scholarly journals Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 521-529 ◽  
Author(s):  
MN Fukuda ◽  
G Klier ◽  
J Yu ◽  
P Scartezzini
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Band 3 ◽  
Precursor Cells ◽  
Erythrocyte Membranes ◽  
Immunogold Electron Microscopy ◽  
Glycophorin A ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Cytoplasmic Area

Abstract Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

scholarly journals Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 521-529 ◽  
Author(s):  
MN Fukuda ◽  
G Klier ◽  
J Yu ◽  
P Scartezzini
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Band 3 ◽  
Precursor Cells ◽  
Erythrocyte Membranes ◽  
Immunogold Electron Microscopy ◽  
Glycophorin A ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Cytoplasmic Area

Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

scholarly journals Glycophorin A in two patients with congenital dyserythropoietic anemia type I and type II is partly unglycosylated.

2000 ◽  
Vol 47 (3) ◽  
pp. 773-779 ◽  
Author(s):  
E Zdebska ◽  
M Adamczyk-Popławska ◽  
J Kościelak
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glycosylation Site ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Type I ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Significant Deficit ◽  
Preliminary Extraction

Glycophorins A from erythrocyte membranes of two patients with congenital dyserythropoietic anemia type I and type II (CDA type I and II) were analyzed for carbohydrate molar composition employing a modification of the recently published method that allowed simultaneous determination of carbohydrates and protein in electrophoretic bands of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Zdebska & Kościelak, 1999, Anal Biochem., 275, 171-179). The modification involved a preliminary extraction of erythrocyte membranes with aqueous phenol, subsequent electrophoresis and analysis of the extracted glycophorins rather than electrophoresis and analysis of the glycophorin from intact erythrocyte membranes. The results showed a large deficit of N-acetylgalactosamine, galactose, and sialic acid residues in glycophorin A from patients with CDA type I and type II amounting to about 45% and 55%, respectively. The results strongly suggest that glycophorin A in these patients is partly unglycosylated with respect to O-linked glycans. In addition, glycophorin A from erythrocytes of a patient with CDA II but not CDA I exhibited a significant deficit of mannose and N-acetylglucosamine suggesting that its N-glycosylation site was also partly unglycosylated.

scholarly journals The cisternae decorating the red blood cell membrane in congenital dyserythropoietic anemia (type II) originate from the endoplasmic reticulum

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4433-4439 ◽  
Author(s):  
N Alloisio ◽  
P Texier ◽  
L Denoroy ◽  
C Berger ◽  
E Miraglia del Giudice ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Sodium Dodecyl ◽  
Immunogold Electron Microscopy ◽  
Major Protein ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Sds Page

We studied 20 individuals from 17 unrelated families with congenital dyserythropoietic anemia (type II; CDAII). The clinical phenotype was mild to moderate. The inheritance pattern was invariably recessive. Coomassie blue stained gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) show that band 3 was thinner and migrated slightly faster than usual. In addition, staining showed two unknown minor bands (in the patients), but not in normal controls, the obligate carrier parents, or in patients with other anemic syndromes. These minor proteins were studied using partial digestion, amino acid sequencing, Western blotting, immunofluorescence, and immunogold electron microscopy. They were identified as the glucose-regulated protein GRP78 and calreticulin that are resident proteins of the endoplasmic reticulum (ER). Using specific antibody, we showed that protein disulfide isomerase (PDI), a third major protein of the ER, was also present on the SDS-PAGE of red blood cell (RBC) ghosts. Immunofluorescence colocalized PDI with the dense discontinuous ring decorating the RBC membrane. Immunogold electron microscopy showed that PDI was localized in the lumen of the cisternae, confirming that these originate from the smooth ER. From a practical point of view, screening the above minor proteins in RBC membranes appears to be a straightforward and reliable diagnostic test for CDAII.

Effect of Integral Proteins on Frozen Membrane Fracture

1980 ◽  
Vol 38 ◽  
pp. 756-759 ◽  
Author(s):  
S. Kirchanski ◽  
D. Branton
Keyword(s):  
Lipid Bilayers ◽  
Freeze Fracture ◽  
Covalent Bond ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Experimental Protocol ◽  
Transmembrane Glycoprotein ◽  
Bond Breakage ◽  
Human Erythrocyte Membranes ◽  
Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

scholarly journals Subcellular Distribution and Relative Expression of Fibrocyte Markers in the CD/1 Mouse Cochlea Assessed by Semiquantitative Immunogold Electron Microscopy

