developed. Based on magnetic resonance imaging of the head, the patient was diagnosed with reversible posterior leukoencephalopathy syndrome. Hypertension persisted despite administration of multiple antihypertensive agents, and the patient experienced a sudden generalized seizure. Computed tomography of the head showed multiple cerebral hemorrhages. However, his blood pressure subsequently decreased and the platelet count increased. TMA remitted following 36 plasma exchange sessions, but renal function was not restored, and maintenance hemodialysis was continued. The patient was discharged on the 119th day of hospitalization. In conclusion, it was shown that TMA, FGN and anti-GBM antibody were closely related. PMID:25873933

  9. Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen.

    PubMed

    Foster, Mary H; Buckley, Elizabeth S; Chen, Benny J; Hwang, Kwan-Ki; Clark, Amy G

    2016-08-01

    Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion. PMID:27450516

  10. Podocytic cytoskeletal disaggregation and basement-membrane detachment in puromycin aminonucleoside nephrosis.

    PubMed Central

    Whiteside, C. I.; Cameron, R.; Munk, S.; Levy, J.

    1993-01-01

    Puromycin aminonucleoside--(PAN) treated rats develop acute nephrotic syndrome, mimicking human minimal lesion disease. In PAN nephrosis, podocyte detachment from the glomerular basement membrane (GBM) is the most likely cause of massive proteinuria in this model. To elucidate further the mechanisms of PAN-induced cellular dysfunction, new methods were employed to visualize podocyte cytoskeletal aggregation and to measure fibrillar attachment to the GBM. Adult Sprague-Dawley rats (n = 4/group) received a single tail-vein injection of PAN (75 mg/kg). On days 1, 2, 3, and 5 following injection, 24-hour urine collections were obtained for creatinine clearance, albuminuria, and total proteinuria. Then kidneys from each group were fixed by perfusion. Podocytic cytoskeleton was visualized by scanning electron microscopy. Subepithelial GBM staining and attachment fiber number, observed on digitized images of transmission electron micrographs, were quantitated with computer-based density analysis. A significant reduction in attachment fiber number in the GBM lamina rara externa occurred by day 5. On scanning electron micrographs, the secondary and tertiary podocytic processes were observed to be formed by highly aggregated cytoskeleton, which became partially disaggregated by day 3, was totally absent by day 5, and normalized by day 20. Immunogold staining revealed that actin and vinculin localized to the tertiary podocytic processes in the normal state were dispersed into the cell body following PAN. Podocyte cytoskeletal disaggregation precedes, and detachment from the GBM occurs simultaneously with, the onset of massive proteinuria in the PAN model. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:8494056

  11. Caenorhabditis elegans Teneurin, ten-1, Is Required for Gonadal and Pharyngeal Basement Membrane Integrity and Acts Redundantly with Integrin ina-1 and Dystroglycan dgn-1

    PubMed Central

    Trzebiatowska, Agnieszka; Topf, Ulrike; Sauder, Ursula; Drabikowski, Krzysztof

    2008-01-01

    The Caenorhabditis elegans teneurin ortholog, ten-1, plays an important role in gonad and pharynx development. We found that lack of TEN-1 does not affect germline proliferation but leads to local basement membrane deficiency and early gonad disruption. Teneurin is expressed in the somatic precursor cells of the gonad that appear to be crucial for gonad epithelialization and basement membrane integrity. Ten-1 null mutants also arrest as L1 larvae with malformed pharynges and disorganized pharyngeal basement membranes. The pleiotropic phenotype of ten-1 mutant worms is similar to defects found in basement membrane receptor mutants ina-1 and dgn-1 as well as in the mutants of the extracellular matrix component laminin, epi-1. We show that the ten-1 mutation is synthetic lethal with mutations of genes encoding basement membrane components and receptors due to pharyngeal or hypodermal defects. This indicates that TEN-1 could act redundantly with integrin INA-1, dystroglycan DGN-1, and laminin EPI-1 in C. elegans development. Moreover, ten-1 deletion sensitizes worms to loss of nidogen nid-1 causing a pharynx unattached phenotype in ten-1;nid-1 double mutants. We conclude that TEN-1 is important for basement membrane maintenance and/or adhesion in particular organs and affects the function of somatic gonad precursor cells. PMID:18632986

  12. Differential expression of epithelial basement membrane components nidogens and perlecan in corneal stromal cells in vitro

    PubMed Central

    Santhanam, Abirami; Torricelli, Andre A. M.; Wu, Jiahui; Marino, Gustavo K.

    2015-01-01

    Purpose The purpose of this study was to examine the expression of corneal epithelial basement membrane (EBM) components in different corneal stromal cell types. In vitro model systems were used to explore the expression of EBM components nidogen-1, nidogen-2, and perlecan that are the primary components in the lamina lucida and the lamina densa that defectively regenerate in corneas with stromal opacity after in −9.0 D photorefractive keratectomy (PRK). Methods Primary rabbit corneal stromal cells were cultured using varying serum concentrations and exogenous growth factors, including fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1, to optimize the growth of each cell type of interest. The expression of the keratocyte-specific marker keratocan and the myofibroblast-specific marker α-smooth muscle actin (α-SMA) were analyzed with real-time PCR, western blot, and immunocytochemical staining to evaluate the specificity of the cell types and select optimal conditions (high keratocan and low α-SMA for keratocytes; low keratocan and high α-SMA for myofibroblasts; low keratocan and low α-SMA for corneal fibroblasts). The expression of the EBM components nidogen-1, nidogen-2, and perlecan was evaluated in each corneal cell type using real-time PCR, immunostaining, and western blotting. In agreement with previous studies, serum-free DMEM was found to be optimal for keratocytes, DMEM with 10% serum and 40 ng/ml FGF-2 yielded the best marker profile for corneal fibroblasts, and DMEM with 1% serum and 2 ng/ml TGF-β1 was found to be optimal for myofibroblasts. Results Nidogen-1 and nidogen-2 mRNAs were highly expressed in keratocytes, whereas perlecan was highly expressed in myofibroblasts. In keratocytes, nidogen-2 and perlecan proteins were expressed predominantly in intracellular compartments, whereas in myofibroblasts expression of both EBM components was observed diffusely throughout the cell. Although the perlecan mRNA levels were high

  13. Interleukin-1 receptor antagonist ameliorates experimental anti-glomerular basement membrane antibody-associated glomerulonephritis.

    PubMed Central

    Tang, W W; Feng, L; Vannice, J L; Wilson, C B

    1994-01-01

    The contribution of IL-1 to leukocyte infiltration in anti-glomerular basement membrane (GBM) antibody (Ab) glomerulonephritis (GN) was examined by the administration of a specific IL-1 receptor antagonist (IL-1ra). Lewis rats received anti-GBM Ab or normal rabbit serum and were treated with either 0.9% saline or 6 mg IL-1ra over a 24-h time period. Plasma IL-1ra concentration was 2,659 +/- 51 ng/ml 4 h after anti-GBM Ab and IL-1ra administration. PMN and monocyte/macrophage infiltration declined 39% (9.8 +/- 1.9 to 6.0 +/- 1.5 PMN/glomerulus, P < 0.001) and 29% (4.9 +/- 0.8 to 3.5 +/- 0.8 ED-1 cells/glomerulus, P = 0.002) with IL-1ra treatment at 4 h, respectively. Similarly, the number of glomerular cells staining for lymphocyte function-associated molecule-1 beta (CD18) declined 39% from 16.7 +/- 1.9 to 10.7 +/- 1.6 cells/glomerulus at 4 h (P = 0.0001). This was associated with a decrease in glomerular intracellular adhesion molecule-1 expression. The mean glomerular intracellular adhesion molecule-1 score in anti-GBM Ab GN rats treated with IL-1ra was less than that of rats administered anti-GBM Ab and 0.9% saline at 4 (2.0 +/- 0.2 vs 2.5 +/- 0.2, P < 0.05) and 24 (2.5 +/- 0.1 vs 3.1 +/- 0.2, P = 0.0001) h. These immunopathologic changes correlated with a 50% reduction in proteinuria from 147 +/- 34 to 75 +/- 25 mg/d (P < 0.002). Treatment with IL-1ra did not affect the steady state mRNA expression of either IL-1 beta or TNF alpha. An increase in the IL-1ra dose to 30 mg given within the initial 4 h provided no additional benefit. The decline in PMN and monocyte/macrophage infiltration of the glomerulus at 4 h was similar to that found in the initial study. Furthermore, the protective benefit of IL-1ra was abrogated by doubling the dose of the anti-GBM Ab GN, despite administering high dose IL-1ra (30 mg). In these studies, detectable IL-1ra was found in the serum of untreated anti-GBM Ab GN controls. These data suggest a positive yet limited role for IL-1ra in

  14. ER stress and basement membrane defects combine to cause glomerular and tubular renal disease resulting from Col4a1 mutations in mice

    PubMed Central

    Jones, Frances E.; Bailey, Matthew A.; Murray, Lydia S.; Lu, Yinhui; McNeilly, Sarah; Schlötzer-Schrehardt, Ursula; Lennon, Rachel; Sado, Yoshikazu; Brownstein, David G.; Mullins, John J.; Kadler, Karl E.; Van Agtmael, Tom

    2016-01-01

    ABSTRACT Collagen IV is a major component of basement membranes, and mutations in COL4A1, which encodes collagen IV alpha chain 1, cause a multisystemic disease encompassing cerebrovascular, eye and kidney defects. However, COL4A1 renal disease remains poorly characterized and its pathomolecular mechanisms are unknown. We show that Col4a1 mutations in mice cause hypotension and renal disease, including proteinuria and defects in Bowman's capsule and the glomerular basement membrane, indicating a role for Col4a1 in glomerular filtration. Impaired sodium reabsorption in the loop of Henle and distal nephron despite elevated aldosterone levels indicates that tubular defects contribute to the hypotension, highlighting a novel role for the basement membrane in vascular homeostasis by modulation of the tubular response to aldosterone. Col4a1 mutations also cause diabetes insipidus, whereby the tubular defects lead to polyuria associated with medullary atrophy and a subsequent reduction in the ability to upregulate aquaporin 2 and concentrate urine. Moreover, haematuria, haemorrhage and vascular basement membrane defects confirm an important vascular component. Interestingly, although structural and compositional basement membrane defects occurred in the glomerulus and Bowman's capsule, no tubular basement membrane defects were detected. By contrast, medullary atrophy was associated with chronic ER stress, providing evidence for cell-type-dependent molecular mechanisms of Col4a1 mutations. These data show that both basement membrane defects and ER stress contribute to Col4a1 renal disease, which has important implications for the development of treatment strategies for collagenopathies. PMID:26839400

  15. Differential binding of fibroblast growth factor-2 and -7 to basement membrane heparan sulfate: comparison of normal and abnormal human tissues.

    PubMed Central

    Friedl, A.; Chang, Z.; Tierney, A.; Rapraeger, A. C.

    1997-01-01

    Fibroblast growth factors (FGFs) play multiple roles during development and in adult tissues as paracrine regulators of growth and differentiation. FGFs signal through transmembrane receptor tyrosine kinases, but heparan sulfate is also required for signaling by members of the FGF family. In addition, heparan sulfate may be involved in determining tissue distribution of FGFs. Using biotinylated FGF-2 and FGF-7 (KGF) as probes, we have identified specific interactions between FGFs and heparan sulfates in human tissues. Both FGF species bind to tissue mast cells and to epithelial cell membranes. Binding to basement membrane heparan sulfate is tissue source dependent and specific. Although FGF-2 strongly binds to basement membrane heparan sulfate in skin and most other tissue sites examined, FGF-7 fails to bind to basement membrane heparan sulfate in most locations. However, in subendothelial matrix in blood vessels and in the basement membrane of a papillary renal cell carcinoma, strong FGF-7 binding is seen. In summary, distinct and specific affinities of heparan sulfates for different FGFs were identified that may affect growth factor activation and local distribution. Heparan sulfate may have a gatekeeper function to either restrict or permit diffusion of heparin-binding growth factors across the basement membrane. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9094999

  16. Recombinant vascular basement-membrane-derived multifunctional peptide inhibits angiogenesis and growth of hepatocellular carcinoma

    PubMed Central

    Wu, You-Hua; Cao, Jian-Guo; Xiang, Hong-Lin; Xia, Hong; Qin, Yong; Huang, A-Ji; Xiao, Di; Xu, Fang

    2009-01-01

    AIM: To investigate the anti-angiogenic and anti-tumor activities of recombinant vascular basement membrane-derived multifunctional peptide (rVBMDMP) in hepatocellular carcinoma (HCC). METHODS: HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines were cultured in vitro and the inhibitory effect of rVBMDMP on proliferation of cells was detected by MTT assay. The in vivo antitumor efficacy of rVBMDMP on HCC was assessed by HepG2 xenografts in nude mice. Distribution of rVBMDMP, mechanism by which the growth of HepG2 xenografts is inhibited, and microvessel area were observed by proliferating cell nuclear antigen (PCNA) and CD31 immunohistochemistry. RESULTS: MTT assay showed that rVBMDMP markedly inhibited the proliferation of human HCC (HepG2, Bel-7402, Hep-3B) cells and human umbilical vein endothelial (HUVE-12) cells in a dose-dependent manner, with little effect on the growth of L-02 cells. When the IC50 was 4.68, 7.65, 8.96, 11.65 and 64.82 μmol/L, respectively, the potency of rVBMDMP to HepG2 cells was similar to 5-fluorouracil (5-FU) with an IC50 of 4.59 μmol/L. The selective index of cytotoxicity to HepG2 cells of rVBMDMP was 13.8 (64.82/4.68), which was higher than that of 5-FU [SI was 1.9 (8.94/4.59)]. The VEGF-targeted recombinant humanized monoclonal antibody bevacizumab (100 mg/L) did not affect the proliferation of HepG2, Bel-7402, Hep-3B and L-02 cells, but the growth inhibitory rate of bevacizumab (100 mg/L) to HUVE-12 cells was 87.6% ± 8.2%. Alternis diebus intraperitoneal injection of rVBMDMP suppressed the growth of HepG2 xenografts in a dose-dependent manner. rVBMDMP (1, 3, 10 mg/kg) decreased the tumor weight by 12.6%, 55.9% and 79.7%, respectively, compared with the vehicle control. Immunohistochemical staining of rVBMDMP showed that the positive area rates (2.2% ± 0.73%, 4.5% ± 1.3% and 11.5% ± 3.8%) in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly higher than that (0.13% ± 0.04%) in the control group (P < 0.01). The positive

  17. A Novel Function for the nm23-Hl Gene: Overexpression in Human Breast Carcinoma Cells Leads to the Formation of Basement Membrane and Growth Arrest

    SciTech Connect

    Howlett, Anthony R; Petersen, Ole W; Steeg, Patricia S; Bissell, Mina J

    1994-01-01

    We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene

  18. Basement membrane heparan sulfate proteoglycan (perlecan) synthesized by ACC3, adenoid cystic carcinoma cells of human salivary gland origin.

    PubMed

    Kimura, S; Cheng, J; Toyoshima, K; Oda, K; Saku, T

    1999-02-01

    The biosynthesis of basement membrane heparan sulfate proteoglycan (HSPG), known as perlecan, in ACC3 cells established from a adenoid cystic carcinoma of the human salivary gland was studied using metabolic labeling and immunoprecipitation with discriminative antibodies specific for HSPG core protein. Treatment of immunoprecipitated HSPG with HNO2, heparitinase, and chondroitinase ABC revealed that ACC3 cells synthesized HSPG molecules composed of 470-kDa core protein and heparan sulfate but not of chondroitin sulfate. The core protein was shown to contain complex type N-linked oligosaccharides by digestion with N-glycanase and endoglycosidase H. Pulse-chase experiments showed that the mature form of HSPG was formed in the cells in 30 min and released into the medium thereafter. Degradation of HSPG was also found in the chase period of 3 h. In time course experiments, HSPG was found to be synthesized maximally at day 4 after plating, deposited in the cell layer maximally at day 6, and secreted maximally at day 8. This was also confirmed by immunofluorescence, Northern blotting, and in-situ hybridization. The results indicate that ACC3 cells synthesize, secrete and degrade basement membrane type HSPG, which is analogous to those produced by other cell types, and that the biosynthesis and secretion of HSPG in ACC3 cells are strictly regulated by the cell growth, that may be reflected in the characteristic histology of adenoid cystic carcinomas. PMID:9990141

  19. Biochemical and immunohistochemical characterization of human type XIX defines a novel class of basement membrane zone collagens.

    PubMed Central

    Myers, J. C.; Li, D.; Bageris, A.; Abraham, V.; Dion, A. S.; Amenta, P. S.

    1997-01-01

    Nineteen types, the product of 33 genes, comprise the collagen family of proteins. Types I, II, III, V, and XI constitute the fibrillar collagens, whereas types IV, VI to X, and XII to XIX represent the structurally diverse, nonfibrillar members. Type XIX collagen was discovered from the sequence of rhabdomyosarcoma cDNA clones. The type XIX chain consists of 1142 amino acids that contribute primarily to a unique five subdomain triple-helical region. To characterize the protein, to determine the tissue distribution, and to provide some insight into its function, we generated two type XIX-specific polyclonal antibodies. One was directed against a recombinant molecule containing amino-terminal sequences, and the second was derived from a synthetic peptide corresponding to most of the short carboxy terminus. These antibodies were used in immunoblot assays of rhabdomyosarcoma cell/matrix homogenates to identify a 165-kd disulfide-bonded and bacterial collagenase-sensitive protein. Immunohistochemical analysis of type XIX collagen was performed for human skeletal muscle, spleen, prostate, kidney, liver, placenta, colon, and skin. In contrast to Northern blot hybridizations, which showed very low levels of the 12-kb transcript in few tissues, the protein was found in all tissues examined. The type XIX collagen distribution was restricted to vascular, neuronal, mesenchymal, and some epithelial basement membrane zones, which is similar to the profile recently established (Ref. 8) and further extended here for type XV collagen. Nevertheless, localization of type XIX exhibited significant differences from type XV collagen that were particularly evident in the kidney, liver, and spleen. This report, in conjunction with the type XV results and other studies of type XVIII collagen, indicates the existence of a new collagen subgroup founded on their widespread presence in basement membrane zones regardless of chain homology. In addition to their role in basement membrane

  20. [The influence of basement membrane matrix on the attachment of human gingival epithelial cell to titanium: a scanning electron microscopic study].

    PubMed

    Wang, J; Liu, B; Lü, C

    1997-02-01

    In order to investigate the factors which could enhance the integrity of the biological seal between implant and epithelial cells, basement membrane matrigel was coated on the surface of smooth pure titanium. SEM observations showed that the spreading of primary cultured human gingival epithelial cells on Matrigel coated titanium surface was better than that of control groups on the third and the sixth day. This result suggests that the attachment of human gingival epithelial cells to titanium can be enhanced by the basement membrane matrigel. PMID:11480046

  1. Increased Water Retention in Polymer Electrolyte Membranes at Elevated Temperatures Assisted by Capillary Condensation

    SciTech Connect

    Park, M.J.; Downing, K.H.; Jackson, A.; Gomez, E.D.; Minor, A.M.; Cookson, D.; Weber, A.Z.; Balsara, N.P.

    2008-10-03

    We establish a new systematic methodology for controlling the water retention of polymer electrolyte membranes. Block copolymer membranes comprising hydrophilic phases with widths ranging from 2 to 5 nm become wetter as the temperature of the surrounding air is increased at constant relative humidity. The widths of the moist hydrophilic phases were measured by cryogenic electron microscopy experiments performed on humid membranes. Simple calculations suggest that capillary condensation is important at these length scales. The correlation between moisture content and proton conductivity of the membranes is demonstrated.

  2. Novel therapy for anti-glomerular basement membrane disease with IgA nephropathy: A case report

    PubMed Central

    XU, DECHAO; WU, JIANXIANG; WU, JUN; XU, CHENGGANG; ZHANG, YUQIANG; MEI, CHANGLIN; GAO, XIANG

    2016-01-01

    Anti-glomerular basement membrane (GBM) disease is characterized by circulating anti-GBM antibodies and deposition of these antibodies in the renal GBM. Renal involvement in anti-GBM is more severe when compared with other types of immune-mediated glomerulonephritis, and the majority of patients manifest progressive renal failure, leading to end-stage renal disease. In a limited number of cases, anti-GBM disease has been shown to be accompanied with other immune-mediated glomerulonephritis. The present study reported the case of a 50-year-old female patient presenting with rapidly progressive glomerulonephritis, who was diagnosed with anti-GBM disease with IgA nephropathy. The patient achieved a relatively good therapeutic outcome with administration of corticosteroids plus mycophenolate mofetil (MMF), which may prove to be a novel treatment option for this rare disease; however, the exact underlying mechanism requires further in-depth investigation. PMID:27168822

  3. A role for PDGF-C/PDGFRα signaling in the formation of the meningeal basement membranes surrounding the cerebral cortex.

    PubMed

    Andrae, Johanna; Gouveia, Leonor; Gallini, Radiosa; He, Liqun; Fredriksson, Linda; Nilsson, Ingrid; Johansson, Bengt R; Eriksson, Ulf; Betsholtz, Christer

    2016-01-01

    Platelet-derived growth factor-C (PDGF-C) is one of three known ligands for the tyrosine kinase receptor PDGFRα. Analysis ofPdgfcnull mice has demonstrated roles for PDGF-C in palate closure and the formation of cerebral ventricles, but redundancy with other PDGFRα ligands might obscure additional functions. In search of further developmental roles for PDGF-C, we generated mice that were double mutants forPdgfc(-/-)andPdgfra(GFP/+) These mice display a range of severe phenotypes including spina bifida, lung emphysema, abnormal meninges and neuronal over-migration in the cerebral cortex. We focused our analysis on the central nervous system (CNS), where PDGF-C was identified as a critical factor for the formation of meninges and assembly of the glia limitans basement membrane. We also present expression data onPdgfa,PdgfcandPdgfrain the cerebral cortex and microarray data on cerebral meninges. PMID:26988758

  4. Rab10-Mediated Secretion Synergizes with Tissue Movement to Build a Polarized Basement Membrane Architecture for Organ Morphogenesis.

    PubMed

    Isabella, Adam J; Horne-Badovinac, Sally

    2016-07-11

    Basement membranes (BMs) are planar protein networks that support epithelial function. Regulated changes to BM architecture can also contribute to tissue morphogenesis, but how epithelia dynamically remodel their BMs is unknown. In Drosophila, elongation of the initially spherical egg chamber correlates with the generation of a polarized network of fibrils in its surrounding BM. Here, we use live imaging and genetic manipulations to determine how these fibrils form. BM fibrils are assembled from newly synthesized proteins in the pericellular spaces between the egg chamber's epithelial cells and undergo oriented insertion into the BM by directed epithelial migration. We find that a Rab10-based secretion pathway promotes pericellular BM protein accumulation and fibril formation. Finally, by manipulating this pathway, we show that BM fibrillar structure influences egg chamber morphogenesis. This work highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape. PMID:27404358

  5. Sequential occurrence of anti-glomerular basement membrane disease 9 years after anti-neutrophil cytoplasmic antibody-associated vasculitis

    PubMed Central

    Chan, Pui Shan Julia; Leung, Moon Ho

    2016-01-01

    We report a case of 63-year-old Chinese man, having a history of anti-myeloperoxidase (MPO) antibody anti-neutrophil cytoplasmic antibody (ANCA)-associated pulmonary-renal syndrome 9 years ago, presented with second episode of rapidly progressive glomerulonephritis (RPGN) and alveolar haemorrhage compatible with anti-glomerular basement membrane (GBM) disease. In first presentation, his anti-GBM antibody was negative. This time, anti-MPO antibody was negative, but anti-GBM antibody was positive. The long interval of sequential development of anti-GBM disease after ANCA-associated vasculitis in this patient may provide clues to the potential immunological links between these two distinct conditions. Clinicians should be aware of such double-positive association. PMID:27123311

  6. A role for PDGF-C/PDGFRα signaling in the formation of the meningeal basement membranes surrounding the cerebral cortex

    PubMed Central

    Andrae, Johanna; Gouveia, Leonor; Gallini, Radiosa; He, Liqun; Fredriksson, Linda; Nilsson, Ingrid; Johansson, Bengt R.; Eriksson, Ulf; Betsholtz, Christer

    2016-01-01

    ABSTRACT Platelet-derived growth factor-C (PDGF-C) is one of three known ligands for the tyrosine kinase receptor PDGFRα. Analysis of Pdgfc null mice has demonstrated roles for PDGF-C in palate closure and the formation of cerebral ventricles, but redundancy with other PDGFRα ligands might obscure additional functions. In search of further developmental roles for PDGF-C, we generated mice that were double mutants for Pdgfc−/− and PdgfraGFP/+. These mice display a range of severe phenotypes including spina bifida, lung emphysema, abnormal meninges and neuronal over-migration in the cerebral cortex. We focused our analysis on the central nervous system (CNS), where PDGF-C was identified as a critical factor for the formation of meninges and assembly of the glia limitans basement membrane. We also present expression data on Pdgfa, Pdgfc and Pdgfra in the cerebral cortex and microarray data on cerebral meninges. PMID:26988758

  7. Reactive oxygen products in heterologous anti-glomerular basement membrane nephritis in rats.

    PubMed Central

    Birtwistle, R. J.; Michael, J.; Howie, A. J.; Adu, D.

    1989-01-01

    The effect of 'scavengers' of reactive oxygen products (ROPs) was studied in the heterologous phase of anti-glomerular basement (anti-GBM) nephritis induced in rats. Glomerulonephritis was induced by the intravenous administration of sheep anti-GBM antibody (5 mg/100 g) to rats on day 0. The intraperitoneal administration of superoxide dismutase (SOD) 30 mg/kg/day or 150 mg/kg/day leads to a significant reduction in proteinuria on day 1 and also on day 3 in animals given SOD 30 mg/kg/day. Proteinuria was not significantly reduced by the intraperitoneal administration of inactivated SOD (150 mg/kg/day). In rats given polyethylene glycol coupled catalase (PEG-catalase) intraperitoneally at a dose of 10,000 iu/kg/day and 100,000 iu/kg/day proteinuria was lower than in rats with unmodified anti-GBM nephritis. These differences were significant on day 1 (P less than 0.05) in rats given PEG-catalase 100,000 iu/kg/day and on days 3 and 5 in rats treated with either dose of PEG-catalase (P less than 0.01). These data suggest a role for superoxide anion and hydrogen peroxide, or a product of their interaction such as hydroxyl radical, in glomerular injury induced by anti-GBM antibody. PMID:2786425

  8. Assessment of a Basement Membrane-Degrading Protease on Dissemination and Secondary Infection of Autographa californica Multiple Nucleopolyhedrovirus in Heliothis virescens L.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, that digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus that expresses ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significant...

  9. Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

    PubMed

    Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

    2009-10-01

    The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions. PMID:19602722

  10. The HPV16 and MusPV1 papillomaviruses initially interact with distinct host components on the basement membrane.

    PubMed

    Day, Patricia M; Thompson, Cynthia D; Lowy, Douglas R; Schiller, John T

    2015-07-01

    To understand and compare the mechanisms of murine and human PV infection, we examined pseudovirion binding and infection of the newly described MusPV1 using the murine cervicovaginal challenge model. These analyses revealed primary tissue interactions distinct from those previously described for HPV16. Unlike HPV16, MusPV1 bound basement membrane (BM) in an HSPG-independent manner. Nevertheless, subsequent HSPG interactions were critical. L2 antibodies or low doses of VLP antibodies, sufficient to prevent infection, did not lead to disassociation of the MusPV1 pseudovirions from the BM, in contrast to previous findings with HPV16. Similarly, furin inhibition did not lead to loss of MusPV1 from the BM. Therefore, phylogenetically distant PV types differ in their initial interactions with host attachment factors, but initiate their lifecycle on the acellular BM. Despite these differences, these distantly related PV types displayed similar intracellular trafficking patterns and susceptibilities to biochemical inhibition of infection. PMID:25771496

  11. Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

    PubMed

    Nagai, N; Nakano, K; Sado, Y; Naito, I; Gunduz, M; Tsujigiwa, H; Nagatsuka, H; Ninomiya, Y; Siar, C H

    2001-10-01

    The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation. PMID:11732842

  12. Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.

    PubMed Central

    Pejler, G; David, G

    1987-01-01

    Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed. PMID:2963617

  13. The HPV16 and MusPV1 papillomaviruses initially interact with distinct host components on the basement membrane

    PubMed Central

    Day, Patricia M.; Thompson, Cynthia D.; Lowy, Douglas R.; Schiller, John T.

    2015-01-01

    To understand and compare the mechanisms of murine and human PV infection, we examined pseudovirion binding and infection of the newly described MusPV1 using the murine cervicovaginal challenge model. These analyses revealed primary tissue interactions distinct from those previously described for HPV16. Unlike HPV16, MusPV1 bound basement membrane (BM) in an HSPG-independent manner. Nevertheless, subsequent HSPG interactions were critical. L2 antibodies or low doses of VLP antibodies, sufficient to prevent infection, did not lead to disassociation of the MusPV1 pseudovirions from the BM, in contrast to previous findings with HPV16. Similarly, furin inhibition did not lead to loss of MusPV1 from the BM. Therefore, phylogenetically distant PV types that differ in their initial interactions with host attachment factors, but initiate their lifecycle on the acellular BM. Despite these differences, these distantly related PV types displayed similar intracellular trafficking patterns and susceptibilities to biochemical inhibition of infection. PMID:25771496

  14. Goodpasture's disease in the absence of circulating anti-glomerular basement membrane antibodies as detected by standard techniques.

    PubMed

    Salama, Alan D; Dougan, Tammy; Levy, Jeremy B; Cook, H Terry; Morgan, Steve H; Naudeer, Sarah; Maidment, Geoff; George, Andrew J T; Evans, David; Lightstone, Liz; Pusey, Charles D

    2002-06-01

    Goodpasture's disease is characterized by rapidly progressive glomerulonephritis, often accompanied by pulmonary hemorrhage, in association with deposition of antibodies in a linear pattern on the glomerular basement membrane (GBM). The diagnosis of Goodpasture's disease in patients with acute renal failure often relies on the use of immunoassays to detect circulating anti-GBM antibodies in serum samples. We describe three cases of Goodpasture's disease in which no circulating anti-GBM antibodies were detectable in serum by well-established enzyme-linked immunosorbent assay or Western blotting techniques. The diagnosis of Goodpasture's disease was confirmed by renal biopsy, with linear deposition of immunoglobulin along the GBM and crescentic glomerulonephritis. In addition, an alternative method of antibody detection using a highly sensitive biosensor system confirmed that circulating antibodies were present in sera from both patients tested. Because this technique is not routinely available for the detection of anti-GBM antibodies, we suggest that diagnosis always be confirmed with a renal biopsy, and despite negative serological test results using immunoassay, the diagnosis of Goodpasture's disease should still be considered in the correct clinical context. PMID:12046026

  15. High recombination between two physically close human basement membrane collagen genes at the distal end of chromosome 13q.

    PubMed Central

    Bowcock, A M; Hebert, J M; Wijsman, E; Gadi, I; Cavalli-Sforza, L L; Boyd, C D

    1988-01-01

    Two basement membrane collagen genes coding for the pro alpha 1 chain and pro alpha 2 chain of type IV collagen map to 13q34 and are linked with a maximum likelihood estimate of recombination of 0.028 at a logarithm of odds (lod) score of 19.98. The single-copy sequence that identifies the locus D13S3 is also closely linked to both collagen genes. Four enzymes reveal polymorphisms with COL4A1, and 10 haplotypes have been observed in Caucasoids. Within COL4A1 a nonrandom association of alleles exists only between alleles defined by Hae III and those defined by the other three enzymes. A random association of alleles of COL4A1 and COL4A2 is observed. Between the two collagen genes were detected three meiotic recombination events that contributed to the estimate of 2.8% recombination. This is higher than expected for two genes that lie within 650 kilobases of each other. The lack of linkage disequilibrium between COL4A1 and COL4A2 is in agreement with the relatively high recombination that is observed. Images PMID:2895928

  16. Dual targeting of Angiopoetin-2 and VEGF potentiates effective vascular normalisation without inducing empty basement membrane sleeves in xenograft tumours

    PubMed Central

    Coutelle, O; Schiffmann, L M; Liwschitz, M; Brunold, M; Goede, V; Hallek, M; Kashkar, H; Hacker, U T

    2015-01-01

    Background: Effective vascular normalisation following vascular endothelial growth factor (VEGF) inhibition is associated with endothelial cell regression leaving empty basement membrane sleeves (BMS). These long-lived BMS permit the rapid regrowth of tumour vasculature upon treatment cessation and promote resistance to VEGF-targeting drugs. Previous attempts at removing BMS have failed. Angiopoietin-2 (Ang2) is a vascular destabilizing factor that antagonises normalisation. We hypothesised that Ang2 inhibition could permit vascular normalisation at significantly reduced doses of VEGF inhibition, avoiding excessive vessel regression and the formation of empty BMS. Methods: Mice xenografted with human colorectal cancer cells (LS174T) were treated with low (0.5 mg kg−1) or high (5 mg kg−1) doses of the VEGF-targeting antibody bevacizumab with or without an Ang2 blocking peptibody L1-10. Tumour growth, BMS formation and normalisation parameters were examined including vessel density, pericyte coverage, adherence junctions, leakiness, perfusion, hypoxia and proliferation. Results: Dual targeting of VEGF and Ang2 achieved effective normalisation at only one-tenth of the dose required with bevacizumab alone. Pericyte coverage, vascular integrity, adherence junctions and perfusion as prerequisites for improved access of chemotherapy were improved without inducing empty BMS that facilitate rapid vascular regrowth. Conclusions: Dual targeting of VEGF and Ang2 can potentiate the effectiveness of VEGF inhibitors and avoid the formation of empty BMS. PMID:25562438

  17. Abnormalities in the basement membrane structure promote basal keratinocytes in the epidermis of hypertrophic scars to adopt a proliferative phenotype

    PubMed Central

    YANG, SHAOWEI; SUN, YEXIAO; GENG, ZHIJUN; MA, KUI; SUN, XIAOYAN; FU, XIAOBING

    2016-01-01

    The majority of studies on scar formation have mainly focused on the dermis and little is known of the involvement of the epidermis. Previous research has demonstrated that the scar tissue-derived keratinocytes are different from normal cells at both the genetic and cell biological levels; however, the mechanisms responsible for the fundamental abnormalities in keratinocytes during scar development remain elusive. For this purpose, in this study, we used normal, wound edge and hypertrophic scar tissue to examine the morphological changes which occur during epidermal regeneration as part of the wound healing process and found that the histological structure of hypertrophic scar tissues differed from that of normal skin, with a significant increase in epidermal thickness. Notably, staining of the basement membrane (BM) appeared to be absent in the scar tissues. Moreover, immunofluorescence staining for cytokeratin (CK)10, CK14, CK5, CK19 and integrin-β1 indicated the differential expression of cell markers in the epidermal keratinocytes among the normal, wound edge and hypertrophic scar tissues, which corresponded with the altered BM structures. By using a panel of proteins associated with BM components, we validated our hypothesis that the BM plays a significant role in regulating the cell fate decision of epidermal keratinocytes during skin wound healing. Alterations in the structure of the BM promote basal keratinocytes to adopt a proliferative phenotype both in vivo and in vitro. PMID:26986690

  18. Suppression of Apoptosis by Basement Membrane Requires three-dimensional Tissue Organization and Withdrawal from the Cell Cycle

    SciTech Connect

    Boudreau, N.; Werb, Z.; Bissell, M.J.

    1995-12-28

    The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce {beta}-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the G1 cyclin kinase inhibitor p21/WAF-I and positive proliferative signals including c-myc and cyclin Dl were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor a and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-{beta}1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.

  19. Complement and Humoral Adaptive Immunity in the Human Choroid Plexus: Roles for Stromal Concretions, Basement Membranes, and Epithelium

    PubMed Central

    Laule, Cornelia; Leung, Esther; Pavlova, Vladimira; Morgan, B. Paul; Esiri, Margaret M.

    2016-01-01

    The choroid plexus (CP) provides a barrier to entry of toxic molecules from the blood into the brain and transports vital molecules into the cerebrospinal fluid. While a great deal is known about CP physiology, relatively little is known about its immunology. Here, we show immunohistochemical data that help define the role of the CP in innate and adaptive humoral immunity. The results show that complement, in the form of C1q, C3d, C9, or C9neo, is preferentially deposited in stromal concretions. In contrast, immunoglobulin (Ig) G (IgG) and IgA are more often found in CP epithelial cells, and IgM is found in either locale. C4d, IgD, and IgE are rarely, if ever, seen in the CP. In multiple sclerosis CP, basement membrane C9 or stromal IgA patterns were common but were not specific for the disease. These findings indicate that the CP may orchestrate the clearance of complement, particularly by deposition in its concretions, IgA and IgG preferentially via its epithelium, and IgM by either mechanism. PMID:26994633

  20. YSZ-Reinforced Alumina Multi-Channel Capillary Membranes for Micro-Filtration

    PubMed Central

    Wang, Bo; Lee, Melanie; Li, Kang

    2015-01-01

    The combined phase-inversion and sintering method not only produces ceramic hollow fibre membranes with much lower fabrication costs than conventional methods, but these membranes can also be designed to have greatly reduced transport resistances for filtration processes. The bottleneck of this technique is the weak mechanical property of the fibres, due to the small dimensions and the brittle nature of the ceramic materials. In this study, yttrium stabilised zirconia (YSZ) reinforced alumina seven-channel capillary microfiltration membranes were prepared with a pore size of ~230 nm and their mechanical property and permeation characteristics were studied. It is found that the addition of YSZ can effectively enhance the mechanical property of the membrane and also increase pure water permeation flux. The Al2O3-YSZ seven-channel capillary membranes could reach a fracture load of 23.4 N and a bending extension of 0.54 mm when being tested with a 6 cm span, to meet the requirements for most industrial microfiltration applications. PMID:26729178

  1. Preparation and characterization of micro-cell membrane chromatographic column with silica-based porous layer open tubular capillary as cellular membrane carrier.

    PubMed

    Zhang, Fugeng; Zhao, Xinchao; Xu, Bei; Cheng, Shuai; Tang, Cheng; Duan, Hongquan; Xiao, Xuefeng; Du, Wuxun; Xu, Liang

    2016-04-01

    Cell membrane chromatography (CMC) is a powerful tool to study membrane protein interactions and to screen active compounds extracted from natural products. Unfortunately, a large amount of cells are typically required for column preparation in order to carry out analyses in an efficient manner. Micro-CMC (mCMC) has recently been developed by using a silica capillary as a membrane carrier. However, a reduced retention of analytes is generally associated with mCMC mostly due to a low ligand (cellular membrane) capacity. To solve this common problem, in this work a silica-based porous layer open tubular (PLOT) capillary was fabricated and, to the best of our knowledge, for the first time applied to mCMC. The mCMC column was prepared by physical adsorption of rabbit red blood cell (rRBC) membranes onto the inner surface of the PLOT capillary. The effects of the PLOT capillaries fabricated by different feed compositions, on the immobilization amount of cellular membranes (represented by the fluorescence intensity of the capillary immobilized with fluorescein isothiocyanate isomer-labeled cellular membranes) and on the dynamic binding capacity (DBC) of verapamil (VP, a widely used calcium antagonist which specific interacts with L-type calcium channel proteins located on cellular membrane of rRBC) have been systematically investigated. The fluorescence intensity of the mCMC column when combined with the PLOT capillary was found to be more than five times higher than the intensity using a bare capillary. This intriguing result indicates that the PLOT capillary exhibits a higher cellular membrane capacity. The DBC of VP in the PLOT column was found to be more than nine times higher than that in the bare capillary. An rRBC/CMC column was also prepared for comparative studies. As a result, mCMC provides similar chromatographic retention factors and stability with common CMC; however, the cellular membrane consumption for mCMC was found to be more than 460 times lower than

  2. Nature of sulphated macromolecules in mouse Reichert's membrane. Evidence for tyrosine O-sulphate in basement-membrane proteins.

    PubMed Central

    Paulsson, M; Dziadek, M; Suchanek, C; Huttner, W B; Timpl, R

    1985-01-01

    Seven different sulphated macromolecules were detected in 6 M-guanidinium chloride extracts of metabolically [35S]sulphate-labelled mouse Reichert's membrane and were partially separated. Polypeptide bands of apparent Mr 50 000, 150 000 (tentatively identified as entactin) and 170 000 contained essentially tyrosine O-sulphate as the labelled component. Most of the radioactive sulphate was incorporated into three different proteoglycans, which could be separated by chromatography and density-gradient centrifugation before and after enzymic degradation. Enzymic analysis of glycosaminoglycans and of protein cores by immunoassays identified these components as low-density and high-density forms of heparan sulphate proteoglycan and a high-density form of chondroitin sulphate or dermatan sulphate proteoglycan. Images Fig. 2. PMID:4074325

  3. Wetting and capillary condensation as means of protein organization in membranes.

    PubMed Central

    Gil, T; Sabra, M C; Ipsen, J H; Mouritsen, O G

    1997-01-01

    Wetting and capillary condensation are thermodynamic phenomena in which the special affinity of interfaces to a thermodynamic phase, relative to the stable bulk phase, leads to the stabilization of a wetting phase at the interfaces. Wetting and capillary condensation are here proposed as mechanisms that in membranes may serve to induce special lipid phases in between integral membrane proteins leading to long-range lipid-mediated joining forces acting between the proteins and hence providing a means of protein organization. The consequences of wetting in terms of protein aggregation and protein clustering are derived both within a simple phenomenological theory as well as within a concrete calculation on a microscopic model of lipid-protein interactions that accounts for the lipid bilayer phase equilibria and direct lipid-protein interactions governed by hydrophobic matching between the lipid bilayer hydrophobic thickness and the length of the hydrophobic membrane domain. The theoretical results are expected to be relevant for optimizing the experimental conditions required for forming protein aggregates and regular protein arrays in membranes. Images FIGURE 2 FIGURE 4 FIGURE 6 PMID:9336169

  4. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish.

    PubMed

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-10-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets. PMID:26451951

  5. First Identification of a Triple Corneal Dystrophy Association: Keratoconus, Epithelial Basement Membrane Corneal Dystrophy and Fuchs’ Endothelial Corneal Dystrophy

    PubMed Central

    Mazzotta, Cosimo; Traversi, Claudio; Raiskup, Frederik; Rizzo, Caterina Lo; Renieri, Alessandra

    2014-01-01

    Purpose To report the observation of a triple corneal dystrophy association consisting of keratoconus (KC), epithelial basement membrane corneal dystrophy (EBMCD) and Fuchs’ endothelial corneal dystrophy (FECD). Methods A 55-year-old male patient was referred to our cornea service for blurred vision and recurrent foreign body sensation. He reported bilateral recurrent corneal erosions with diurnal visual fluctuations. He underwent corneal biomicroscopy, Scheimpflug tomography, in vivo HRT confocal laser scanning microscopy and genetic testing for TGFBI and ZEB1 mutations using direct DNA sequencing. Results Biomicroscopic examination revealed the presence of subepithelial central and paracentral corneal opacities. The endothelium showed a bilateral flecked appearance, and the posterior corneal curvature suggested a possible concomitant ectatic disorder. Corneal tomography confirmed the presence of a stage II KC in both eyes. In vivo confocal laser scanning microscopy revealed a concomitant bilateral EBMCD with hyperreflective deposits in basal epithelial cells, subbasal Bowman's layer microfolds and ridges with truncated subbasal nerves as pseudodendritic elements. Stromal analysis revealed honeycomb edematous areas, and the endothelium showed a strawberry surface configuration typical of FECD. The genetic analysis resulted negative for TGFBI mutations and positive for a heterozygous mutation in exon 7 of the gene ZEB1. Conclusion This is the first case reported in the literature in which KC, EBMCD and FECD are present in the same patient and associated with ZEB1 gene mutation. The triple association was previously established by means of morphological analysis of the cornea using corneal Scheimpflug tomography and in vivo HRT II confocal laser scanning microscopy. PMID:25408666

  6. Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

    PubMed

    Liu, Kui; Li, Quan-Zhen; Delgado-Vega, Angelica M; Abelson, Anna-Karin; Sánchez, Elena; Kelly, Jennifer A; Li, Li; Liu, Yang; Zhou, Jinchun; Yan, Mei; Ye, Qiu; Liu, Shenxi; Xie, Chun; Zhou, Xin J; Chung, Sharon A; Pons-Estel, Bernardo; Witte, Torsten; de Ramón, Enrique; Bae, Sang-Cheol; Barizzone, Nadia; Sebastiani, Gian Domenico; Merrill, Joan T; Gregersen, Peter K; Gilkeson, Gary G; Kimberly, Robert P; Vyse, Timothy J; Kim, Il; D'Alfonso, Sandra; Martin, Javier; Harley, John B; Criswell, Lindsey A; Wakeland, Edward K; Alarcón-Riquelme, Marta E; Mohan, Chandra

    2009-04-01

    Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus. PMID:19307730

  7. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish

    PubMed Central

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-01-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets. PMID:26451951

  8. Genome Wide Analysis Indicates Genes for Basement Membrane and Cartilage Matrix Proteins as Candidates for Hip Dysplasia in Labrador Retrievers

    PubMed Central

    Lavrijsen, Ineke C. M.; Leegwater, Peter A. J.; Martin, Alan J.; Harris, Stephen J.; Tryfonidou, Marianna A.; Heuven, Henri C. M.; Hazewinkel, Herman A. W.

    2014-01-01

    Hip dysplasia, an abnormal laxity of the hip joint, is seen in humans as well as dogs and is one of the most common skeletal disorders in dogs. Canine hip dysplasia is considered multifactorial and polygenic, and a variety of chromosomal regions have been associated with the disorder. We performed a genome-wide association study in Dutch Labrador Retrievers, comparing data of nearly 18,000 single nucleotide polymorphisms (SNPs) in 48 cases and 30 controls using two different statistical methods. An individual SNP analysis based on comparison of allele frequencies with a χ2 statistic was used, as well as a simultaneous SNP analysis based on Bayesian variable selection. Significant association with canine hip dysplasia was observed on chromosome 8, as well as suggestive association on chromosomes 1, 5, 15, 20, 25 and 32. Next-generation DNA sequencing of the exons of genes of seven regions identified multiple associated alleles on chromosome 1, 5, 8, 20, 25 and 32 (p<0.001). Candidate genes located in the associated regions on chromosomes 1, 8 and 25 included LAMA2, LRR1 and COL6A3, respectively. The associated region on CFA20 contained candidate genes GDF15, COMP and CILP2. In conclusion, our study identified candidate genes that might affect susceptibility to canine hip dysplasia. These genes are involved in hypertrophic differentiation of chondrocytes and extracellular matrix integrity of basement membrane and cartilage. The functions of the genes are in agreement with the notion that disruptions in endochondral bone formation in combination with soft tissue defects are involved in the etiology of hip dysplasia. PMID:24498183

  9. Basement membrane and vascular remodelling in smokers and chronic obstructive pulmonary disease: a cross-sectional study

    PubMed Central

    2010-01-01

    Background Little is known about airway remodelling in bronchial biopsies (BB) in smokers and chronic obstructive pulmonary disease (COPD). We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm) fragmentation and altered vessel distribution in COPD. Methods To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects. Results Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p < 0.02); smoking and COPD seemed to have additive effects. Rbm fragmentation correlated with smoking history in COPD but not with age. There were more vessels in the Rbm and fewer vessels in the lamina propria in current smokers compared to healthy nonsmokers (p < 0.05). The number of vessels staining for vascular endothelial growth factor (VEGF) in the Rbm was higher in both current smoker groups and ex-smoker COPD compared to healthy nonsmokers (p < 0.004). In current smoker COPD VEGF vessel staining correlated with FEV1% predicted (r = 0.61, p < 0.02). Conclusions Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences. PMID:20670454

  10. Genome wide analysis indicates genes for basement membrane and cartilage matrix proteins as candidates for hip dysplasia in Labrador Retrievers.

    PubMed

    Lavrijsen, Ineke C M; Leegwater, Peter A J; Martin, Alan J; Harris, Stephen J; Tryfonidou, Marianna A; Heuven, Henri C M; Hazewinkel, Herman A W

    2014-01-01

    Hip dysplasia, an abnormal laxity of the hip joint, is seen in humans as well as dogs and is one of the most common skeletal disorders in dogs. Canine hip dysplasia is considered multifactorial and polygenic, and a variety of chromosomal regions have been associated with the disorder. We performed a genome-wide association study in Dutch Labrador Retrievers, comparing data of nearly 18,000 single nucleotide polymorphisms (SNPs) in 48 cases and 30 controls using two different statistical methods. An individual SNP analysis based on comparison of allele frequencies with a χ(2) statistic was used, as well as a simultaneous SNP analysis based on Bayesian variable selection. Significant association with canine hip dysplasia was observed on chromosome 8, as well as suggestive association on chromosomes 1, 5, 15, 20, 25 and 32. Next-generation DNA sequencing of the exons of genes of seven regions identified multiple associated alleles on chromosome 1, 5, 8, 20, 25 and 32 (p<0.001). Candidate genes located in the associated regions on chromosomes 1, 8 and 25 included LAMA2, LRR1 and COL6A3, respectively. The associated region on CFA20 contained candidate genes GDF15, COMP and CILP2. In conclusion, our study identified candidate genes that might affect susceptibility to canine hip dysplasia. These genes are involved in hypertrophic differentiation of chondrocytes and extracellular matrix integrity of basement membrane and cartilage. The functions of the genes are in agreement with the notion that disruptions in endochondral bone formation in combination with soft tissue defects are involved in the etiology of hip dysplasia. PMID:24498183

  11. Characterization and mechanisms of photoageing-related changes in skin. Damages of basement membrane and dermal structures.

    PubMed

    Amano, Satoshi

    2016-08-01

    Sun-exposed skin is characterized by superficial changes such as wrinkles, sagging and pigmentary changes, and also many internal changes in the structure and function of epidermis, basement membrane (BM) and dermis. These changes (so-called photoageing) are predominantly induced by the ultraviolet (UV) component of sunlight. Epidermis of UV-irradiated skin produced several enzymes such as matrix metalloproteinases (MMPs), urinary plasminogen activator (uPA)/plasmin and heparanase, which degrade dermal collagen fibres and elastic fibres in the dermis, and components of epidermal BM. The BM at the dermal-epidermal junction (DEJ) controls dermal-epidermal signalling and plays an important role in the maintenance of a healthy epidermis and dermis. BM is repetitively damaged in sun-exposed skin compared with unexposed skin, leading to epidermal and dermal deterioration and accelerated skin ageing. UV exposure also induces an increase in vascular endothelial growth factor (VEGF), an angiogenic factor, while thrombospondin-1 (TSP-1), an anti-angiogenic factor, is decreased; these changes induce angiogenesis in papillary dermis with increased migration of elastase-positive leucocytes, leading to dermal elastic fibre damage. Elastic fibres, such as oxytalan fibres in papillary dermis, are associated with not only skin resilience, but also skin surface texture, and elastic fibre formation by fibroblasts is facilitated by increased expression of fibulin-5. Thus, induction of fibulin-5 expression is a damage-repair mechanism, and fibulin-5 is an early marker of photoaged skin. UV-induced skin damage is cumulative and leads to premature ageing of skin. However, appropriate daily skincare may ameliorate photoageing by inhibiting processes causing damage and enhancing repair processes. PMID:27539897

  12. Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography

    PubMed Central

    Gallagher, Elyssia S.; Adem, Seid M.; Baker, Christopher A.; Ratnayaka, Saliya N.; Jones, Ian W.; Hall, Henry K.; Saavedra, S. Scott; Aspinwall, Craig A.

    2015-01-01

    The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2′,4′-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50mM triton X-100 or 50% acetonitrile) and physical (15 min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed > 44% increase in retention times (P < 0.0001) for all analytes on poly(bis-SorbPC)-functionalized stationary phase compared to bare silica microspheres, suggesting a lipophilic retention mechanism. Phospholipid membrane-functionalized stationary phases that withstand the chemical and physical rigors of capillary LC conditions can substantially increase the efficacy of lipid membrane affinity chromatography, and represents a key advance towards the development of robust membrane protein-functionalized chromatographic stationary phases. PMID:25670414

  13. NanoCapillary Network Proton Conducting Membranes for High Temperature Hydrogen/Air Fuel Cells

    SciTech Connect

    Pintauro, Peter

    2012-07-09

    The objective of this proposal is to fabricate and characterize a new class of NanoCapillary Network (NCN) proton conducting membranes for hydrogen/air fuel cells that operate under high temperature, low humidity conditions. The membranes will be intelligently designed, where a high density interconnecting 3-D network of nm-diameter electrospun proton conducting polymer fibers is embedded in an inert (uncharged) water/gas impermeable polymer matrix. The high density of fibers in the resulting mat and the high ion-exchange capacity of the fiber polymer will ensure high proton conductivity. To further enhance water retention, molecular silica will be added to the sulfonated polymer fibers. The uncharged matrix material will control water swelling of the high ion-exchange capacity proton conducting polymer fibers and will impart toughness to the final nanocapillary composite membrane. Thus, unlike other fuel cell membranes, the role of the polymer support matrix will be decoupled from that of the proton-conducting channels. The expected final outcome of this 5-year project is the fabrication of fuel cell membranes with properties that exceed the DOE’s technical targets, in particular a proton conductivity of 0.1 S/cm at a temperature less than or equal to120°C and 25-50% relative humidity.

  14. Reconstruction of hepatic stellate cell-incorporated liver capillary structures in small hepatocyte tri-culture using microporous membranes.

    PubMed

    Kasuya, Junichi; Sudo, Ryo; Masuda, Genta; Mitaka, Toshihiro; Ikeda, Mariko; Tanishita, Kazuo

    2015-03-01

    In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC-incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri-culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell-cell interactions, which are essential to form HSC-EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d, l-lactide-co-glycolide) (PLGA) microporous membranes in SH-HSC-EC tri-culture to reconstruct the HSC-incorporated liver capillary structures in vitro. First, the formation of EC capillary-like structures was induced on Matrigel-coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary-like structures. This process was mediated in part by platelet-derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary-like structures with their long cytoplasmic processes. In the HSC-incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro. PMID:23086892

  15. A novel, post-column micro-membrane reactor for fluorescent analysis of protein in capillary electrophoresis.

    PubMed

    Liu, Fan; Zhang, Lingyi; Qian, Junhong; Ren, Jun; Gao, Fangyuan; Zhang, Weibing

    2013-11-01

    Based on the semipermeability of hollow fiber membranes, a post-column membrane reactor was developed for capillary electrophoresis (CE)-laser induced fluorescence (LIF) analysis of proteins by using a hollow fiber membrane to connect the separation and detection capillaries. The membrane length between the separation and detection capillaries was 1 mm. Driven by the chemical potential difference between the separation buffer inside the membrane and the fluorescence derivatization solution outside the membrane, the derivatization reagent can be easily drawn into hollow fiber membrane to react with proteins. Also, the separation buffer can be adjusted by the derivatization solution to match the conditions of derivatization without sample loss. The effect of the separation buffer on the derivatization reaction was investigated and the results showed that even a strong acidic solution and multiple additives can be adopted in the separation buffer without destroying the post-column derivatization of proteins. Under the optimized conditions, the highly sensitive detection of BSA was achieved with a detection limit of 3.3 nmol L(-1) and a linear calibration range from 0.007 to 0.1 mg mL(-1). The proposed CE-LIF system with a post-column membrane reactor was also successfully applied to the separation and detection of proteins in rat liver and loach muscle. PMID:24015400

  16. Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition

    SciTech Connect

    Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Villadsen, Rene; Rank, Fritz; Bissell, Mina J.; Petersen, Ole William

    2001-10-04

    The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. We have shown previously that luminal epithelial cells can be polarized when cultured within a reconstituted basement membrane gel. We reasoned that such cues in vivo may be given by myoepithelial cells. Accordingly, we used an assay where luminal epithelial cells are incorrectly polarized to test this hypothesis. We show that culturing human primary luminal epithelial cells within collagen-I gels leads to formation of structures with no lumina and with reverse polarity as judged by dual stainings for sialomucin, epithelial specific antigen or occludin. No basement membrane is deposited, and {beta}4-integrin staining is negative. Addition of purified human myoepithelial cells isolated from normal glands corrects the inverse polarity, and leads to formation of double-layered acini with central lumina. Among the laminins present in the human breast basement membrane (laminin-1, -5 and -10/11), laminin-1 was unique in its ability to substitute for myoepithelial cells in polarity reversal. Myoepithelial cells were purified also from four different breast cancer sources including a biphasic cell line. Three out of four samples either totally lacked the ability to interact with luminal epithelial cells, or conveyed only correction of polarity in a fraction of acini. This behavior was directly related to the ability of the tumor myoepithelial cells to produce {alpha}-1 chain of laminin. In vivo, breast carcinomas were either negative for laminin-1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Dual staining with myoepithelial markers revealed that tumorassociated myoepithelial cells were either negative or weakly positive for expression of laminin-1, establishing a strong correlation between loss of laminin-1 and breast cancer. We conclude that the double-layered breast acinus may be

  17. Basement Insulation

    SciTech Connect

    Not Available

    2002-01-01

    This is one of a series of technology fact sheets created to help housing designers and builders adopt a whole-house design approach and energy efficient design practices. The fact sheet advises how to create a comfortable basement environment that is free of moisture problems and easy to condition.

  18. Collagen metabolism and basement membrane formation in cultures of mouse mammary epithelial cells: Induction of assembly on fibrillar type I collagen substrata

    SciTech Connect

    David, G.; van der Schueren, B.; van den Berghe, H. ); Nusgens, B.; Van Cauwenberge, D.; Lapiere, C. )

    1987-06-01

    Collagen metabolism was compared in cultures of mouse mammary epithelial cells maintained on plastic or fibrillar type I collagen gel substrata. The accumulation of dialysable and non-dialysable ({sup 3}H)hydroxyproline and the identification of the collagens produced suggest no difference between substrata in the allover rates of collagen synthesis and degradation. The proportion of the ({sup 3}H)collagen which accumulates in the monolayers of cultures on collagen, however, markedly exceeds that of cultures on plastic. Cultures on collagen deposit a sheet-like layer of extracellular matrix materials on the surface of the collagen fibers. Transformed cells on collagen produce and accumulate more ({sup 3}H)collage, yet are less effective in basement membrane formation than normal cells, indicting that the accumulation of collagen alone and the effect of interstitial collagen thereupon do not suffice. Thus, exogenous fibrillar collagen appears to enhance, but is not sufficient for proper assembly of collagenous basement membrane components near the basal epithelial cell surface.

  19. Performance Validation and Scaling of a Capillary Membrane Solid-Liquid Separation System

    SciTech Connect

    Rogers, S; Cook, J; Juratovac, J; Goodwillie, J; Burke, T

    2011-10-25

    Algaeventure Systems (AVS) has previously demonstrated an innovative technology for dewatering algae slurries that dramatically reduces energy consumption by utilizing surface physics and capillary action. Funded by a $6M ARPA-E award, transforming the original Harvesting, Dewatering and Drying (HDD) prototype machine into a commercially viable technology has required significant attention to material performance, integration of sensors and control systems, and especially addressing scaling issues that would allow processing extreme volumes of algal cultivation media/slurry. Decoupling the harvesting, dewatering and drying processes, and addressing the rate limiting steps for each of the individual steps has allowed for the development individual technologies that may be tailored to the specific needs of various cultivation systems. The primary performance metric used by AVS to assess the economic viability of its Solid-Liquid Separation (SLS) dewatering technology is algae mass production rate as a function of power consumption (cost), cake solids/moisture content, and solids capture efficiency. An associated secondary performance metric is algae mass loading rate which is dependent on hydraulic loading rate, area-specific hydraulic processing capacity (gpm/in2), filter:capillary belt contact area, and influent algae concentration. The system is capable of dewatering 4 g/L (0.4%) algae streams to solids concentrations up to 30% with capture efficiencies of 80+%, however mass production is highly dependent on average cell size (which determines filter mesh size and percent open area). This paper will present data detailing the scaling efforts to date. Characterization and performance data for novel membranes, as well as optimization of off-the-shelf filter materials will be examined. Third party validation from Ohio University on performance and operating cost, as well as design modification suggestions will be discussed. Extrapolation of current productivities

  20. Successful Treatment of Dual-Positive Anti-Myeloperoxidase and Anti-Glomerular Basement Membrane Antibody Vasculitis with Pulmonary-Renal Syndrome

    PubMed Central

    Huang, Jinxian; Wu, Ling; Huang, Xiaoyan; Xie, Yan; Yu, Jinquan; Yang, Jin; Fang, Huiqiong; Zhang, Lijun

    2016-01-01

    Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis and anti-glomerular basement membrane (GBM) disease are two separate diseases, while sometimes they can coexist together. The exact mechanisms are not clear, but due to the rapid progression and poor prognosis, prompt and aggressive treatment is usually required. We treated with steroids combined with cyclophosphamide and rituximab an 84-year-old man with ANCA-associated vasculitis and anti-GBM disease who had prior pulmonary fibrosis and a coexisting anterosuperior mediastinal mass. Conventional therapy including steroids, plasmapheresis and cyclophosphamide failed to attenuate the anti-GBM disease, yet he responded to an alternative treatment of rituximab. This case suggests the efficacy of steroids and immunosuppressant for the treatment of a dual-positive case with an anterosuperior mediastinal mass. PMID:26889474

  1. A microRNA from infectious spleen and kidney necrosis virus modulates expression of the virus-mock basement membrane component VP08R.

    PubMed

    Yan, Muting; He, Jianhui; Zhu, Weibin; Zhang, Jing; Xia, Qiong; Weng, Shaoping; He, Jianguo; Xu, Xiaopeng

    2016-05-01

    Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. Infection of ISKNV is characterized by a unique pathological phenomenon in that the infected cells are attached by lymphatic endothelial cells (LECs). ISKNV mediates the formation of a virus-mock basement membrane (VMBM) structure on the surface of infected cells to provide attaching sites for LECs. The viral protein VP08R is an important component of VMBM. In this study, a novel ISKNV-encoded microRNA, temporarily named ISKNV-miR-1, was identified. ISKNV-miR-1 is complementary to the VP08R-coding sequence and can modulate VP08R expression through reducing its mRNA level. This suggests that formation of VMBM may be under fine regulation by ISKNV. PMID:26896933

  2. Case with Brunsting-Perry-like localized subepidermal blister formations and immunoglobulin G antibodies against unidentified basement membrane zone antigen.

    PubMed

    Sato-Shibuya, Mami; Dainichi, Teruki; Egawa, Gyohei; Honda, Tetsuya; Otsuka, Atsushi; Ishii, Norito; Hashimoto, Takashi; Miyachi, Yoshiki; Kabashima, Kenji

    2016-04-01

    Brunsting-Perry type bullous pemphigoid is defined by the blister formation limited to the head and neck, and autoantibodies to type VII collagen are detected in several cases. However, the pathomechanisms and autoantigens in this condition remain unknown. We report a 20-year-old female patient with a more than 2-year history of recurrent tense blisters localized on the face with no distinct atrophic scar formation. The patient had neither extensive sun exposure nor a history suggestive of contact dermatitis. Oral betamethasone was effective on the skin lesions. Histopathology revealed subepidermal blister formation with dermal infiltrates of neutrophils. Although direct and indirect immunofluorescence tests detected immunoglobulin G antibodies to the basement membrane zone (BMZ), no known dermal or epidermal autoantigens were detected in immunoblot analyses. Therefore, this case may be a rare variant of Brunsting-Perry type localized bullous pemphigoid with autoantibodies to an undetermined BMZ antigen. PMID:26362108

  3. Effect of equine herpesvirus type 1 (EHV-1) infection of nasal mucosa epithelial cells on integrin alpha 6 and on different components of the basement membrane.

    PubMed

    Bannazadeh Baghi, Hossein; Nauwynck, Hans J

    2016-01-01

    The respiratory mucosa is the common port of entry of equine herpesvirus type 1 (EHV-1) and several other alphaherpesviruses. An important prerequisite for successful host invasion of the virus is to cross the epithelial cell layer and the underlying basement membrane barrier. In the present study, an analysis was performed to see if an EHV-1 infection of nasal mucosa epithelial cells leads to damage of the underlying extracellular matrix proteins. Nasal mucosa explants were inoculated with EHV-1 and collected at 0, 24 and 48 hours post-inoculation (hpi). Then, double immunofluorescence staining was performed to detect viral-antigen-positive cells on the one hand and integrin alpha 6, laminin, collagen IV and collagen VII on the other hand. The area of these extracellular matrix proteins was measured in regions of interest (ROIs) at a magnification of 200X by means of the software imaging system ImageJ. ROIs were defined beneath uninfected and infected regions. In uninfected regions, 22-28 % of the ROI was stained for integrin alpha 6, 18-37 % for laminin, 14-38 % for collagen IV and 18-26 % for collagen VII. In infected regions, the percentage positive for integrin alpha 6 was significantly decreased to 0.1-9 % and 0.1-6 % after 24 and 48 hours of inoculation, respectively. Infection did not alter the percentages for laminin and collagen IV. For collagen VII, an increase in the percentage (from 18-26 % to 28-39 %) could be observed underneath EHV-1-infected plaques at 48 hours of inoculation. In conclusion, the results revealed a substantial impact of EHV-1 infection on integrin alpha 6 and collagen VII, two important components of the extracellular matrix, which are associated with the basement membrane and may facilitate virus penetration via hijacked leukocytes to underlying tissues. PMID:26497179

  4. NC1 domain of collagen α3(IV) derived from the basement membrane regulates Sertoli cell blood-testis barrier dynamics.

    PubMed

    Wong, Elissa W P; Cheng, C Yan

    2013-04-01

    The blood-testis barrier (BTB) is an important ultrastructure for spermatogenesis. Delay in BTB formation in neonatal rats or its irreversible damage in adult rats leads to meiotic arrest and failure of spermatogonial differentiation beyond type A. While hormones, such as testosterone and FSH, are crucial to BTB function, little is known if there is a local regulatory mechanism in the seminiferous epithelium that modulates BTB function. Herein, we report that collagen α3(IV) chain, a component of the basement membrane in the rat testis, could generate a noncollagenous (NC1) domain peptide [Colα3(IV) NC1] via limited proteolysis by matrix metalloproteinase-9 (MMP-9), and that the expression of MMP-9 was upregulated by TNFα. While recombinant Colα3(IV) NC1 protein produced in E. coli failed to perturb Sertoli cell tight junction (TJ)-permeability barrier function, possibly due to the lack of glycosylation, Colα3(IV) NC1 recombinant protein produced in mammalian cells and purified to apparent homogeneity by affinity chromatography was found to reversibly perturb the Sertoli cell TJ-barrier function. Interestingly, Colα3(IV) NC1 recombinant protein did not perturb the steady-state levels of several TJ- (e.g., occludin, CAR, JAM-A, ZO-1) and basal ectoplasmic specialization- (e.g., N-cadherin, α-catenin, β-catenin) proteins at the BTB but induced changes in protein localization and/or distribution at the Sertoli cell-cell interface in which these proteins moved from the cell surface into the cell cytosol, thereby destabilizing the TJ function. These findings illustrate the presence of a local regulatory axis known as the BTB-basement membrane axis that regulates BTB restructuring during spermatogenesis. PMID:23885308

  5. Reciprocal interactions between Beta1-integrin and epidermal growth factor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology

    SciTech Connect

    Wang, F.; Weaver, V.M.; Petersen, O.W.; Larabell, C.A.; Dedhar, S.; Briand, P.; Lupu, R.; Bissell, M.J.

    1998-09-30

    Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of {beta}1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4-2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a threedimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of {beta}1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogenactivated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibodymediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant downregulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Crossmodulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and {beta}1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of {beta}1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be 'normalized' by manipulating either pathway.

  6. Glycosylation of human glomerular basement membrane collagen: increased content of hexose in ketoamine linkage and unaltered hydroxylysine-O-glycosides in patients with diabetes.

    PubMed

    Uitto, J; Perejda, A J; Grant, G A; Rowold, E A; Kilo, C; Williamson, J R

    1982-01-01

    To study the glycosylation of glomerular basement membrane collagen (GBMC) in diabetes, kidneys were obtained at autopsy from 5 patients with insulin-requiring diabetes of long duration and diabetic complications, and from 5 control subjects. Glomeruli were prepared by sieving and collagen was isolated by limited pepsin proteolysis followed by salt precipitations. Amino acid analyses of the collagen preparations, after acid hydrolysis, indicated a composition consistent with that of type IV collagen. No differences in the relative contents of various amino acids, and in particular, 3-hydroxyproline, 4-hydroxyproline and hydroxylysine, were noted between diabetic and control samples. Non-enzymatic glucosylation was assessed by measuring hexose in ketoamine linkage with thiobarbituric acid after conversion to 5-hydroxymethylfurfural. In 4 of the 5 patients studied, glucosylation values exceeded the mean +2 S.D. of the controls; in the fifth subject glucosylation was in the high normal range. No correlation between the severity of diabetes and hexose content of GBMC was noted, however. In further studies, enzymatic glycosylation of GBMC was assayed after alkaline hydrolysis by separation of glucosylgalactosyl-O-hydroxylysine, galactosyl-O-hydroxylysine, and unsubstituted hydroxylysine in an amino acid analyzer. No differences in the relative contents of hydroxylysine-O-glycosides were evident between diabetic and control GBMC. The results suggest that non-enzymatic glucosylation, but not glycosylation catalyzed by collagen glucosyl and galactosyl transferases, is increased in diabetes. The increased carbohydrate content of collagen may lead to decreased turnover and/or excessive accumulations of basement membrane collagen thus contributing to the vascular complications of diabetes. PMID:6218960

  7. Relationship of structural to functional impairment during alveolar-capillary membrane development.

    PubMed

    Ahlfeld, Shawn K; Gao, Yong; Conway, Simon J; Tepper, Robert S

    2015-04-01

    Bronchopulmonary dysplasia is a chronic lung disease of extreme preterm infants and results in impaired gas exchange. Although bronchopulmonary dysplasia is characterized histologically by alveolar-capillary simplification in animal models, it is clinically defined by impaired gas diffusion. With the use of a developmentally relevant model, we correlated alveolar-capillary structural simplification with reduced functional gas exchange as measured by the diffusing factor for carbon monoxide (DFCO). Neonatal mouse pups were exposed to >90% hyperoxia or room air during postnatal days 0 to 7, and then all pups were returned to room air from days 7 to 56. At day 56, DFCO was measured as the ratio of carbon monoxide uptake to neon dilution, and lungs were fixed for histologic assessment of alveolar-capillary development. Neonatal hyperoxia exposure inhibited alveolar-capillary septal development as evidenced by significantly increased mean linear intercept, increased airspace-to-septal ratio, decreased nodal density, and decreased pulmonary microvasculature. Importantly, alveolar-capillary structural deficits in hyperoxia-exposed pups were accompanied by a significant 28% decrease in DFCO (0.555 versus 0.400; P < 0.0001). In addition, DFCO was highly and significantly correlated with structural measures of reduced alveolar-capillary growth. Simplification of alveolar-capillary structure is highly correlated with impaired gas exchange function. Current mechanistic and therapeutic animal models of inhibited alveolar development may benefit from application of DFCO as an alternative physiologic indicator of alveolar-capillary development. PMID:25661110

  8. Bilirubin removal from human plasma by Cibacron Blue F3GA using immobilized microporous affinity membranous capillary method.

    PubMed

    Zhang, Lei; Jin, Gu

    2005-07-01

    A novel affinity sorbent system for direct bilirubin removal from human plasma was developed. These new adsorbents comprise Cibacron Blue F3GA as the specific ligand, and microporous membranous poly(tetrafluoroethylene) capillary (modified by coating with a hydrophilic layer of poly(vinyl alcohol) after activation) as the carrier matrix. The affinity adsorbents carrying 126.5 micromol Cibacron Blue F3GA/g polymer was then used to remove bilirubin in a flow-injection system. Non-specific adsorption on the poly(vinyl alcohol) coated capillary remains low, and higher affinity adsorption capacity, of up to 76.2 mg/g polymer was obtained after dye immobilization. The bilirubin adsorption capacity of the affinity capillary decreased with increase in the recirculation rate of plasma. The adsorption capacity increased with increase the temperature while decreased with increase the ionic strength. The maximum adsorption was only observed in neutral solution (pH 6-7). The adsorption isotherm fitted the Langmuir model well. These new adsorbents have higher velocity of mass transfer, better adsorption capacity, less fouling, longer service life and good reusability. The results of blood tests suggested the dye affinity capillary has good blood compatibility. PMID:15894520

  9. Basement membrane protein ladinin-1 and the MIF-CD44-β1 integrin signaling axis are implicated in laryngeal cancer metastasis.

    PubMed

    Klobučar, Marko; Sedić, Mirela; Gehrig, Peter; Grossmann, Jonas; Bilić, Mario; Kovač-Bilić, Lana; Pavelić, Krešimir; Kraljević Pavelić, Sandra

    2016-10-01

    Laryngeal squamous cell carcinoma (LSCC) is the most common form of malignant disease in the head and neck region characterized by frequent occurrence of metastases in the neck lymph nodes early in the disease onset. In the presented study, we performed quantitative proteomic profiling of patient-matched primary tumor and adjacent non-tumorous tissues derived from metastatic LSCC as to identify new protein candidates with potential diagnostic and therapeutic significance. Obtained results revealed for the first time involvement of the basement membrane protein ladinin-1 in laryngeal cancer metastases. Alterations in the cellular microenvironment that propel metastatic events in laryngeal cancer include activation of MIF-CD44-β1 integrin signal transduction pathway and induction of downstream signaling mediated by NF-κB and Src tyrosine kinase, which ultimately impinge on cytoskeletal dynamics and architecture resulting in increased cellular motility and invasiveness. In this context, particularly interesting finding is upregulation of several actin-binding proteins novel to laryngeal cancer pathogenesis including coronin-1C and plastin-2, whose functional significance in laryngeal carcinogenesis has yet to be established. We also detected for the first time a complete loss of afamin in metastatic laryngeal cancer tissues, which warrants further studies into its use as a possible marker for monitoring disease progression and/or treatment outcome. PMID:27460703

  10. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

    SciTech Connect

    Collier, I.E.; Wilhelm, S.M.; Eisen, A.Z.; Marmer, B.L.; Grant, G.A.; Seltzer, J.L.; Kronberger, A.; He, C.; Bauer, E.A.; Goldberg, G.I.

    1988-05-15

    H-ras transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on this ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors. Type IV collagenase consists of three domains. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.

  11. A Two-Dimensional Model of the Colonic Crypt Accounting for the Role of the Basement Membrane and Pericryptal Fibroblast Sheath

    PubMed Central

    Dunn, Sara-Jane; Appleton, Paul L.; Nelson, Scott A.; Näthke, Inke S.; Gavaghan, David J.; Osborne, James M.

    2012-01-01

    The role of the basement membrane is vital in maintaining the integrity and structure of an epithelial layer, acting as both a mechanical support and forming the physical interface between epithelial cells and the surrounding connective tissue. The function of this membrane is explored here in the context of the epithelial monolayer that lines the colonic crypt, test-tube shaped invaginations that punctuate the lining of the intestine and coordinate a regular turnover of cells to replenish the epithelial layer every few days. To investigate the consequence of genetic mutations that perturb the system dynamics and can lead to colorectal cancer, it must be possible to track the emerging tissue level changes that arise in the crypt. To that end, a theoretical crypt model with a realistic, deformable geometry is required. A new discrete crypt model is presented, which focuses on the interaction between cell- and tissue-level behaviour, while incorporating key subcellular components. The model contains a novel description of the role of the surrounding tissue and musculature, based upon experimental observations of the tissue structure of the crypt, which are also reported. A two-dimensional (2D) cross-sectional geometry is considered, and the shape of the crypt is allowed to evolve and deform. Simulation results reveal how the shape of the crypt may contribute mechanically to the asymmetric division events typically associated with the stem cells at the base. The model predicts that epithelial cell migration may arise due to feedback between cell loss at the crypt collar and density-dependent cell division, an hypothesis which can be investigated in a wet lab. This work forms the basis for investigation of the deformation of the crypt structure that can occur due to proliferation of cells exhibiting mutant phenotypes, experiments that would not be possible in vivo or in vitro. PMID:22654652

  12. Comparing The Efficacy of Hematoxylin and Eosin, Periodic Acid Schiff and Fluorescent Periodic Acid Schiff-Acriflavine Techniques for Demonstration of Basement Membrane in Oral Lichen Planus: A Histochemical Study

    PubMed Central

    Pujar, Ashwini; Pereira, Treville; Tamgadge, Avinash; Bhalerao, Sudhir; Tamgadge, Sandhya

    2015-01-01

    Background: Basement membrane (BM) is a thick sheet of extracellular matrix molecules, upon which epithelial cells attach. Various immunohistochemical studies in the past have been carried out but these advanced staining techniques are expensive and not feasible in routine laboratories. Although hematoxylin and eosin (H-E) is very popular among pathologists for looking at biopsies, the method has some limitations. This is where special stains come handy. Aims and Objectives: The aim of the present study was to demonstrate and compare the efficacy of H-E, periodic acid Schiff (PAS) and fluorescent periodic acid–acriflavine staining techniques for the basement membrane and to establish a histochemical stain which could be cost effective, less time consuming, and unambiguous for observation of the basement membrane zone. Materials and Methods: A total number of 40 paraffin-embedded tissue sections of known basement membrane containing tissues including 10 – Normal oral mucosa (NOM) and 30 – oral lichen planus (OLP) were considered in the study. Four-micron-thick sections of each block were cut and stained with H-E stain, PAS and fluorescent periodic acid–acriflavine stain. Sections were evaluated by three oral pathologists independently for continuity, contrast and pattern. Results: Though all the three stains showed favorable features at different levels, acriflavine stain was better than the other stains in demonstrating BM continuity, contrast and also the pattern followed by PAS stain. Acriflavine stain was the better in demonstrating a fibrillar pattern of a BM. Acriflavine stains a BM distinctly and is less time consuming and easy to carry out using readily available dyes as compared to other stains. Conclusion: The continuity and contrast along with the homogenous pattern and the afibrillar pattern of the BM was better demonstrated by acriflavine followed by the PAS stain. PMID:26538690

  13. Genetic interaction between Caenorhabditis elegans teneurin ten-1 and prolyl 4-hydroxylase phy-1 and their function in collagen IV–mediated basement membrane integrity during late elongation of the embryo

    PubMed Central

    Topf, Ulrike; Chiquet-Ehrismann, Ruth

    2011-01-01

    Teneurins are a family of phylogenetically conserved proteins implicated in pattern formation and morphogenesis. The sole orthologue in Caenorhabditis elegans, ten-1, is important for hypodermal cell migration, neuronal migration, path finding and fasciculation, gonad development, and basement membrane integrity of some tissues. However, the mechanisms of TEN-1 action remain to be elucidated. Using a genome-wide RNA interference approach, we identified phy-1 as a novel interaction partner of ten-1. phy-1 codes for the catalytic domain of collagen prolyl 4-hydroxylase. Loss of phy-1 significantly enhanced the embryonic lethality of ten-1 null mutants. Double-mutant embryos arrested during late elongation with epidermal defects, disruption of basement membranes, and detachment of body wall muscles. We found that deletion of phy-1 caused aggregation of collagen IV in body wall muscles in elongated embryos and triggered the loss of tissue integrity in ten-1 mutants. In addition, phy-1 and ten-1 each genetically interact with genes encoding collagen IV. These findings support a functional mechanism in which loss of ten-1, together with a reduction of assembled and secreted basement membrane collagen IV protein, leads to detachment of the epidermis from muscle cells during late elongation of the embryo when mechanical stress is generated by muscle contractions. PMID:21795395

  14. Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling.

    PubMed

    Gkantidis, Nikolaos; Katsaros, Christos; Chiquet, Matthias

    2012-10-01

    Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo. PMID:22688677

  15. Large introns in the 3' end of the gene for the pro. cap alpha. 1(IV) chain of human basement membrane collagen

    SciTech Connect

    Soininen, R.; Tikka, L.; Chow, L.; Pihlajaniemi, T.; Kurkinen, M.; Prockop, D.J.; Boyd, C.D.; Tryggvason, K.

    1986-03-01

    Using a recently characterized cDNA clone (HT-21) coding for the pro..cap alpha..1(IV) chain of human type IV procollagen, the authors have isolated three clones from a bacteriophage lambda Charon 4A library of human genomic DNA. The intron/exon structure of the pro..cap alpha..1(IV) genomic clones was analyzed by heteroduplex electron microscopy and nucleotide sequencing. The analysis showed that the introns separating exons 2-9 are large and have a total length of over 12,000 base pairs (bp). Six of seven exons at the 3' end of the gene coded for -Gly-Xaa-Yaa-repeats of the collagenous part of the chain. Five of the -Gly-Xaa-Yaa-coding exons (numbers 5-9) varied in size between 72 bp and 134 bp, and none of them were 54 bp or multiples thereof. A sixth exon (exon 4) was a junction exon containing 71 bp coding for-Gly-Xaa-Yaa-sequences and 142 bp coding for the carboxyl-terminal noncollagenous domain (NC-1). The seventh exon (exon 3, 178 bp) coded for sequences of the NC-1 domain. Five of the six-Gly-Xaa-Yaa- coding exons began with the second base coding for glycine, and only one exon began with a complete glycine codon at the 5' end. The results (i) suggest that the gene for the pro..cap alpha..1(IV) chain of human basement membrane collagen is significantly larger than the genes for fibrillar collagens and (ii) show that it lacks the 54-bp exon repeats characteristic of fibrillar collagen genes.

  16. Erythropoietin protects the tubular basement membrane by promoting the bone marrow to release extracellular vesicles containing tPA-targeting miR-144.

    PubMed

    Zhou, Yang; Fang, Li; Yu, Yanting; Niu, Jing; Jiang, Lei; Cao, Hongdi; Sun, Qi; Zen, Ke; Dai, Chunsun; Yang, Junwei

    2016-01-01

    Renal fibrosis is an inevitable outcome of chronic kidney disease (CKD). Erythropoietin (EPO) has been recently reported to be able to mitigate renal fibrosis. The mechanism underlying the protective effect of EPO, however, remains elusive. In the present study, employing a mouse model of renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO), we demonstrated that EPO markedly reduced the disruption of the tubular basement membrane (TBM) through attenuating the activation of tissue plasminogen activator (tPA) and matrix metalloproteinase 9 (MMP9), the major matrix proteolytic network in the obstructed kidney. Instead of acting directly on tPA in the kidney, EPO strongly increased the level of circulating microRNA (miR)-144, which was delivered to the injured renal fibroblasts via extracellular vesicles (EVs) to target the tPA 3'-untranslated region and suppress tPA expression. The protective effect of EPO on mouse TBM was inhibited by miR-144 antagomir. Furthermore, in vitro results confirmed that EPO could stimulate bone marrow-derived Sca-1(+)CD44(+)CD11b(-)CD19(-) cells to secrete miR-144-containing EVs, which markedly suppressed tPA expression, as well as metalloproteinase 9 (MMP9) level and activity, in cultured renal fibroblasts. In conclusion, our study provides the first evidence that EPO protects mouse renal TBM through promoting bone marrow cells to generate and secrete miR-144, which, in turn, is efficiently delivered into the mouse kidney via EVs to inhibit the activation of the tPA/MMP9-mediated proteolytic network. This finding thus suggests that EPO, a hormone widely used to treat anemia in CKD, is a potential therapeutic strategy for renal fibrosis. PMID:26469975

  17. Symposium: Role of the extracellular matrix in mammary development. Regulation of milk protein and basement membrane gene expression: The influence of the extracellular matrix

    SciTech Connect

    Aggeler, J.; Park, C.S.; Bissell, M.J.

    1988-10-01

    Synthesis and secretion of milk proteins ({alpha}-casein, {beta}-casein, {gamma}-casein, and transferrin) by cultured primary mouse mammary epithelial cells is modulated by the extracellular matrix. In cells grown on released or floating type I collagen gels, mRNA for {beta}-casein and transferrin is increased as much as 30-fold over cells grown on plastic. Induction of {beta}-casein expression depends strongly on the presence of lactogenic hormones, especially prolactin, in the culture. When cells are plated onto partially purified reconstituted basement membrane, dramatic changes in morphology and milk protein gene expression are observed. Cells cultured on the matrix for 6 to 8 d in the presence of prolactin, insulin, and hydrocortisone form hollow spheres and duct-like structures that are completely surrounded by matrix. The cells lining these spheres appear actively secretory and are oriented with their apices facing the lumen. Hybridization experiments indicate that mRNA for {beta}-casein can be increased as much as 70-fold in these cultures. Because > 90% of the cultured cells synthesize immunoreactive {beta}-casein, as compared with only 40% of cells in the late pregnant gland, the matrix appears to be able to induce protein expression in previously silent cells. Synthesis of laminin and assembly of a mammary-specific basal lamina by cells cultured on different extracellular matrices also appears to depend on the presence of lactogenic hormones. These studies provide support for the concept of dynamic reciprocity in which complex interactions between extracellular matrix and the cellular cytoskeleton contribute to the induction and maintenance of tissue-specific gene expression in the mammary gland.

  18. Regulation of basement membrane-reactive B cells in BXSB, (NZBxNZW)F1, NZB, and MRL/lpr lupus mice

    PubMed Central

    Clark, Amy G.; Fan, Qihua; Brady, Graham F.; Mackin, Katherine M.; Coffman, Evan D.; Weston, Melissa L.; Foster, Mary H.

    2013-01-01

    Autoantibodies to diverse antigens escape regulation in systemic lupus erythematosus under the influence of a multitude of predisposing genes. To gain insight into the differential impact of diverse genetic backgrounds on tolerance mechanisms controlling autoantibody production in lupus, we established a single lupus-derived nephritis associated anti-basement membrane Ig transgene on each of four inbred murine lupus strains, including BXSB, (NZBxNZW)F1, NZB, and MRL/lpr, as approved by the Duke University and the Durham Veterans Affairs Medical Centers’ Animal Care and Use Committees. In nonautoimmune C57BL/6 mice, B cells bearing this anti-laminin Ig transgene are stringently regulated by central deletion, editing, and anergy. Here, we show that tolerance is generally intact in unmanipulated Ig transgenic BXSB, (NZBxNZW)F1, and NZB mice, based on absence of serum transgenic anti-laminin autoantibodies and failure to recover spontaneous anti-laminin monoclonal antibodies. Four- to six-fold depletion of splenic B cells in transgenic mice of these strains, as well as in MRL/lpr transgenic mice, and reduced frequency of IgM+ bone marrow B cells suggest that central deletion is grossly intact. Nonetheless the four strains demonstrate distinct transgenic B cell phenotypes, including endotoxin-stimulated production of anti-laminin antibodies by B cells from transgenic NZB mice, and in vitro hyperproliferation of both endotoxin- and BCR-stimulated B cells from transgenic BXSB mice, which are shown to have an enrichment of CD21-high marginal zone cells. Rare anti-laminin transgenic B cells spontaneously escape tolerance in MRL/lpr mice. Further study of the mechanisms underlying these strain-specific B cell fates will provide insight into genetic modification of humoral autoimmunity in lupus. PMID:23157336

  19. Targeted Expression of Stromelysin-1 in Mammary Gland Provides Evidence for a Role of Proteinases in Branching Morphogenesis and the Requirement for an Intact Basement Membrane for Tissue-specific Gene Expression

    SciTech Connect

    Sympson, Carolyn J; Talhouk, Rabih S; Alexander, Caroline M; Chin, Jennie R; Cliff, Shirley M; Bissell, Mina J; Werb, Zena

    1994-05-01

    The extracellular matrix (ECM) is an important regulator of the differentiated phenotype of mammary epithelial cells in culture. Despite the fact that ECM-degrading enzymes have been implicated in morphogenesis and tissue remodeling, there is little evidence for a direct role for such regulation in vivo. We generated transgenic mice that express autoactivated isoforms of the matrix metalloproteinase stromelysin-1, under the control of the whey acidic protein gene promoter, to examine the effect of inappropriate expression of this enzyme. Stromelysin-1 is implicated as the primary player in the loss of basement membrane and loss of function in the mammary gland during involution. The transgene was expressed at low levels in mammary glands of virgin female mice, leading to an unexpected phenotype: The primary ducts had supernumerary branches and showed precocious development of alveoli that expressed beta-casein at levels similar to that of an early- to mid-pregnant gland. Lactating glands showed high levels of transgene expression, with accumulation at the basement membrane, and a decrease in laminin and collagen IV, resulting in a loss of basement membrane integrity; this was accompanied by a dramatic alteration of alveolar morphology, with decreased size and shrunken lumina containing little beta-casein. During pregnancy, expression of endogenous whey acidic protein and beta-casein was reduced in transgenic glands, confirming the observed dependence of milk protein transcription of ECM in mammary epithelial cells in culture. These data provide direct evidence that stromelysin-1 activity can be morphogenic for mammary epithelial cells, inducing hyperproliferation and differentiation in virgin animals, and that its lytic activity can, indeed, disrupt membrane integrity and reduce mammary-specific function. We conclude that the balance of ECM-degrading enzymes with their inhibitors, and the associated regulation of ECM structure, is crucial for tissue-specific gene

  20. On the factors which contribute to thinning of the villous membrane in human placentae at high altitude. II. An increase in the degree of peripheralization of fetal capillaries.

    PubMed

    Jackson, M R; Mayhew, T M; Haas, J D

    1988-01-01

    The contribution made by fetal capillary peripheralization to the thinning of the villous membrane seen in human placentae from high-altitude pregnancies is examined by stereological methods. Variables characterizing the shape of the villous core and the spatial relationships between trophoblast and capillaries are quantified. They shed light on the relative importance of dynamic versus mechanistic processes of villous membrane attenuation. Highland villi differ from lowland villi in several ways. On average, they possess a thinner barrier due to closer approximation of capillaries to overlying trophoblast; in consequence, the villous core is more irregular in outline and its surface (that of the inner aspect of the trophoblast) exceeds in area that of the outer aspect of the trophoblast. These results suggest that the dynamic process (protoplasmic streaming within syncytiotrophoblast) cannot alone explain thinning of the villous membrane. A mechanistic process (capillary peripheralization and obtrusion into the trophoblastic epithelium) is sufficient to account for the differences observed, although the possibility that both processes operate concurrently cannot be discounted. This report completes a study into factors contributing to villous membrane thinning at high altitude. PMID:3362794

  1. CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes.

    PubMed Central

    Knutson, J R; Iida, J; Fields, G B; McCarthy, J B

    1996-01-01

    Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion

  2. Proteolysis breaks tolerance toward intact α345(IV) collagen, eliciting novel anti-glomerular basement membrane autoantibodies specific for α345NC1 hexamers.

    PubMed

    Olaru, Florina; Wang, Xu-Ping; Luo, Wentian; Ge, Linna; Miner, Jeffrey H; Kleinau, Sandra; Geiger, Xochiquetzal J; Wasiluk, Andrew; Heidet, Laurence; Kitching, A Richard; Borza, Dorin-Bogdan

    2013-02-15

    Goodpasture disease is an autoimmune kidney disease mediated by autoantibodies against noncollagenous domain 1 (NC1) monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis (GN). We identified a novel type of human IgG4-restricted anti-GBM autoantibodies associated with mild nonprogressive GN, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti-GBM autoantibodies were investigated in mouse models recapitulating this phenotype. Wild-type and FcγRIIB(-/-) mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoantibodies specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause GN. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3(-/-) Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG Abs specific for α345NC1 hexamers, which were not subclass restricted. As heterologous Ag in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a, and IgG2b autoantibodies specific for α345NC1 hexamers and induced anti-GBM Ab GN. These findings indicate that tolerance toward autologous intact α345(IV) collagen is established in hosts expressing this Ag, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α345NC1 hexamers. This provides a mechanism eliciting autoantibodies specific for α345NC1 hexamers, which are restricted to noninflammatory IgG subclasses and are nonnephritogenic. In Alport syndrome, lack of tolerance toward α345(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including proinflammatory IgG subclasses that mediate posttransplant anti-GBM nephritis. PMID:23303673

  3. Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning

    PubMed Central

    Pietilä, Ilkka; Prunskaite-Hyyryläinen, Renata; Kaisto, Susanna; Tika, Elisavet; van Eerde, Albertien M.; Salo, Antti M.; Garma, Leonardo; Miinalainen, Ilkka; Feitz, Wout F.; Bongers, Ernie M. H. F.; Juffer, André; Knoers, Nine V. A. M.; Renkema, Kirsten Y.; Myllyharju, Johanna; Vainio, Seppo J.

    2016-01-01

    The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to Wnt5a knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the Wnt5a knock out when compared to control. Expression of a panel of BM components such as laminin and of type IV collagen was also reduced due to the Wnt5a knock out. The P4ha1 gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in Wnt5a function from control. The compound Wnt5a+/-;P4ha1+/- embryos demonstrated Wnt5a-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling in vitro. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease. PMID:26794322

  4. Antigen and epitope specificity of anti-glomerular basement membrane antibodies in patients with goodpasture disease with or without anti-neutrophil cytoplasmic antibodies.

    PubMed

    Yang, Rui; Hellmark, Thomas; Zhao, Juan; Cui, Zhao; Segelmark, Marten; Zhao, Ming-Hui; Wang, Hai-Yan

    2007-04-01

    Goodpasture disease (GP) is defined by the presence of anti-glomerular basement membrane (anti-GBM) antibodies and rapidly progressive glomerulonephritis. Besides anti-GBM, many patients with GP produce anti-neutrophil cytoplasmic antibodies (ANCA). For elucidation of the pathophysiologic significance of ANCA in this setting, epitope and antigen specificity of the anti-GBM antibodies and antigen specificity of ANCA were studied. Bovine testis alpha(IV)NC1 (tNC1); recombinant human alpha1, alpha3, alpha4, and alpha5(IV)NC1 (ralpha1 through ralpha5); and three chimeric proteins that contain previously defined epitope regions designated E(A), E(B), and S2 were used to examine the anti-GBM antibodies by ELISA in 205 Chinese patients with GP with or without ANCA. In the 205 anti-GBM antibody-positive sera, 63 (30.7%) were also ANCA positive (61 myeloperoxidase-ANCA and six proteinase 3-ANCA, four being triple positive). All 205 sera recognized tNC1 and ralpha3(IV)NC1. In the double-positive group, 54.0, 66.7, 71.4% of the sera could recognize ralpha1, ralpha4, and ralpha5, respectively, compared with 49.3, 60.6, and 55.6% for patients with anti-GBM antibodies alone. The levels of the antibodies to ralpha3, tNC1, and the alpha3/alpha1 ratio were lower in the double-positive group than that in patients with anti-GBM antibody alone (P < 0.05). Most of the sera could recognize the epitope regions E(A), E(B), and S2, but the absorbance values to E(A), E(B), and S2 were lower in double-positive group (P < 0.05). Double-positive patients had a broader spectrum of anti-GBM antibodies and lower levels of antibodies against alpha3(IV)NC1 compared with that of patients with anti-GBM antibodies alone. PMID:17329569

  5. Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning.

    PubMed

    Pietilä, Ilkka; Prunskaite-Hyyryläinen, Renata; Kaisto, Susanna; Tika, Elisavet; van Eerde, Albertien M; Salo, Antti M; Garma, Leonardo; Miinalainen, Ilkka; Feitz, Wout F; Bongers, Ernie M H F; Juffer, André; Knoers, Nine V A M; Renkema, Kirsten Y; Myllyharju, Johanna; Vainio, Seppo J

    2016-01-01

    The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to Wnt5a knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the Wnt5a knock out when compared to control. Expression of a panel of BM components such as laminin and of type IV collagen was also reduced due to the Wnt5a knock out. The P4ha1 gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in Wnt5a function from control. The compound Wnt5a+/-;P4ha1+/- embryos demonstrated Wnt5a-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling in vitro. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease. PMID:26794322

  6. Hollow fiber membranes for advanced life support systems. [permeable capillaries for medical filtration

    NASA Technical Reports Server (NTRS)

    Roebelen, G. J., Jr.; Lysaght, M. J.

    1977-01-01

    This paper describes an investigation of the practicability of utilizing hollow fiber membranes in vehicular and portable life support system applications. A preliminary screening of potential advanced life support applications resulted in the selection of five applications for feasibility study and testing. As a result of the feasibility study and testing, three applications, heat rejection, deaeration, and bacteria filtration, were chosen for breadboard development testing. Breadboard hardware has been manufactured and tested, and the physical properties of the three hollow fiber membrane assemblies applicable to use aboard future spacecraft have been characterized.

  7. Lung membrane conductance and capillary volume derived from the NO and CO transfer in high-altitude newcomers.

    PubMed

    Martinot, Jean-Benoît; Mulè, Massimiliano; de Bisschop, Claire; Overbeek, Maria J; Le-Dong, Nhat-Nam; Naeije, Robert; Guénard, Hervé

    2013-07-15

    Acute exposure to high altitude may induce changes in carbon monoxide (CO) membrane conductance (DmCO) and capillary lung volume (Vc). Measurements were performed in 25 lowlanders at Brussels (D0), at 4,300 m after a 2- or 3-day exposure (D2,3) without preceding climbing, and 5 days later (D7,8), before and after an exercise test, under a trial with two arterial pulmonary vasodilators or a placebo. The nitric oxide (NO)/CO transfer method was used, assuming both infinite and finite values to the NO blood conductance (θNO). Doppler echocardiography provided hemodynamic data. Compared with sea level, lung diffusing capacity for CO increased by 24% at D2,3 and is returned to control at D7,8. The acute increase in lung diffusing capacity for CO resulted from increases in DmCO and Vc with finite and infinite θNO assumptions. The alveolar volume increased by 16% at D2,3 and normalized at D7,8. The mean increase in systolic arterial pulmonary pressure at rest at D2,3 was minimal. In conclusion, the acute increase in Vc may be related to the increase in alveolar volume and to the increase in capillary pressure. Compared with the infinite θNO value, the use of a finite θNO value led to about a twofold increase in DmCO value and to a persistent increase in DmCO at D7,8 compared with D0. After exercise, DmCO decreased slightly less in subjects treated by the vasodilators, suggesting a beneficial effect on interstitial edema. PMID:23599397

  8. Dynamic supported liquid membrane tip extraction of glyphosate and aminomethylphosphonic acid followed by capillary electrophoresis with contactless conductivity detection.

    PubMed

    See, Hong Heng; Hauser, Peter C; Sanagi, M Marsin; Ibrahim, Wan Aini Wan

    2010-09-10

    A dynamic supported liquid membrane tip extraction (SLMTE) procedure for the effective extraction and preconcentration of glyphosate (GLYP) and its metabolite aminomethylphosphonic acid (AMPA) in water has been investigated. The SLMTE procedure was performed in a semi-automated dynamic mode and demonstrated a greater performance against a static extraction. Several important extraction parameters such as donor phase pH, cationic carrier concentration, type of membrane solvent, type of acceptor stripping phase, agitation and extraction time were comprehensively optimized. A solution of Aliquat-336, a cationic carrier, in dihexyl ether was selected as the supported liquid incorporated into the membrane phase. Quantification of GLYP and AMPA was carried out using capillary electrophoresis with contactless conductivity detection. An electrolyte solution consisting of 12 mM histidine (His), 8 mM 2-(N-morpholino)ethanesulfonic acid (MES), 75 microM cetyltrimethylammonium bromide (CTAB), 3% methanol, pH 6.3, was used as running buffer. Under the optimum extraction conditions, the method showed good linearity in the range of 0.01-200 microg/L (GLYP) and 0.1-400 microg/L (AMPA), acceptable reproducibility (RSD 5-7%, n=5), low limits of detection of 0.005 microg/L for GLYP and 0.06 microg/L for AMPA, and satisfactory relative recoveries (90-94%). Due to the low cost, the SLMTE device was disposed after each run which additionally eliminated the possibility of carry-over between runs. The validated method was tested for the analysis of both analytes in spiked tap water and river water with good success. PMID:20696433

  9. Down-Regulation of the miRNA-200 Family at the Invasive Front of Colorectal Cancers with Degraded Basement Membrane Indicates EMT Is Involved in Cancer Progression12

    PubMed Central

    Paterson, Emily L; Kazenwadel, Jan; Bert, Andrew G; Khew-Goodall, Yeesim; Ruszkiewicz, Andrew; Goodall, Gregory J

    2013-01-01

    Cancer progression is a complex series of events thought to incorporate the reversible developmental process of epithelial-to-mesenchymal transition (EMT). In vitro, the microRNA-200 family maintains the epithelial phenotype by posttranscriptionally inhibiting the E-cadherin repressors, ZEB1 and ZEB2. Here, we used in situ hybridization and immunohistochemistry to assess expression of miR-200 and EMT biomarkers in formalin-fixed paraffin-embedded human colorectal adenocarcinomas. In addition, laser capture microdissection and quantitative real-time polymerase chain reaction were employed to quantify levels of miR-200 in the normal epithelium, tumor core, invasive front, and stroma. We find that miR-200 is downregulated at the invasive front of colorectal adenocarcinomas that have destroyed and invaded beyond the basement membrane. However, regional lymph node metastases and vascular carcinoma deposits show strong expression of miR-200, suggesting this family of miRNAs is involved in the recapitulation of the primary tumor phenotype at metastatic sites. In contrast, adenomas and adenocarcinomas with intact basement membranes showed uniform miR-200 expression from the tumor core to the tumor-host interface. Taken together, these data support the involvement of EMT and mesenchymal-to-epithelial transition (MET) in the metastasis cascade and show that miR-200 is downregulated in the initial stages of stromal invasion but is restored at metastatic sites. PMID:23441132

  10. Seismic basement in Poland

    NASA Astrophysics Data System (ADS)

    Grad, Marek; Polkowski, Marcin

    2015-09-01

    The area of contact between Precambrian and Phanerozoic Europe in Poland has complicated structure of sedimentary cover and basement. The thinnest sedimentary cover in the Mazury-Belarus anteclize is only 0.3-1 km thick, increases to 7-8 km along the East European Craton margin, and 9-12 km in the Trans-European Suture Zone (TESZ). The Variscan domain is characterized by a 1- to 2-km-thick sedimentary cover, while the Carpathians are characterized by very thick sediments, up to c. 20 km. The map of the basement depth is created by combining data from geological boreholes with a set of regional seismic refraction profiles. These maps do not provide data about the basement depth in the central part of the TESZ and in the Carpathians. Therefore, the data set is supplemented by 32 models from deep seismic sounding profiles and a map of a high-resistivity (low-conductivity) layer from magnetotelluric soundings, identified as a basement. All of these data provide knowledge about the basement depth and of P-wave seismic velocities of the crystalline and consolidated type of basement for the whole area of Poland. Finally, the differentiation of the basement depth and velocity is discussed with respect to geophysical fields and the tectonic division of the area.

  11. Seismic basement in Poland

    NASA Astrophysics Data System (ADS)

    Grad, Marek; Polkowski, Marcin

    2016-06-01

    The area of contact between Precambrian and Phanerozoic Europe in Poland has complicated structure of sedimentary cover and basement. The thinnest sedimentary cover in the Mazury-Belarus anteclize is only 0.3-1 km thick, increases to 7-8 km along the East European Craton margin, and 9-12 km in the Trans-European Suture Zone (TESZ). The Variscan domain is characterized by a 1- to 2-km-thick sedimentary cover, while the Carpathians are characterized by very thick sediments, up to c. 20 km. The map of the basement depth is created by combining data from geological boreholes with a set of regional seismic refraction profiles. These maps do not provide data about the basement depth in the central part of the TESZ and in the Carpathians. Therefore, the data set is supplemented by 32 models from deep seismic sounding profiles and a map of a high-resistivity (low-conductivity) layer from magnetotelluric soundings, identified as a basement. All of these data provide knowledge about the basement depth and of P-wave seismic velocities of the crystalline and consolidated type of basement for the whole area of Poland. Finally, the differentiation of the basement depth and velocity is discussed with respect to geophysical fields and the tectonic division of the area.

  12. Capillary Hemangioma

    MedlinePlus

    ... Why do capillary hemangiomas on the eyelids cause vision problems? Capillary Hemangiomas of the eyelid can cause ... a capillary hemangioma in the eye socket cause vision problems? A capillary hemangioma in the eye socket ( ...

  13. Glomerular adaptations to renal injury or ablation. Role of capillary hypertension in the pathogenesis of progressive glomerulosclerosis.

    PubMed

    Rennke, H G

    1988-01-01

    A reduction in the effective glomerular capillary surface available for filtration by renal ablation or acquired renal disease results in functional and structural adaptations which lead to endothelial, mesangial and epithelial cell dysfunction. The structural lesions that ensue include capillary microthrombosis, mesangial expansion, microaneurysm formation, epithelial cell detachment with hyalin deposition, and basement membrane thickening. These alterations lead to segmental and eventually global glomerulosclerosis, closing the loop of a positive feedback mechanism that perpetuates and accelerates the microvascular damage. Dietary, hormonal, and as yet poorly identified constitutional factors can modulate the hemodynamic response to the original insult and offer an opportunity to influence this process therapeutically. PMID:3061399

  14. Interwoven Four-Compartment Capillary Membrane Technology for Three-Dimensional Perfusion with Decentralized Mass Exchange to Scale Up Embryonic Stem Cell Culture

    PubMed Central

    Gerlach, Jörg C.; Lübberstedt, Marc; Edsbagge, Josefina; Ring, Alexander; Hout, Mariah; Baun, Matt; Rossberg, Ingrid; Knöspel, Fanny; Peters, Grant; Eckert, Klaus; Wulf-Goldenberg, Annika; Björquist, Petter; Stachelscheid, Harald; Urbaniak, Thomas; Schatten, Gerald; Miki, Toshio; Schmelzer, Eva; Zeilinger, Katrin

    2010-01-01

    We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent ‘arteriovenous’ flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 × 109 cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining ‘stemness’ markers during cell expansion. PMID:20197653

  15. Lab-on-a-brane: nanofibrous polymer membranes to recreate organ-capillary interfaces

    NASA Astrophysics Data System (ADS)

    Budhwani, Karim I.; Thomas, Vinoy; Sethu, Palaniappan

    2016-03-01

    Drug discovery is a complex and time consuming process involving significant basic research and preclinical evaluation prior to testing in patients. Preclinical studies rely extensively on animal models which often fail in human trials. Biomimetic microphysiological systems (MPS) using human cells can be a promising alternative to animal models; where critical interactions between different organ systems are recreated to provide physiologically relevant in vitro human models. Central here are blood-vessel networks, the interface controlling transport of cellular and biomolecular components between the circulating fluid and underlying tissue. Here we present a novel lab-on-a-brane (or lab-on-a-membrane) nanofluidics MPS that combines the elegance of lab-on-a-chip with the more realistic morphology of 3D fibrous tissue-engineering constructs. Our blood-vessel lab-on-a-brane effectively simulates in vivo vessel-tissue interface for evaluating transendothelial transport in various pharmacokinetic and nanomedicine applications. Attributes of our platform include (a) nanoporous barrier interface enabling transmembrane molecular transport, (b) transformation of substrate into nanofibrous 3D tissue matrix, (c) invertible-sandwich architecture, and (d) simple co-culture mechanism for endothelial and smooth muscle layers to accurately mimic arterial anatomy. Structural, mechanical, and transport characterization using scanning electron microscopy, stress/strain analysis, infrared spectroscopy, immunofluorescence, and FITC-Dextran hydraulic permeability confirm viability of this in vitro system. Thus, our lab-on-a-brane provides an effective and efficient, yet considerably inexpensive, physiologically relevant alternative for pharmacokinetic evaluation; possibly reducing animals used in preclinical testing, costs from false starts, and time-to-market. Furthermore, it can be configured in multiple simultaneous arrays for personalized and precision medicine applications and for

  16. Membrane-assisted capillary isoelectric focusing coupling with matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry for neuropeptide analysis.

    PubMed

    Zhang, Zichuan; Wang, Junhua; Hui, Limei; Li, Lingjun

    2011-08-01

    Herein we report a highly efficient and reliable membrane-assisted capillary isoelectric focusing (MA-CIEF) system being coupled with MALDI-FTMS for the analysis of complex neuropeptide mixtures. The new interface consists of two membrane-coated joints made near each end of the capillary for applying high voltage, while the capillary ends were placed in the two reservoirs which were filled with anolyte (acid) and catholyte (base) to provide pH difference. Optimizations of CIEF conditions and comparison with conventional CIEF were carried out by using bovine serum albumin (BSA) tryptic peptides. It was shown that the MA-CIEF could provide more efficient, reliable and faster separation with improved sequence coverage when coupled to MALDI-FTMS. Analyses of orcokinin family neuropeptides from crabs Cancer borealis and Callinectes sapidus brain extracts have been conducted using the established MA-CIEF/MALDI-FTMS platform. Increased number of neuropeptides was observed with significantly enhanced MS signal in comparison with direct analysis by MALDI-FTMS. The results highlighted the potential of MA-CIEF as an efficient fractionation tool for coupling to MALDI MS for neuropeptide analysis. PMID:21696746

  17. Miniaturized supported liquid membrane device for selective on-line enrichment of basic drugs in plasma combined with capillary zone electrophoresis.

    PubMed

    Pálmarsdóttir, S; Thordarson, E; Edholm, L E; Jönsson, J A; Mathiasson, L

    1997-05-01

    A hollow fiber miniaturized supported liquid membrane (SLM) device for sample preparation is connected on-line with capillary electrophoresis and used for determination of a basic drug, bambuterol, in human plasma. The analyte is extracted from the outside of the hollow fiber (donor) through the liquid membrane (pores of the fiber impregnated with organic solvent) into the acceptor solution in the fiber lumen. The process is driven by differences in pH between the donor and acceptor solution. The whole volume of the acceptor solution can then be injected into the CZE capillary by using the double-stacking procedure for large volume-injection. Very clean extracts of low ionic strength are obtained from the SLM treatment, making this sample pretreatment method compatible with the CZE double-stacking procedure, which in turn makes it possible to inject large volumes of sample onto the separation capillary. Good performance of the whole procedure is demonstrated, and detection limits in the low nanomolar range were obtained in spite of the relatively weak UV absorbance of bambuterol. Extractions through the miniaturized SLM unit can be performed for 5-6 h without regenerating the fiber. The regeneration procedure was tested, and no relevant changes in the performance of the extraction could be found after seven regenerations, allowing the same fiber to be used for a week. PMID:9145027

  18. Capillary electrophoresis

    SciTech Connect

    Warner, M.

    1988-10-15

    Rapid instrumental methods for performing electrophoretic separations in capillary tubes have recently been developed, making capillary electrophoresis one of the most exciting new techniques available to analytical chemists. This article discusses detection methods, applications, and the future of capillary electrophoresis.

  19. Capillary sample

    MedlinePlus

    ... using capillary blood sampling. Disadvantages to capillary blood sampling include: Only a limited amount of blood can be drawn using this method. The procedure has some risks (see below). Capillary ...

  20. Age and diabetes related changes of the retinal capillaries: An ultrastructural and immunohistochemical study.

    PubMed

    Bianchi, Enrica; Ripandelli, Guido; Taurone, Samanta; Feher, Janos; Plateroti, Rocco; Kovacs, Illes; Magliulo, Giuseppe; Orlando, Maria Patrizia; Micera, Alessandra; Battaglione, Ezio; Artico, Marco

    2016-03-01

    Normal human aging and diabetes are associated with a gradual decrease of cerebral flow in the brain with changes in vascular architecture. Thickening of the capillary basement membrane and microvascular fibrosis are evident in the central nervous system of elderly and diabetic patients. Current findings assign a primary role to endothelial dysfunction as a cause of basement membrane (BM) thickening, while retinal alterations are considered to be a secondary cause of either ischemia or exudation. The aim of this study was to reveal any initial retinal alterations and variations in the BM of retinal capillaries during diabetes and aging as compared to healthy controls. Moreover, we investigated the potential role of vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines in diabetic retina.Transmission electron microscopy (TEM) was performed on 46 enucleated human eyes with particular attention to alterations of the retinal capillary wall and Müller glial cells. Inflammatory cytokines expression in the retina was investigated by immunohistochemistry.Our electron microscopy findings demonstrated that thickening of the BM begins primarily at the level of the glial side of the retina during aging and diabetes. The Müller cells showed numerous cytoplasmic endosomes and highly electron-dense lysosomes which surrounded the retinal capillaries. Our study is the first to present morphological evidence that Müller cells start to deposit excessive BM material in retinal capillaries during aging and diabetes. Our results confirm the induction of pro-inflammatory cytokines TNF-α and IL-1β within the retina as a result of diabetes.These observations strongly suggest that inflammatory cytokines and changes in the metabolism of Müller glial cells rather than changes in of endothelial cells may play a primary role in the alteration of retinal capillaries BM during aging and diabetes. PMID:26604209

  1. Anti-glomerular basement membrane glomerulonephritis (anti-GBM GN) in the mouse: BrdU-labelling indices and histological damage.

    PubMed Central

    Wheeler, J.; Robertson, H.; Morley, A. R.; Appleton, D. R.

    1993-01-01

    In-vivo BrdU incorporation and visualization by immunohistochemistry, previously reported in normal mouse kidney, were applied to a mouse model of anti-GBM GN, induced by immunization with rabbit anti-mouse GBM antiserum, to assess the contribution of capsular cell proliferation in the development of crescents. A significant increase (P = 0.003) in the BrdU-labelling index (LI) for capsular cells was observed, as compared to normal mice (5.76 +/- 1.1 vs 0.70% +/- 0.12%). Elevated LI were also observed for tuft and tubular cells but these increases were not statistically significant. It was concluded that, in this model, capsular cell proliferation is a major contributory factor to the formation of cellular crescents. In addition, other pathological features, indicative of glomerular damage, were assessed semi-quantitatively alongside numbers of labelled capsular cells per glomerulus. It was found that podocyte vacuolation is strongly associated with, and may precede, proliferation, suggesting some common causative factor. Fibrin, when present, was confined within the tuft capillary loops and was only weakly associated with either podocyte vacuolation or capsular cell proliferation. It was concluded that this protein does not play a major role in the initiation of pathological damage. Finally, glomerular lesions were found to be randomly distributed. Thus, the idea of intraglomerular signalling, resulting in 'clustering' of damaged glomeruli, is not supported. Images Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:8471539

  2. Direct determination of chlorophenols present in liquid samples by using a supported liquid membrane coupled in-line with capillary electrophoresis equipment.

    PubMed

    Almeda, S; Nozal, L; Arce, L; Valcárcel, M

    2007-03-21

    Actually there is a great trend on the development of effective analytical methods for monitoring trace levels of various phenols which can indicate, among others compounds, the water quality. A simple, inexpensive supported liquid membrane (SLM) device was used in combination with commercially available capillary electrophoresis (CE) equipment for the direct determination of chlorophenols in surface water samples. The manifold was used simultaneously to extract and preconcentrate the analytes from liquid samples. In the extraction set-up, the donor phase (4 mL) was placed in the CE vial, where a micro-membrane extraction unit (MMEU) accommodating the acceptor phase (100 microL) in its lumen was immersed. The supported liquid membrane was constructed by impregnating a porous Fluoropore Teflon (PTFE) membrane with a water-immiscible organic solvent (dihexyl ether). The extraction process was optimized with regard to the pH of the donor and acceptor phases, membrane liquid, extraction time and voltage applied to the inlet or outlet vial during extraction. The chlorinated phenols pentachlorophenol (PCP), 2,3,6 trichlorophenol (TCP) and 2,6 dichlorophenol (DCP) were thus efficiently separated by CE, using tris(hydroxymethyl)aminomethane (Tris) and an NaH(2)PO(4) solution containing 1% (v/v) methanol at pH 10.5 as running buffer. PMID:17386759

  3. UNDERSTANDING THE EFFECT OF DYNAMIC FEED CONDITIONS ON WATER RECOVERY FROM IC ENGINE EXHAUST BY CAPILLARY CONDENSATION WITH INORGANIC MEMBRANES

    SciTech Connect

    DeBusk, Melanie Moses; Bischoff, Brian L; Hunter, James A; Klett, James William; Nafziger, Eric J; Daw, C Stuart

    2014-01-01

    An inorganic membrane water recovery concept is evaluated as a method to recovering water from the exhaust of an internal combustion engine. Integrating the system on-board a vehicle would create a self-sustaining water supply that would make engine water injection technologies consumer transparent . In laboratory experiments, water recovery from humidified air was measured to evaluate how different operating parameters affect the membrane system s efficiency. The observed impact of transmembrane pressure and gas flow rate suggest that gas residence time is more important than water flux through the membrane. Heat transfer modeling suggests that increasing membrane length can be used to improve efficiency and allow greater flow per membrane, an important parameter for practical applications where space is limited. The membrane water recovery concept was also experimentally validated by extracting water from diesel exhaust coming from a stationary generator. The insight afforded by these studies provides a basis for developing improved membrane designs that balance both efficiency and cost.

  4. Reactivity of human anti-alpha-galactosyl IgG antibody with alpha(1-->3)-linked galactosyl epitopes exposed on basement membranes and on glomerular epithelial cells: an in vitro and in vivo study in the mouse.

    PubMed Central

    Vecchi, M L; Davin, J C; Castronovo, V; Foidart, J M; Malaise, M; Foidart, J B; Dechene, C; Sangiorgi, G B; Mahieu, P

    1989-01-01

    Anti-alpha-galactosyl antibody (a-Gal Ab) is a human natural antibody belonging to the IgG class, found in high titres in all normal sera regardless of blood group, and specifically recognizing alpha (1-->3)-linked galactosyl residues. We have observed by radioimmunoassay, ELISA, passive haemagglutination and immunofluorescence blocking studies that affinity-purified a-Gal Ab reacted with mouse laminin, but not with the other mouse basement membrane proteins tested; it was able to fix complement in vitro. When injected intravenously into mice, the a-Gal Ab was found to mainly accumulate in kidneys, liver, spleen and lungs. No acute respiratory distress syndrome was observed shortly after the i.v. injection of 100 or 200 microg of antibodies. These doses of a-Gal Ab were also unable to induce acute glomerular injury. However, in primary cultures, the a-Gal Ab (100 or 200 microg per ml of medium) was shown to impair the attachment of mouse glomerular epithelial cells to mouse laminin and to elicit complement-dependent cell damage. The data indicate that the a-Gal Ab can interact in vitro and/or in vivo with alpha (1-->3)-linked galactosyl residues exposed on murine laminin or on murine cultured glomerular epithelial cells. Although this antibody fails to be pathogenic when administered at low doses in the intact animal, similar doses can alter some metabolic properties of these cells in vitro. PMID:12412761

  5. Complete primary structure of the sixth chain of human basement membrane collagen, alpha 6(IV). Isolation of the cDNAs for alpha 6(IV) and comparison with five other type IV collagen chains.

    PubMed

    Zhou, J; Ding, M; Zhao, Z; Reeders, S T

    1994-05-01

    Basement membranes were previously believed to contain five distinct type IV collagen subunits. We have recently isolated part of the cDNA for a novel type IV collagen, alpha 6(IV), and shown that COL4A6, the gene encoding this new chain, is deleted in Alport syndrome-associated leiomyomatosis (Zhou, J., Mochizuki, T., Smeets, H., Antignac, C., Laurila, P., de Paepe, A., Tryggvason, K., and Reeders, S. T. (1993) Science 261, 1167-1169). Here, we describe the entire human alpha 6(IV) cDNA and show that the gene encodes a classical type IV collagen with homology throughout its length to all the other five chains. There is a 21-residue signal peptide, a 1417-residue collagenous domain interrupted at 25 points, and a 228-residue carboxyl-terminal non-collagenous domain. When the complete primary structure of this new chain was compared with all the other known chains, it became clear that alpha 6(IV) has the most resemblance to alpha 2(IV) and alpha 4(IV). The evolution of the six chains was deduced, allowing a new classification of the type IV collagen family. The alpha 6(IV) chain is a candidate gene for X-linked Alport syndrome; knowledge of the complete structure of the chain will permit us to screen systematically for mutations in patients and to generate recombinant proteins and synthetic peptides for further study of cell-matrix interactions involving the alpha 6(IV) chain. PMID:8175748

  6. Supported liquid membrane extraction coupled in-line to commercial capillary electrophoresis for rapid determination of formate in undiluted blood samples.

    PubMed

    Pantůčková, Pavla; Kubáň, Pavel; Boček, Petr

    2013-07-19

    A cheap, disposable sample pretreatment device with planar supported liquid membrane (SLM) was proposed, assembled and placed into an autosampler carousel of a commercial capillary electrophoresis (CE) instrument for automated pretreatment and analysis of formate in undiluted whole blood and serum samples. All analytical procedures except for filling the pretreatment device with donor and acceptor solutions, i.e., extraction across SLM, injection of the extracted sample and CE-UV determination of formate, were performed fully automatically. The pretreatment device required only μL volumes of blood sample and organic solvent per extraction and was disposed off after each extraction. Good repeatability of peak areas (≤7.7%) and migration times (≤1.5%), linear relationship (r(2)=0.998-0.999) and limits of detection (≤35μM) were achieved. The overall analytical process including blood withdrawal, filling the SLM device with respective solutions, extraction of blood sample, injection into separation capillary and CE separation of formate from other anions took less than 4min. The method was proved useful by direct determination of elevated formate concentrations in undiluted serum samples of a methanol intoxicated patient. Due to its compatibility with currently commercially available CE instrumentation, disposability of extraction devices, minimum sample handling/consumption, and short extraction/analysis times, the developed method might be attractive for rapid diagnosis of methanol poisoning in clinical and toxicological laboratories. PMID:23777836

  7. Rapid and simple pretreatment of human body fluids using electromembrane extraction across supported liquid membrane for capillary electrophoretic determination of lithium.

    PubMed

    Strieglerová, Lenka; Kubáň, Pavel; Boček, Petr

    2011-05-01

    Electromembrane extraction was used for simultaneous sample cleanup and preconcentration of lithium from untreated human body fluids. The sample of a body fluid was diluted 100 times with 0.5 mM Tris solution and lithium was extracted by electromigration through a supported liquid membrane composed of 1-octanol into 100 mM acetic acid acceptor solution. Matrix compounds, such as proteins, red blood cells, and other high-molecular-weight compounds were efficiently retained on the supported liquid membrane. The liquid membrane was anchored in pores of a short segment of a polypropylene hollow fiber, which represented a low cost, single use, disposable extraction unit and was discarded after each use. Acceptor solutions were analyzed using capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4) D) and baseline separation of lithium was achieved in a background electrolyte solution consisting of 18 mM L-histidine and 40 mM acetic acid at pH 4.6. Repeatability of the electromembrane extraction-CE-C(4) D method was evaluated for the determination of lithium in standard solutions and real samples and was better than 0.6 and 8.2% for migration times and peak areas, respectively. The concentration limit of detection of 9 nM was achieved. The developed method was applied to the determination of lithium in urine, blood serum, blood plasma, and whole blood at both endogenous and therapeutic concentration levels. PMID:21500213

  8. Monoclonal gammopathy associated membranous glomerulonephritis: A rare entity

    PubMed Central

    Gowda, K. K.; Joshi, K.; Ramachandran, R.; Nada, R.

    2015-01-01

    A 40-year-old male presented with nephrotic syndrome. Light microscopic analysis of the renal biopsy showed thickening of the glomerular capillary wall. Immunofluorescence examination revealed granular deposition of monoclonal immunoglobulin (Ig) G3-kappa and complement C3 along the glomerular basement membrane. Electron microscopy showed subepithelial electron dense deposits, thus confirming membranous glomerulonephritis (MGN) with monoclonal gammopathy. MGN with monoclonal gammopathy is an extremely rare but distinctive entity. This patient was treated with a combination of bortezomib, thalidomide and dexamethasone and showed partial remission of his nephrotic state and dysproteinemia. PMID:25684873

  9. 7. VIEW OF BASEMENT, LOOKING NORTH ALONG EAST BASEMENT WALL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. VIEW OF BASEMENT, LOOKING NORTH ALONG EAST BASEMENT WALL TOWARD TURBINES. AT RIGHT IS A WATER-POWERED EAR CORN CRUSHER (manufacturer unknown), WHICH PERFORMED THE INITIAL COARSE GRINDING OF EAR CORN Photographer: Jet T. Lowe, 1985 - Alexander's Grist Mill, Lock 37 on Ohio & Erie Canal, South of Cleveland, Valley View, Cuyahoga County, OH

  10. Occlusion of retinal capillaries caused by glial cell proliferation in chronic ocular inflammation.

    PubMed

    Bianchi, E; Ripandelli, G; Feher, J; Plateroti, A M; Plateroti, R; Kovacs, I; Plateroti, P; Taurone, S; Artico, M

    2015-01-01

    The inner blood-retinal barrier is a gliovascular unit in which glial cells surround capillary endothelial cells and regulate retinal capillaries by paracrine interactions. During chronic ocular inflammation, microvascular complications can give rise to vascular proliferative lesions, which compromise visual acuity. This pathologic remodelling caused by proliferating Müller cells determines occlusion of retinal capillaries. The aim of the present study was to identify qualitative and quantitative alterations in the retinal capillaries in patients with post-traumatic chronic ocular inflammation or post-thrombotic vascular glaucoma. Moreover, we investigated the potential role of vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines in retinal inflammation. Our electron microscopy findings demonstrated that during chronic ocular inflammation, thickening of the basement membrane, loss of pericytes and endothelial cells and proliferation of Müller cells occur with irreversible occlusion of retinal capillaries. Angiogenesis takes place as part of a regenerative reaction that results in fibrosis. We believe that VEGF and pro-inflammatory cytokines may be potential therapeutic targets in the treatment of this disease although further studies are required to confirm these findings. PMID:25792393

  11. Brain capillaries in Alzheimer's disease.

    PubMed

    Baloyannis, Stavros J

    2015-01-01

    , silver impregnation techniques revealed a marked tortuosity of the capillaries in early cases of AD. In addition, the distance between two branch points is longer in capillaries of AD brains, whereas the branch point density as well as the ratio of the branch point density to astrocytic density is substantially decreased in AD in comparison with age matched normal controls. EM revealed, that the most frequent morphological alterations of the brain capillaries in AD consist of thickness, splitting and duplication of the basement membrane, reduction of the length of tight junctions, decrease of the number of tight junctions per vessel length, associated as a rule, with morphological alterations of the mitochondria of the endothelial cells, the pericytes and the perivascular astrocytic processes. The number of the pinocytotic vesicles is substantially increase in the endothelium of the brain capillaries in AD in comparison with age matched normal controls. Endothelial cells play a very important role in the transport systems in the brain. Subsequently, the dysfunction of the endothelial cells and the disruption of the BBB may induce serious impairment in the transport system. The dysfunction of the brain capillaries may result in releasing neurotoxic factors, such as thrombin, pro-inflammatory cytokines, nitric oxide and leukocyte adhesion molecules, and in abnormal regulation of Aβ-peptide homeostasis in the brain. The impairment of the brain capillaries in structures of the brain, which are crucial for the homeostatic equilibrium, such as the hypothalamic nuclei, may induce autonomic dysfunction, which usually occur in the advanced stages of AD, affecting dramatically the viability of the patients. Degeneration of the pericytes is also observed emphasizing even more the importance of the vascular factor in AD. Pericytes may serve as integrators, coordinators and effectors of blood-brain barrier structure and maintenance, and play a key role in microvascular stability

  12. 22. VIEW OF THE BASEMENT FLOOR PLAN. THE BASEMENT TUNNELS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    22. VIEW OF THE BASEMENT FLOOR PLAN. THE BASEMENT TUNNELS WERE DESIGNED AS FALLOUT SHELTERS AND USED FOR STORAGE. THE ORIGINAL DRAWING HAS BEEN ARCHIVED ON MICROFILM. THE DRAWING WAS REPRODUCED AT THE BEST QUALITY POSSIBLE. LETTERS AND NUMBERS IN THE CIRCLES INDICATE FOOTER AND/OR COLUMN LOCATIONS. - Rocky Flats Plant, General Manufacturing, Support, Records-Central Computing, Southern portion of Plant, Golden, Jefferson County, CO

  13. Basement plan. ("Alter COC Bldg 2605, Basement Plan and Architectural ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Basement plan. ("Alter COC Bldg 2605, Basement Plan and Architectural Details.") Strategic Air Command, Riverside, California, March Air Force Base. Drawing no. B-973, sheet no. 1 of 6, 14 April 1966; project no. MAR-267-5; CE-353; file drawer 1308. Last revised 20 October 1966. Various scales. 28x40 inches. pencil on paper - March Air Force Base, Strategic Air Command, Combat Operations Center, 5220 Riverside Drive, Moreno Valley, Riverside County, CA

  14. Measure Guideline: Basement Insulation Basics

    SciTech Connect

    Aldrich, R.; Mantha, P.; Puttagunta, S.

    2012-10-01

    This guideline is intended to describe good practices for insulating basements in new and existing homes, and is intended to be a practical resources for building contractors, designers, and also to homeowners.

  15. In-line coupling of microextractions across polymer inclusion membranes to capillary zone electrophoresis for rapid determination of formate in blood samples.

    PubMed

    Pantůčková, Pavla; Kubáň, Pavel; Boček, Petr

    2015-08-01

    Polymer inclusion membranes (PIMs) have several important features, i.e., PIMs are dry and non-porous membranes, which can be prepared ahead of use and stored without noticeable deterioration in extraction performance. In this contribution, in-line coupling of microextractions across PIMs to a separation method for clinical purposes was demonstrated for the first time. Formate (the major metabolite in methanol poisoning) was determined in undiluted human serum and whole blood by capillary zone electrophoresis (CZE) with simultaneous capacitively coupled contactless conductivity detection (C(4)D) and UV-Vis detection. A purpose-made microextraction device with PIM was coupled to a commercial CZE instrument in order to ensure complete automation of the entire analytical procedure, i.e., of formate extraction, injection, CZE separation and quantification. PIMs for formate extractions consisted of 60% (w/w) cellulose triacetate as base polymer and 40% (w/w) Aliquat™ 336 as anion carrier. The method was characterized by good repeatability of peak areas (≤7.0%) and migration times (≤0.8%) and by good linearity of calibration curves (r(2) = 0.993-0.999). Limits of detection in various matrices ranged from 15 to 54 μM for C(4)D and from 200 to 635 μM for UV-Vis detection and were sufficiently low to clearly distinguish between endogenous and toxic levels of formate in healthy and methanol intoxicated individuals. In addition, PIMs proved that they may act as phase interfaces with excellent long-term stability since once prepared, they retained their extractions properties for, at least, two months of storage. PMID:26320792

  16. 20. VIEW OF THE BASEMENT FLOOR PLAN. THE BASEMENT AREA ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. VIEW OF THE BASEMENT FLOOR PLAN. THE BASEMENT AREA INCLUDES A UTILITY ROOM, PROCESS WASTE STORAGE AND MAINTENANCE AREAS, AND THE ENTRANCE TO AN UNDERGROUND TUNNEL LEADING TO BUILDING 881. THE ORIGINAL DRAWING HAS BEEN ARCHIVED ON MICROFILM. THE DRAWING WAS REPRODUCED AT THE BEST QUALITY POSSIBLE. LETTERS AND NUMBERS IN THE CIRCLES INDICATE FOOTER AND/OR COLUMN LOCATIONS. - Rocky Flats Plant, Uranium Rolling & Forming Operations, Southeast section of plant, southeast quadrant of intersection of Central Avenue & Eighth Street, Golden, Jefferson County, CO

  17. Ivory Basements and Ivory Towers

    ERIC Educational Resources Information Center

    Fitzgerald, Tanya

    2012-01-01

    The metaphors of the ivory tower and ivory basement are used in this chapter to reflect how many women understand and experience the academy. The ivory tower signifies a place that is protected, a place of privilege and authority and a place removed from the outside world (and consequently the rigours of the market place). The ivory tower, by…

  18. Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay.

