scholarly journals Assembly of the glomerular filtration surface. Differentiation of anionic sites in glomerular capillaries of newborn rat kidney.

1980 ◽  
Vol 85 (3) ◽  
pp. 735-753 ◽  
Author(s):  
W H Reeves ◽  
Y S Kanwar ◽  
M G Farquhar
Keyword(s):  
Sialic Acid ◽  
Heparan Sulfate ◽  
Rat Kidney ◽  
Ruthenium Red ◽  
Basement Membranes ◽  
Cationized Ferritin ◽  
Cell Surfaces ◽  
Anionic Sites ◽  
Newborn Rat ◽  
Shaped Body

Glomerular development was studied in the newborn rat kidney by electron microscopy and cytochemistry. Glomerular structure at different developmental stages was related to the permeability properties of its components and to the differentiation of anionic sites in the glomerular basement membrane (GBM) and on endothelial and epithelia cell surfaces. Cationic probes (cationized ferritin, ruthenium red, colloidal iron) were used to determine the time of appearance and distribution of anionic sites, and digestion with specific enzymes (neuraminidase, heparinase, chondroitinases, hyaluronidases) was used to determine their nature. Native (anionic) ferritin was used to investigate glomerular permeability. The main findings were: (a) The first endothelial fenestrae (which appear before the GBM is fully assembled) possess transient, negatively charged diaphragms that bind cationized ferritin and are impermeable to native ferritin. (b). Two types of glycosaminoglycan particles can be identified by staining with ruthenium red. Large (30-nm) granules are seen only in the cleft of the S-shaped body at the time of mesenchymal migration into the renal vesicle. They consist of hyaluronic acid and possibly also chondroitin sulfate. Smaller (10-15-nm) particles are seen in the earliest endothelial and epithelial basement membranes (S-shaped body stage), become concentrated in the laminae rarae after fusion of these two membranes to form the GBM, and contain heparan sulfate. They are assumed to be precursors of the heparan sulfate-rich granules present in the mature GBM. (c) Distinctive sialic acid-rich, and sialic acid-poor plasmalemmal domains have been delineated on both the epithelial and endothelial cell surfaces. (d) The appearance of sialoglycoproteins on the epithelial cell surface concides with the development of foot processes and filtration slits. (e) Initially the GBM is loosely organized and quite permeable to native ferritin ;it becomes increasinly impermeable to ferritin as the lamina densa becomes more compact. (f) The number of endothelial fenestrae and open epithelial slits increases as the GBM matures and becomes organized into an effective barrier to the passage of native ferritin.

scholarly journals Structural features of alveolar wall basement membrane in the adult rat lung.

1981 ◽  
Vol 91 (2) ◽  
pp. 427-437 ◽  
Author(s):  
C A Vaccaro ◽  
J S Brody
Keyword(s):  
Basement Membrane ◽  
Ruthenium Red ◽  
Basement Membranes ◽  
Alveolar Wall ◽  
Type I ◽  
Type Ii ◽  
Rat Lung ◽  
Anionic Sites ◽  
Lamina Densa ◽  
Adult Rat

The ultrastructural characteristics of alveolar (ABM) and capillary (CBM) basement membranes in the adult rat lung have been defined using tannic acid fixation, ruthenium red staining, or incubation in guanidine HCl. ABM is dense and amorphous, has 3- to 5-nm filaments in the lamina rara externa (facing the alveolus) that run between the lamina densa and the basal cell surface of the epithelium, has an orderly array of ruthenium red-positive anionic sites that appear predominantly (79%) on the lamina rara externa, and has discontinuities beneath alveolar type II cells but not type I cells that allow penetration of type II cytoplasmic processes into the interstitium of the alveolar wall. The CBM is fibrillar and less compact than ABM, has no lamina rara filaments, and has one fifth the number of ruthenium red-positive anionic sites of ABM that appear predominantly (64%) overlying the lamina densa. Incubation of lung tissue with Flavobacterium heparinum enzyme or with chondroitinase has shown that ABM anionic sites represent heparan sulfate proteoglycans, whereas CBM anionic sites contain this and other sulfated proteoglycans. The CBM fuses in a local fashion with ABM, compartmentalizing the alveolar wall into a thick and thin side and establishing a thin, single, basement-membrane gas-exchange surface between alveolar air, and capillary blood. The potential implications of ABM and CBM ultrastructure for permeability, cell differentiation, and repair and morphogenesis of the lung are discussed.

scholarly journals Biochemical and ultrastructural studies of proteoheparan sulfates synthesized by PYS-2, a basement membrane-producing cell line.

1982 ◽  
Vol 92 (2) ◽  
pp. 357-367 ◽  
Author(s):  
A Oohira ◽  
T N Wight ◽  
J McPherson ◽  
P Bornstein
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Cell Line ◽  
Molecular Size ◽  
Sulfated Polysaccharide ◽  
Ruthenium Red ◽  
Basement Membranes ◽  
Gel Chromatography ◽  
Ultrastructural Studies ◽  
Type Iv

The mouse teratocarcinoma-derived cell line, PYS-2, has been shown to produce laminin, a basement membrane-specific glycoprotein. In these studies we demonstrate that PYS-2 cells synthesize and secrete into the culture medium a proteoglycan which contains only heparan sulfate as its sulfated polysaccharide side chains, as well as type IV procollagen and laminin. The apparent molecular weights of the proteoglycan and its heparan sulfate side chain were estimated to be 400,000 and 25,000, respectively, by gel chromatography. A proteoheparan sulfate with properties closely similar, if not identical, to those of the proteoglycan in the medium, together with two heparan sulfate single chains of different molecular size, were extracted from the cell layer with 2% SDS in the presence of protease inhibitors. Ultrastructurally, a fine fibrillar intercellular matrix was recognized which contained discrete 100-200 A diameter ruthenium red-positive granules interspersed throughout the filamentous meshwork. The PYS-2 cultures were shown by immunofluorescence to react with antibodies against the heparan sulfate-containing proteoglycan isolated from the mouse EHS sarcoma (Hassell, J. R., P. G. Robey, H. J. Barrach, J. Wilczek, S. I. Rennard, and G. R. Martin. 1980. Proc. Natl. Acad. Sci. U. S. A. 77:4494-4498). Immunoelectron microscopic examination, using the same antibodies, revealed that the proteoheparan sulfate was located not only at the edges but also within the interstices of the matrix. These findings indicate that PYS-2 cells synthesize and secrete a proteoglycan with properties similar to those of basement membrane proteoglycan. These cells may therefore serve as a useful model system for the study of the biosynthesis and structure of basement membranes.

scholarly journals Alterations in alveolar basement membranes during postnatal lung growth.

1982 ◽  
Vol 95 (2) ◽  
pp. 394-402 ◽  
Author(s):  
J S Brody ◽  
C A Vaccaro ◽  
P J Gill ◽  
J E Silbert
Keyword(s):  
Heparan Sulfate ◽  
Ruthenium Red ◽  
Basement Membranes ◽  
Alveolar Type ◽  
Age Related ◽  
Enzyme Degradation ◽  
Alveolar Formation