2011 ◽  
Vol 59 (11) ◽  
pp. 984-1000 ◽  
Author(s):  
Shanthini Mahendrasingam ◽  
Catherine Bebb ◽  
Ella Shepard ◽  
David N. Furness
Keyword(s):  
Electron Microscopy ◽  
Tissue Growth ◽  
Immunogold Electron Microscopy ◽  
Spiral Ligament ◽  
Type Ii ◽  
Relative Expression ◽  
Aquaporin 1 ◽  
Cellular Transplantation ◽  
S 100 ◽  
Type V

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

Freeze-fracture Electron Microscopic observations on the effects of sulphydryl group reagents on human erythrocyte membranes

1990 ◽  
Vol 48 (3) ◽  
pp. 524-525 ◽  
Author(s):  
Gheorghe Benga ◽  
Anthony Brain ◽  
Victor I. Pop ◽  
John Wrigglesworth
Keyword(s):  
Human Erythrocyte ◽  
Freeze Fracture ◽  
Band 3 ◽  
Erythrocyte Membranes ◽  
Electron Microscopic ◽  
Experimental Conditions ◽  
Water Permeation ◽  
Human Erythrocyte Membranes ◽  
Erythrocyte Membrane Proteins

The intra-membrane particles (IMPs) observed on the fracture face of frozen erythrocyte membranes are thought to correspond primarily to “band 3” tetramers or dimers. Some recent studies correlating the inhibition of water diffusion in erythrocytes by p-chloromercuribenzene sulfonate (PCMBS) with the binding of 203Hg to erythrocyte membrane proteins has enabled band 3 and the polypeptides in band 4.5 to be identified as the proteins associated with the channels for water permeation in human erythrocytes. A further characterization of the effects of the incubation of human erythrocyte membranes with PCMBS and N-ethylmaleimide (NEM) has been performed as previously described. Experimental conditions have been previously described.A comparison was made of the appearance of freeze-etched membranes of control erythrocytes and erythrocytes with the sulphydryl reagents. It was found that on many of the control and NEM-treated cells, small (50-100 nm) elevated patches could be seen (Fig. 1, 2 and 3). These are present on both fracture and etch faces and are devoid of any intramembrane particles. The patch elevations were never observed in the membranes of PCMBS-treated cells (Fig. 4).

Aggregation of band 3 in hereditary ovalocytic red blood cell membranes. Electron microscopy and protein rotational diffusion studies

1993 ◽  
Vol 105 (3) ◽  
pp. 655-660
Author(s):  
A. Che ◽  
R.J. Cherry ◽  
L.H. Bannister ◽  
A.R. Dluzewski
Keyword(s):  
Electron Microscopy ◽  
Rotational Diffusion ◽  
Freeze Fracture ◽  
Band 3 ◽  
Normal Band ◽  
Cytoskeletal Proteins ◽  
Rotational Mobility ◽  
Normal Cells ◽  
Membrane Bound ◽  
Red Blood Cell Membranes

Microaggregation of band 3 proteins in hereditary ovalocytic membranes was investigated by rotational diffusion measurements and by electron microscopy. It was previously shown that band 3 in ovalocytic membranes has decreased rotational mobility compared with band 3 in normal cells (Tilley, L., Nash, G.B., Jones, G.L. and Sawyer, W.L. (1991) J. Membr. Biol. 121, 59–66). This result could arise from either altered interactions with cytoskeletal proteins or from band 3 microaggregation. In the present study it was found that removal of spectrin and actin from the membrane had no effect on the rotational mobility of ovalocytic band 3. Additional removal of ankyrin and band 4.1, as well as cleavage of the cytoplasmic domain of band 3 with trypsin, did enhance band 3 mobility, as is the case in the membranes from normal cells. However, the rotational mobility of ovalocytic band 3 was always considerably less than that of normal band 3 under the same conditions. Scanning electron microscopy and low power electron micrographs of freeze-fracture replicas revealed that the surfaces of ovalocytes were more irregular than those of normal erythrocytes. At higher magnification, numerous linearly arranged intramembranous particles were observed on the P-faces of freeze-fractured ovalocytes but not on normal cells. These clusters consist of straight or slightly curved lines of 10–15 particles in single rows. From these results it is deduced that the reduced rotational mobility of band 3 in ovalocytes is a consequence of the formation of microaggregates, which are very probably induced by the mutation in the membrane-bound domain of ovalocytic band 3.

Internalization of proximal tubular type II Na-Pi cotransporter by PTH: immunogold electron microscopy

2000 ◽  
Vol 278 (1) ◽  
pp. F148-F154 ◽  
Author(s):  
M. Traebert ◽  
J. Roth ◽  
J. Biber ