scholarly journals Studies on the localization of the glycoprotein GP-2 within the renal glomerulus in vivo and in cultured kidney cell strains in vitro.

1981 ◽  
Vol 29 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
T D Oberley ◽  
A E Chung ◽  
J E Murphy-Ullrich ◽  
D F Mosher
Keyword(s):  
Basement Membrane ◽  
Basement Membranes ◽  
Molecular Organization ◽  
Electron Microscopic Level ◽  
Positive Staining ◽  
Electron Microscopic ◽  
Antibody Technique ◽  
Glomerular Cell ◽  
Kidney Glomerulus

Positive staining for the glycoprotein GP-2 was demonstrated in the kidney glomerulus by use of the indirect peroxidase-labeled antibody technique. At the ultrastructural level, heaviest staining for GP-2 was demonstrated along the lamina rara externa and lamina rara interna of the glomerular and tubular basement membranes, demonstrating definite molecular organization for structures which appear amorphous even at the electron microscopic level. However, GP-2 was also present in the lamina densa of the glomerular basement membrane, but not of the tubular basement membrane. The staining for GP-2 is in contrast to the predominantly mesangial staining for fibronectin. Using the indirect immunoperoxidase techniques for kidney cells cultured in vitro, it was demonstrated that cell surfaces of specific subpopulations of glomerular cells stained heavily for both fibronectin and GP-2, while renal medullary cells did not stain at all using specific antiserum to these molecules. GP-2 was present extracellularly and showed moderate staining in glomerular cell culture, while fibronectin showed heavy staining in this location.

scholarly journals Nidogen-1: Expression and Ultrastructural Localization During the Onset of Mesoderm Formation in the Early Mouse Embryo

2000 ◽  
Vol 48 (2) ◽  
pp. 229-237 ◽  
Author(s):  
Nicolai Miosge ◽  
Fabio Quondamatteo ◽  
Christina Klenczar ◽  
Rainer Herken
Keyword(s):  
Basement Membrane ◽  
Collagen Type ◽  
Mesenchymal Cells ◽  
Basement Membranes ◽  
Electron Microscopic Level ◽  
Collagen Type Iv ◽  
Electron Microscopic ◽  
Type Iv ◽  
Mesoderm Formation ◽  
Nidogen 1

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo.

scholarly journals POLYMORPHONUCLEAR LEUKOCYTES IN IMMUNOLOGIC REACTIONS

1966 ◽  
Vol 124 (4) ◽  
pp. 733-752 ◽  
Author(s):  
Charles G. Cochrane ◽  
Barbara S. Aikin
Keyword(s):  
Basement Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Electron Microscopic Observation ◽  
Severe Damage ◽  
Electron Microscopic ◽  
Cationic Proteins ◽  
Vascular Basement Membrane ◽  
Arthus Phenomenon ◽  
Immunologic Reactions

Vascular basement membrane was disrupted in the presence of polymorphonuclear leukocytes (PMN's) during two immunologic reactions: The Arthus phenomenon and the reaction to locally injected antibody to vascular basement membrane. This disruption was evidenced by (a) the inability of the basement membrane to retain circulating carbon, by (b) loss of antigenic constituents, and by (c) electron microscopic observation showing actual gaps in the structure of the vascular basement membrane. The factors within PMN's responsible for damage to isolated glomerular basement membrane in vitro were found by isolation procedures to be cathepsins D and E. Cationic proteins of PMN's were separable from the cathepsins. While inducing vascular permeability upon injection, these basic proteins failed to inflict the severe damage to the basement membrane observed in Arthus and antibasement membrane reactions. It is concluded that the full expression of these immunologic lesions requires destruction of the basement membrane possibly brought about by cathepsins D and E. Some of the physicochemical properties of these pathologically active leukocytic factors are given.

scholarly journals A New Method to Enhance Contrast of Ultrathin Cryosections for Immunoelectron Microscopy

2003 ◽  
Vol 51 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Clark L. Anderson ◽  
John M. Robinson
Keyword(s):  
Immunoelectron Microscopy ◽  
Staining Method ◽  
Microscopic Level ◽  
New Method ◽  
Electron Microscopic Level ◽  
Positive Staining ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Morphological Detail ◽  
Ultrathin Cryosections

Adequate contrast of ultrathin cryosections is crucial for evaluating morphological detail to assess immunocytochemical localization at the electron microscopic level. We have developed a positive staining method for achieving contrast in ultrathin cryosections, from tissue fixed only in paraformaldehyde, that provides excellent contrast at the electron microscopic level.

scholarly journals cDNA Cloning of the Basement Membrane Chondroitin Sulfate Proteoglycan Core Protein, Bamacan: A Five Domain Structure Including Coiled-Coil Motifs

1997 ◽  
Vol 136 (2) ◽  
pp. 433-444 ◽  
Author(s):  
Rong-Rong Wu ◽  
John R. Couchman
Keyword(s):  
Basement Membrane ◽  
Chondroitin Sulfate ◽  
Core Protein ◽  
Coiled Coil ◽  
Basement Membranes ◽  
Cdna Clones ◽  
Molecular Architecture ◽  
Unusual Structure ◽  
Extracellular Matrix Molecule

Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions. This molecular architecture is novel for an extracellular matrix molecule, but it resembles that of a group of intracellular proteins, including some proposed to stabilize the mitotic chromosome scaffold. We have previously proposed a similar stabilizing role for bamacan in the basement membrane matrix. The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser-Gly dipeptides are unfavorable for substitution. Fusion protein antibodies stained basement membranes in a pattern commensurate with bamacan, and they also Western blotted bamacan core protein from rat L2 cell cultures. The antibodies could also specifically immunoprecipitate an in vitro transcription/translation product from a full-length bamacan cDNA. The unusual structure of this proteoglycan is indicative of specific functional roles in basement membrane physiology, commensurate with its distinct expression in development and changes in disease models.

scholarly journals Biotinylphallotoxins: preparation and use as actin probes.

1989 ◽  
Vol 37 (7) ◽  
pp. 1035-1045 ◽  
Author(s):  
H Faulstich ◽  
S Zobeley ◽  
U Bentrup ◽  
B M Jockusch
Keyword(s):  
Actin Filaments ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Permeabilized Cells ◽  
High Affinity ◽  
Double Label ◽  
The One

We describe the synthesis of four phalloidin derivatives conjugated with biotin. An aminomethyldithiolane derivative of ketophalloidin was used as a reactive starter compound, and biotin residues were coupled to this molecule either directly, separated by spacer chains comprised of one or two glycyl residues, or of a 12-atom long chain constructed from succinic acid and hexamethylendiamine. Although all products still displayed a high affinity for F-actin, as seen in competition experiments with [3H]-demethylphalloidin, only the one with the longest spacer (BHPP) showed specific and high-affinity decoration of actin filaments in permeabilized cells, in conjunction with FITC-coupled avidin and fluorescence microscopy. Combined with gold-streptavidin, BHPP decorated the actin filament system at the light and electron microscopic level faithfully and with satisfactory density. Actin filaments polymerized in vitro from purified protein were not as densely labeled as had been expected. However, in all these experiments the new phalloidin probe, when combined with avidin or streptavidin, yielded clear and highly specific labeling of F-actin. Therefore, this system is useful to identify and localize actin unambiguously in microfilaments, independent of actin antibodies, and should facilitate double-label experiments on cytoskeletal components at the ultrastructural level.

scholarly journals ENZYME-LABELED ANTIBODIES FOR THE LIGHT AND ELECTRON MICROSCOPIC LOCALIZATION OF TISSUE ANTIGENS

1967 ◽  
Vol 33 (2) ◽  
pp. 307-318 ◽  
Author(s):  
Paul K. Nakane ◽  
G. Barry Pierce
Keyword(s):  
Acid Phosphatase ◽  
Basement Membrane ◽  
Fluorescent Antibody ◽  
Enzymatic Reaction ◽  
Immunohistochemical Localization ◽  
Fluorescent Antibody Technique ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Antibody Technique ◽  
Tissue Antigens

Enzymes, either acid phosphatase or horseradish peroxidase, were conjugated to antibodies with bifunctional reagents. The conjugates, enzymatically and immunologically active, were employed in the immunohistochemical localization of tissue antigens utilizing the reaction product of the enzymatic reaction as the marker. Tissues reacted with acid phosphatase-labeled antibodies directed against basement membrane were stained for the enzyme with Gomori's method, and those reacted with peroxidase-labeled antibody were stained with Karnovsky's method. The reaction products of the enzymes localized in the basement membrane. Unlike the preparations of the fluorescent antibody technique, enzyme-labeled antibody preparations were permanent, could be observed with an ordinary microscope, and could be examined with the electron microscope. In the latter, specific localization of antibody occurred in the basement membrane and in the endoplasmic reticulum of cells known to synthesize basement membrane antigens. The method is sensitive because of the amplifying effect of the enzymatic activity. The ultrastructural preservation and localization were better with acid phosphatase-labeled antibody than with peroxidase-labeled antibody, but acid phosphatase conjugated antibody was unstable and difficult to prepare. Peroxidase-antibody conjugates were stable and could be stored for several months at 4°C, or indefiniely in a frozen state.

scholarly journals ELECTRON MICROSCOPIC LOCALIZATION OF THE NEPHROTOXIC ANTIBODY IN THE GLOMERULI OF THE RAT AFTER INTRAVENOUS APPLICATION OF PURIFIED NEPHRITOGENIC ANTIBODY-FERRITIN CONJUGATES

1968 ◽  
Vol 127 (5) ◽  
pp. 867-878 ◽  
Author(s):  
Arnold Vogt ◽  
Hermann Bockhorn ◽  
Keniti Kozima ◽  
Masamichi Sasaki
Keyword(s):  
Electron Microscopy ◽  
Basement Membrane ◽  
Intravenous Injection ◽  
Fluorescent Antibody ◽  
Fluorescent Antibody Technique ◽  
Electron Microscopic ◽  
Antibody Technique ◽  
Disappearance Time ◽  
Filtration Surface ◽  
Electron Microscopic Localization

Nephritis in rats was induced by intravenous injection of purified ferritin-conjugated rabbit and duck nephrotoxic globulin. Using the fluorescent antibody technique, the same capillary pattern was found as that in glomeruli of rats receiving uncoupled nephrotoxic globulin. Electron microscopy revealed a heavy accumulation of the basement membrane-fixed antibody almost exclusively at the endothelial side. A higher concentration of ferritin was demonstrable in the peripheral basement membrane. The once-fixed antibody remained at the site of reaction though decreasing with time. The half-disappearance time seemed to be shorter than that of the uncoupled nephrotoxic globulin. No difference in localization was observed between rabbit and duck antibody. At least 40 basement membrane-fixed antibody molecules from the rabbit per 3000 mµ2 of filtration surface were needed to cause immediate nephritis. To induce nephritis using duck antibody, a greater number of basement membrane-fixed antibody seemed to be necessary. No evidence of specific reaction with constituents of glomerular cells was obtained.

scholarly journals EFFECTS OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS ON SHEEP KIDNEYS

2021 ◽  
Vol 25 (1) ◽  
pp. 173-179
Author(s):  
Majid N. Hussain
Keyword(s):  
Cell Proliferation ◽  
Basement Membrane ◽  
Corynebacterium Pseudotuberculosis ◽  
Caseous Lymphadenitis ◽  
Electron Microscopic ◽  
Glomerular Cell

Light and electron microscopic examinations were performed on kidneys of sheep infected with caseous lymphadenitis ( CLA ) caused by Corynebacterium pseudotuberculosis Membranproliferative glomerulonephritis was demonstrated. There were irregular thickenings of the glomeruelar basement membrane and glomerular cell proliferation, in contrast, no such lesions were found in the control lambs, indicating that CLA play a role in inducing glomerulorephritis

scholarly journals Basement membrane proteoglycans in glomerular morphogenesis: chondroitin sulfate proteoglycan is temporally and spatially restricted during development.

1993 ◽  
Vol 41 (3) ◽  
pp. 401-414 ◽  
Author(s):  
K J McCarthy ◽  
K Bynum ◽  
P L St John ◽  
D R Abrahamson ◽  
J R Couchman
Keyword(s):  
Basement Membrane ◽  
Chondroitin Sulfate ◽  
Basement Membranes ◽  
Chondroitin Sulfate Proteoglycan ◽  
Sulfate Proteoglycan ◽  
Electron Microscopic ◽  
Ureteric Buds ◽  
Membrane Components ◽  
Almost All ◽  
Basement Membrane Components

We previously reported the presence of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG) in basement membranes of almost all adult tissues. However, an exception to this ubiquitous distribution was found in the kidney, where BM-CSPG was absent from the glomerular capillary basement membrane (GBM) but present in other basement membranes of the nephron, including collecting ducts, tubules, Bowman's capsule, and the glomerular mesangium. In light of this unique pattern of distribution and of the complex histoarchitectural reorganization occurring during nephrogenesis, the present study used light and electron microscopic immunohistochemistry to examine the distribution of BM-CSPG and basement membrane heparan sulfate proteoglycan (BM-HSPG) during prenatal and postnatal renal development in the rat. Our results show that the temporal and spatial pattern of expression of BM-CSPG during nephrogenesis is unlike that reported for other basement membrane components such as laminin, fibronectin, and BM-HSPG, all of which can be found in the earliest formed basement membranes of the vesicle-stage nephron. Although BM-CSPG is present in the basement membranes of the invading vasculature and ureteric buds, its first appearance in nephron basement membrane occurs during the late comma stage. In capillary loop-stage glomeruli of prenatal animals, BM-CSPG is present in the presumptive mesangial matrix but undetectable in the GBM. However, as postnatal glomerular maturation progresses BM-CSPG is also found in both the lamina rara interna and lamina densa of the GBM in progressively increasing amounts, being most evident in the GBM of 21-day-old animals. Micrographs of glomeruli from 42-day-old animals show that BM-CSPG gradually disappears from the GBM and, by 56 days after birth, appears to be completely absent from the GBM, its pattern of distribution resembling that of the adult animal. Our results show that BM-CSPG is not required for the initial assembly of basement membranes but may in fact serve to stabilize basement membrane structure after histoarchitectural reorganization is completed.

Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier

1992 ◽  
Vol 103 (1) ◽  
pp. 23-37 ◽  
Author(s):  
N.J. Abbott ◽  
C.C. Hughes ◽  
P.A. Revest ◽  
J. Greenwood
Keyword(s):  
Endothelial Cells ◽  
Rat Brain ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Ulex Europaeus ◽  
Blood Brain ◽  
Microcarrier Beads

Primary culture of rat brain endothelial cells is described, based on the method of C. C. W. Hughes and P. L. Lantos. The cells have been characterised using morphological and immunocytochemical techniques, and systematic studies undertaken to determine the optimal culture medium and conditions required to grow the cells at high purity on a variety of substrata. The endothelial cells have a spindle-shaped morphology, and proliferate as plaques from small clusters of cells associated with capillary fragments in the starting material. Tight junction-like cell:cell appositions are seen at the electron-microscopic level. The cells show characteristic staining for antigens recognized by antibodies against von Willebrand factor (Factor VIII-related antigen), angiotensin-converting enzyme (ACE), the transferrin receptor (Ox-26), actin and vimentin. They also show binding of the lectin from Ulex europaeus (UEA I). Potential contaminating cells include smooth muscle, fibroblasts, pericytes and meningeal cells. Contaminants can be kept to < ca. 5% by careful removal of large vessels and meninges during dissection, by brief treatment with Ca(2+)- and Mg(2+)-free saline, by growth in medium supplemented with plasma-derived serum treated for removal of platelet-derived growth factor (PDGF), and by occasional use of medium in which D-valine is substituted for L-valine. Cells attach well to collagen-coated plastic, less well to glass. Cells can be grown on transparent collagen filters (ICN, Cellagen and Costar, Transwell-Col), and on microcarrier beads (Pharmacia, Cytodex-3). The culture has proved to be a useful preparation for studies of cellular physiology, pharmacology and biochemistry of the brain endothelium, and represents a first step in producing an in vitro model of the rat blood-brain barrier.

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