We studied the ultrastructural characteristics of alveolar basement membranes (ABM) and capillary basement membranes (CBM) in rat lungs at birth, at 8-10 d of age, during alveolar formation, and at 6-10 wk of age, after most alveoli have formed. We also measured in vitro lung proteoglycan and heparan sulfate synthesis at each age. We noted three major age-related changes in pulmonary basement membranes. (a) Discontinuities in the ABM through which basilar cytoplasmic foot processes extend are present beneath alveolar type-2 cells but not alveolar type-1 cells. These discontinuities are most prevalent at birth but also exist in the adult. (b) Discontinuities are also present in CBM at the two earliest time points but are maximal at 8 d of age rather than at birth. Fusions between ABM and CBM are often absent at 8 d of age, but CBM and CBM/ABM fusions were complete in the adult. (c) Heparan sulfate proteoglycans identified with ruthenium red and selective enzyme degradation are distributed equally on epithelial and interstitial sides of the ABM lamina densa at birth, but decrease on the interstitial side with age. In vitro proteoglycan and heparan sulfate accumulation at birth was two times that at 8 d and five times that in the adult. Discontinuities in ABM allow epithelial-mesenchymal interactions that may influence type-2 cells cytodifferentiation. Discontinuities in CBM suggest that capillary proliferation and neovascularization are associated with alveolar formation at 8 d. When CBM becomes complete and forms junctions with ABM, lung neovascularization likely ends as does the ability to form new alveoli.

scholarly journals Basement membrane-like matrix of teratocarcinoma-derived endodermal cells: presence of laminin and heparan sulfate in the matrix at points of attachment to cells.

1983 ◽  
Vol 31 (1) ◽  
pp. 35-45 ◽  
Author(s):  
I Leivo
Keyword(s):  
Cell Membrane ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Type Iv Collagen ◽  
Heparan Sulfate Proteoglycan ◽  
Ruthenium Red ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Type Iv ◽  
The Matrix

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.

scholarly journals Location and chemical composition of anionic sites in Bruch's membrane of the rat.

1982 ◽  
Vol 30 (3) ◽  
pp. 245-252 ◽  
Author(s):  
R M Pino ◽  
E Essner ◽  
L C Pino
Keyword(s):  
Chemical Composition ◽  
Heparan Sulfate ◽  
Basal Lamina ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ruthenium Red ◽  
Morphological Evidence ◽  
Anionic Sites ◽  
Bruch's Membrane ◽  
Bruch’S Membrane

The location and chemical composition of anionic sites in Bruch's membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.

Localization of cell-surface and basement-membrane heparan-sulfate proteoglycans using the malaria circumsporozoite protein

1994 ◽  
Vol 52 ◽  
pp. 294-295
Author(s):  
U. Frevert ◽  
S. Sinnis ◽  
C. Cerami ◽  
V. Nussenzweig
Keyword(s):  
Heparan Sulfate ◽  
Protein A ◽  
Kidney Tissue ◽  
Plasmodium Species ◽  
Its Region ◽  
Basement Membranes ◽  
Heparan Sulfate Proteoglycans ◽  
Infected Mosquito ◽  
Complement Components ◽  
Region Ii

Malaria sporozoites, which invade hepatocytes within minutes after transmission by an infected mosquito, are covered with the circumsporozoite (CS) protein, which in all Plasmodium species contains the conserved region II-plus. This region is also found as a cell-adhesive motif in a variety of host proteins like thrombospondin, properdin and the terminal complement components.The CS protein with its region II-plus specifically binds to heparan sulfate proteoglycans (HSPG) on the basolateral surface of hepatocytes in the space of Disse (FIG. 1), to certain basolateral cell membranes and basement membranes of the kidney (FIG. 2) as well as to heparin in the granules of connective tissue mast cells. The distribution of the HSPG receptors for the CS protein was examined by incubation of Lowicryl K4M or LR White sections of liver and kidney tissue with the recombinant CS ligand, whose binding sites were detected with a monoclonal anti-CS antibody and protein A gold.

Interaction between parathyroid hormone and prostaglandin E1 on cyclic AMP metabolism in rat kidney

1980 ◽  
Vol 93 (3) ◽  
pp. 339-345 ◽  
Author(s):  
Naokazu Nagata ◽  
Yuriko Ono ◽  
Narimichi Kimura
Keyword(s):  
Parathyroid Hormone ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Rat Kidney ◽  
Prostaglandin E ◽  
Cortical Tissue ◽  
Newborn Rat ◽  
Simultaneous Addition ◽  
Renal Cortical Tissue ◽  
Subsequent Addition

Abstract. The interaction between parathyroid hormone (PTH) and prostaglandin E1 (PGE1) in influencing cyclic AMP metabolism in rat renal cortical tissue was examined. PTH and PGE1 stimulated additively the adenylate cyclase activity in the homogenate of the tissue. Both PTH and PGE1 enhanced the level of cyclic AMP in the incubated renal cortical tissue, but the effect of their simultaneous addition did not exceed the effect induced by PTH alone. Cyclic AMP accumulated in the incubation medium by stimulation by PTH was decreased by the simultaneous addition of PGE1. When the tissue was pre-incubated for 30 min with 2 to 10 μg/ml of PGE1, the magnitude of the increase of cyclic AMP caused by PTH subsequently added was lessened. However, the response to PTH of adenylate cyclase preparation obtained from the homogenate of PGE1-pre-treated tissue was not decreased. When first PTH was added to the incubating renal cortical tissue, the subsequent addition of PGE1 accelerated the decrease of cyclic AMP content in the tissue and decreased the amount of cyclic AMP released from the tissue. The interaction of PTH and PGE1 on cyclic AMP metabolism in the renal cortical tissue was in contrast to that seen in newborn rat calvaria where PGE1 and PTH acted additively in enhancing the level of cyclic AMP.

scholarly journals Glycan-protein cross-linking mass spectrometry reveals sialic acid-mediated protein networks on cell surfaces

2021 ◽  
Author(s):  
Yixuan Xie ◽  
Siyu Chen ◽  
Qiongyu Li ◽  
Ying Sheng ◽  
Michael R Alvarez ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sialic Acid ◽  
Membrane Proteins ◽  
Cell Membranes ◽  
Sialic Acids ◽  
Cross Linking ◽  
Protein Networks ◽  
Cell Surfaces ◽  
Protein Cross Linking

A cross-linking method is developed to elucidate the glycan-mediated interactions between membrane proteins through sialic acids. The method provides previously unknown extensive glycomic interactions on cell membranes. The vast majority...

SEM localization of cell-surface-associated fibronectin in the cranium of chick embryos utilizing immunolatex microspheres

Development ◽  
1984 ◽  
Vol 80 (1) ◽  
pp. 175-195
Author(s):  
Stephen Meier ◽  
Christopher Drake
Keyword(s):  
Basement Membranes ◽  
Chick Embryos ◽  
Preimmune Serum ◽  
Fluorescent Staining ◽  
Cell Surfaces ◽  
Cell Soma ◽  
Rabbit Igg ◽  
Neural Ectoderm ◽  
Cranial Neural Crest Cells ◽  
Testicular Hyaluronidase

Fibronectin has been localized to basement membranes and cell surfaces with the light microscope by fluorescent staining of thick sections, and with the TEM by immunoperoxidase reaction. However, these methods are limited because it is difficult to appreciate the patterned distribution of fibronectin from sectioned material. We have developed a probe for fibronectin that facilitates its identification with the SEM. Our probe consists of two parts; the first component is a derivatized methacrylate microsphere 90 nm in diameter, linked to purified sheep anti-rabbit IgG. The second component is anti-fibronectin IgG raised in rabbits. Stage-3 to -12 chick embryos were fixed and the ectoderm covering the cranial mesoderm was removed. Embryos were treated with testicular hyaluronidase, exposed to rabbit antifibronectin IgG and finally to sheep anti-rabbit IgG conjugated microspheres. As expected, the basal lamina of surface and neural ectoderm as well as the remaining fibrous ECM were heavily decorated with microspheres, whereas control embryos treated with preimmune serum were beadless. Fibronectin was localized on the cell soma and processes of primary mesenchyme as early as stage 3. In addition, it was possible to decorate to various extents, populations of prosencephalic, mesencephalic, and rhombencephalic cranial neural crest cells. Our studies suggest that fibronectin is present in the cranium of chick embryos at earlier times than heretofore realized, and that fibronectin accumulates in a cranial to caudal gradient that reflects the sequential differentiation of the embryonic axis.

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