    PubMed Central

    Garrido, T.; Riese, H. H.; Aracil, M.; Pérez-Aranda, A.

    1995-01-01

    We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen. This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F10 murine melanoma or MDA-MD-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16F0 cell line. Replacement of tumour cell CM by 10 ng ml-1 basic fibroblast growth factor (bFGF) also results in capillary-like structure formation by BAE cells. An anti-bFGF antibody blocks this effect, showing that bFGF is one of the factors responsible for the angiogenic response induced by B16F10 CM in our assay. Addition of an anti-laminin antibody reduces significantly the formation of capillary-like structures, probably by blocking the attachment of BAE cells to laminin present in Matrigel. The anti-angiogenic compound suramin inhibits in a dose-dependent manner (complete inhibition with 100 microM suramin) the migration and differentiation of BAE cells on type I collagen in response to B16F10 CM. This assay represents a new model system to study tumour-induced angiogenesis in vitro. Images Figure 2 Figure 3 PMID:7536021

  19. Hindered transport of macromolecules in isolated glomeruli. I. Diffusion across intact and cell-free capillaries.

    PubMed Central

    Edwards, A; Deen, W M; Daniels, B S

    1997-01-01

    The filtrate formed by renal glomerular capillaries must pass through a layer of endothelial cells, the glomerular basement membrane (GBM), and a layer of epithelial cells, arranged in series. To elucidate the relative resistances of the GBM and cell layers to movement of uncharged macromolecules, we measured the diffusional permeabilities of intact and cell-free capillaries to narrow fractions of Ficoll with Stokes-Einstein radii ranging from 3.0 to 6.2 nm. Glomeruli were isolated from rat kidneys, and diffusion of fluorescein-labeled Ficoll across the walls of single capillary loops was monitored with a confocal microscopy technique. In half of the experiments the glomeruli were treated first to remove the cells, leaving skeletons that retained the general shape of the glomerulus and consisted almost entirely of GBM. The diffusional permeability of cell-free capillaries to Ficoll was approximately 10 to 20 times that of intact capillaries, depending on molecular size. Taking into account the blockage of much of the GBM surface by cells, the contribution of the GBM to the diffusional resistance of the intact barrier was calculated to be 13% to 26% of the total, increasing with molecular size. Thus, the GBM contribution, although smaller than that of the cells, was not negligible. The structure that is most likely to be responsible for the cellular part of the diffusional resistance is the slit diaphragm, which spans the filtration slit between epithelial foot processes. A novel hydrodynamic model was developed to relate the diffusional resistance of the slit diaphragm to its structure, which was idealized as a single layer of cylindrical fibers in a ladder-like arrangement. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 5 PMID:8994605

  20. EphrinB2 Reverse Signaling Protects against Capillary Rarefaction and Fibrosis after Kidney Injury

    PubMed Central

    Kida, Yujiro; Ieronimakis, Nicholas; Schrimpf, Claudia; Reyes, Morayma

    2013-01-01

    Microvascular disease, a characteristic of acute and chronic kidney diseases, leads to rarefaction of peritubular capillaries (PTCs), promoting secondary ischemic injury, which may be central to disease progression. Bidirectional signaling by EphB4 receptor and ephrinB2 ligand is critical for angiogenesis during murine development, suggesting that ephrinB2 reverse signaling may have a role in renal angiogenesis induced by injury or fibrosis. Here, we found that ephrinB2 reverse signaling is activated in the kidney only after injury. In mice lacking the PDZ intracellular signaling domain of ephrinB2 (ephrinB2 ΔV), angiogenesis was impaired and kidney injury led to increased PTC rarefaction and fibrosis. EphrinB2 ΔV primary kidney pericytes migrated more than wild-type pericytes and were less able to stabilize capillary tubes in three-dimensional culture and less able to stimulate synthesis of capillary basement membrane. EphrinB2 ΔV primary kidney microvascular endothelial cells migrated and proliferated less than wild-type microvascular endothelial cells in response to vascular endothelial growth factor A and showed less internalization and activation of vascular endothelial growth factor receptor-2. Taken together, these results suggest that PDZ domain-dependent ephrinB2 reverse signaling protects against PTC rarefaction by regulating angiogenesis and vascular stability during kidney injury. Furthermore, this signaling in kidney pericytes protects against pericyte-to-myofibroblast transition and myofibroblast activation, thereby limiting fibrogenesis. PMID:23492730

  1. High lung volume increases stress failure in pulmonary capillaries

    NASA Technical Reports Server (NTRS)

    Fu, Z.; Costello, M. L.; Tsukimoto, K.; Prediletto, R.; Elliott, A. R.; Mathieu-Costello, O.; West, J. B.

    1992-01-01

    We previously showed that when pulmonary capillaries in anesthetized rabbits are exposed to a transmural pressure (Ptm) of approximately 40 mmHg, stress failure of the walls occurs with disruption of the capillary endothelium, alveolar epithelium, or sometimes all layers. The present study was designed to test whether stress failure occurred more frequently at high than at low lung volumes for the same Ptm. Lungs of anesthetized rabbits were inflated to a transpulmonary pressure of 20 cmH2O, perfused with autologous blood at 32.5 or 2.5 cmH2O Ptm, and fixed by intravascular perfusion. Samples were examined by both transmission and scanning electron microscopy. The results were compared with those of a previous study in which the lung was inflated to a transpulmonary pressure of 5 cmH2O. There was a large increase in the frequency of stress failure of the capillary walls at the higher lung volume. For example, at 32.5 cmH2O Ptm, the number of endothelial breaks per millimeter cell lining was 7.1 +/- 2.2 at the high lung volume compared with 0.7 +/- 0.4 at the low lung volume. The corresponding values for epithelium were 8.5 +/- 1.6 and 0.9 +/- 0.6. Both differences were significant (P less than 0.05). At 52.5 cmH2O Ptm, the results for endothelium were 20.7 +/- 7.6 (high volume) and 7.1 +/- 2.1 (low volume), and the corresponding results for epithelium were 32.8 +/- 11.9 and 11.4 +/- 3.7. At 32.5 cmH2O Ptm, the thickness of the blood-gas barrier was greater at the higher lung volume, consistent with the development of more interstitial edema. Ballooning of the epithelium caused by accumulation of edema fluid between the epithelial cell and its basement membrane was seen at 32.5 and 52.5 cmH2O Ptm. At high lung volume, the breaks tended to be narrower and fewer were oriented perpendicular to the axis of the pulmonary capillaries than at low lung volumes. Transmission and scanning electron microscopy measurements agreed well. Our findings provide a physiological

  2. Capillary muscle

    PubMed Central

    Cohen, Caroline; Mouterde, Timothée; Quéré, David; Clanet, Christophe

    2015-01-01

    The contraction of a muscle generates a force that decreases when increasing the contraction velocity. This “hyperbolic” force–velocity relationship has been known since the seminal work of A. V. Hill in 1938 [Hill AV (1938) Proc R Soc Lond B Biol Sci 126(843):136–195]. Hill’s heuristic equation is still used, and the sliding-filament theory for the sarcomere [Huxley H, Hanson J (1954) Nature 173(4412):973–976; Huxley AF, Niedergerke R (1954) Nature 173(4412):971–973] suggested how its different parameters can be related to the molecular origin of the force generator [Huxley AF (1957) Prog Biophys Biophys Chem 7:255–318; Deshcherevskiĭ VI (1968) Biofizika 13(5):928–935]. Here, we develop a capillary analog of the sarcomere obeying Hill’s equation and discuss its analogy with muscles. PMID:25944938

  3. Capillary muscle.

    PubMed

    Cohen, Caroline; Mouterde, Timothée; Quéré, David; Clanet, Christophe

    2015-05-19

    The contraction of a muscle generates a force that decreases when increasing the contraction velocity. This "hyperbolic" force-velocity relationship has been known since the seminal work of A. V. Hill in 1938 [Hill AV (1938) Proc R Soc Lond B Biol Sci 126(843):136-195]. Hill's heuristic equation is still used, and the sliding-filament theory for the sarcomere [Huxley H, Hanson J (1954) Nature 173(4412):973-976; Huxley AF, Niedergerke R (1954) Nature 173(4412):971-973] suggested how its different parameters can be related to the molecular origin of the force generator [Huxley AF (1957) Prog Biophys Biophys Chem 7:255-318; Deshcherevskiĭ VI (1968) Biofizika 13(5):928-935]. Here, we develop a capillary analog of the sarcomere obeying Hill's equation and discuss its analogy with muscles. PMID:25944938

  4. Short-term reversibility of ultrastructural changes in pulmonary capillaries caused by stress failure

    NASA Technical Reports Server (NTRS)

    Elliott, A. R.; Fu, Z.; Tsukimoto, K.; Prediletto, R.; Mathieu-Costello, O.; West, J. B.

    1992-01-01

    We previously showed that when the pulmonary capillaries in anesthetized rabbits are exposed to a transmural pressure (Ptm) of approximately 40 mmHg, stress failure of the walls occurs with disruption of the capillary endothelium, alveolar epithelium, or sometimes all layers. The present study was designed to determine whether some of the ultrastructural changes are rapidly reversible when the capillary pressure is reduced. To test this, the Ptm was raised to 52.5 cmH2O for 1 min of blood perfusion and then reduced to 12.5 cmH2O for 3 min of saline-dextran perfusion, followed by intravascular fixation at the same pressure. In another group of animals, the pressure was elevated for 1 min of blood and 3 min of saline-dextran before being reduced. The results were compared with previous studies in which the capillary pressures were maintained elevated at 52.5 cmH2O during the entire procedure. Control studies were also done at sustained low pressures. The results showed that the number of endothelial and epithelial breaks per millimeter and the total fraction area of the breaks were reduced when the pressure was lowered. For example, the number of endothelial breaks per millimeter decreased from 7.1 +/- 2.1 to 2.4 +/- 0.7, and the number of epithelial breaks per millimeter fell from 11.4 +/- 3.7 to 3.4 +/- 0.7. There was evidence that the breaks that closed were those that were initially small and were associated with an intact basement membrane. The results suggest that cells can move along their underlying matrix by rapid disengagement and reattachment of cell adhesion molecules, causing breaks to open or close within minutes.(ABSTRACT TRUNCATED AT 250 WORDS).

  5. Suppressed conductometric capillary electrophoresis separation systems

    SciTech Connect

    Dasgupta, P.K.; Bao, L. )

    1993-04-15

    A tubular cation-exchange membrane is installed at the end of a 60-cm-long 75-[mu]m-bore fused-silica capillary. A static dilute acid regenerant solution surrounds the membranes that functions as a suppressor. With positive high voltage applied to the capillary inlet and the regenerant solution grounded, effective suppression of electrolytes such as solutions of alkalic metal borate, glycinate, or cyanide is observed. Electroosmotic flow carries the capillary effluent past the suppressor into a conductivity detection cell constituted by two platinum wires inserted through the wall of a poly(vinyl chloride) capillary. The system provides detection limits in the 10--20 [mu]g/L range for a variety of anions; a typical separation requires 15 min. Applicability to a variety of real samples is demonstrated. 26 refs., 10 figs.

  6. Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

    1996-08-01

    Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

  7. Capillary ultrastructure and mitochondrial volume density in skeletal muscle in relation to reduced exercise capacity of patients with intermittent claudication.

    PubMed

    Baum, Oliver; Torchetti, Eleonora; Malik, Corinna; Hoier, Birgitte; Walker, Meegan; Walker, Philip J; Odriozola, Adolfo; Graber, Franziska; Tschanz, Stefan A; Bangsbo, Jens; Hoppeler, Hans; Askew, Christopher D; Hellsten, Ylva

    2016-05-15

    Intermittent claudication (IC) is the most commonly reported symptom of peripheral arterial disease (PAD). Impaired limb blood flow is a major casual factor of lower exercise tolerance in PAD but cannot entirely explain it. We hypothesized that IC is associated with structural changes of the capillary-mitochondria interface that could contribute to the reduction of exercise tolerance in IC patients. Capillary and mitochondrial morphometry were performed after light and transmission electron microscopy using vastus lateralis muscle biopsies of 14 IC patients and 10 age-matched controls, and peak power output (PPO) was determined for all participants using an incremental single-leg knee-extension protocol. Capillary density was lower (411 ± 90 mm(-2) vs. 506 ± 95 mm(-2); P ≤ 0.05) in the biopsies of the IC patients than in those of the controls. The basement membrane (BM) around capillaries was thicker (543 ± 82 nm vs. 423 ± 97 nm; P ≤ 0.01) and the volume density of mitochondria was lower (3.51 ± 0.56% vs. 4.60 ± 0.74%; P ≤ 0.01) in the IC patients than the controls. In the IC patients, a higher proportion of capillaries appeared with collapsed slit-like lumen and/or swollen endothelium. PPO was lower (18.5 ± 9.9 W vs. 33.5 ± 9.4 W; P ≤ 0.01) in the IC patients than the controls. We suggest that several structural alterations in skeletal muscle, either collectively or separately, contribute to the reduction of exercise tolerance in IC patients. PMID:27009051

  8. Membranous glomerulonephropathy and nephrotic syndrome associated with iatrogenic metallic mercury poisoning in a cat.

    PubMed

    Shull, R M; Stowe, C M; Osborne, C A; O'Leary, T P; Vernier, R L; Hammer, R F

    1981-02-01

    The nephrotic syndrome, characterized by nonselective proteinuria, hypoproteinemia, hypoalbuminemia, and ascites, was observed in a 10-month-old male cat. Profound glomerular changes and renal tubular changes appear to have been induced by iatrogenic chronic exposure to metallic mercury originally contained in a rectal thermometer. Large concentrations of mercury were present in the kidneys, liver, spleen, and urine. Evaluation of glomeruli by immunofluorescent microscopy revealed interrupted granular deposition of immuno-globulin G and the third component of complement in glomerular capillary walls and the mesangium. Electron microscopic evaluation of glomeruli revealed diffuse alterations in glomerular basement membranes and visceral epithelial cells. Small electron dense deposits were observed in capillary walls, but they were not characteristic of immune complexes. The mechanism(s) responsible for the mercury induced glomerulonephropathy in this patient could not be determined on the basis of available data. PMID:7257162

  9. Versatile microanalytical system with porous polypropylene capillary membrane for calibration gas generation and trace gaseous pollutants sampling applied to the analysis of formaldehyde, formic acid, acetic acid and ammonia in outdoor air.

    PubMed

    Coelho, Lúcia H G; Melchert, Wanessa R; Rocha, Flavio R; Rocha, Fábio R P; Gutz, Ivano G R

    2010-11-15

    The analytical determination of atmospheric pollutants still presents challenges due to the low-level concentrations (frequently in the μg m(-3) range) and their variations with sampling site and time. In this work, a capillary membrane diffusion scrubber (CMDS) was scaled down to match with capillary electrophoresis (CE), a quick separation technique that requires nothing more than some nanoliters of sample and, when combined with capacitively coupled contactless conductometric detection (C(4)D), is particularly favorable for ionic species that do not absorb in the UV-vis region, like the target analytes formaldehyde, formic acid, acetic acid and ammonium. The CMDS was coaxially assembled inside a PTFE tube and fed with acceptor phase (deionized water for species with a high Henry's constant such as formaldehyde and carboxylic acids, or acidic solution for ammonia sampling with equilibrium displacement to the non-volatile ammonium ion) at a low flow rate (8.3 nL s(-1)), while the sample was aspirated through the annular gap of the concentric tubes at 2.5 mL s(-1). A second unit, in all similar to the CMDS, was operated as a capillary membrane diffusion emitter (CMDE), generating a gas flow with know concentrations of ammonia for the evaluation of the CMDS. The fluids of the system were driven with inexpensive aquarium air pumps, and the collected samples were stored in vials cooled by a Peltier element. Complete protocols were developed for the analysis, in air, of NH(3), CH(3)COOH, HCOOH and, with a derivatization setup, CH(2)O, by associating the CMDS collection with the determination by CE-C(4)D. The ammonia concentrations obtained by electrophoresis were checked against the reference spectrophotometric method based on Berthelot's reaction. Sensitivity enhancements of this reference method were achieved by using a modified Berthelot reaction, solenoid micro-pumps for liquid propulsion and a long optical path cell based on a liquid core waveguide (LCW). All

  10. Implicit mechanistic role of the collagen, smooth muscle, and elastic tissue components in strengthening the air and blood capillaries of the avian lung

    PubMed Central

    Maina, John N; Jimoh, Sikiru A; Hosie, Margo

    2010-01-01

    To identify the forces that may exist in the parabronchus of the avian lung and that which may explain the reported strengths of the terminal respiratory units, the air capillaries and the blood capillaries, the arrangement of the parabronchial collagen fibers (CF) of the lung of the domestic fowl, Gallus gallus variant domesticus was investigated by discriminatory staining, selective alkali digestion, and vascular casting followed by alkali digestion. On the luminal circumference, the atrial and the infundibular CF are directly connected to the smooth muscle fibers and the elastic tissue fibers. The CF in this part of the parabronchus form the internal column (the axial scaffold), whereas the CF in the interparabronchial septa and those associated with the walls of the interparabronchial blood vessels form the external, i.e. the peripheral, parabronchial CF scaffold. Thin CF penetrate the exchange tissue directly from the interparabronchial septa and indirectly by accompanying the intraparabronchial blood vessels. Forming a dense network that supports the air and blood capillaries, the CF weave through the exchange tissue. The exchange tissue, specifically the air and blood capillaries, is effectively suspended between CF pillars by an intricate system of thin CF, elastic and smooth muscle fibers. The CF course through the basement membranes of the walls of the blood and air capillaries. Based on the architecture of the smooth muscle fibers, the CF, the elastic muscle fibers, and structures like the interparabronchial septa and their associated blood vessels, it is envisaged that dynamic tensional, resistive, and compressive forces exist in the parabronchus, forming a tensegrity (tension integrity) system that gives the lung rigidity while strengthening the air and blood capillaries. PMID:20819116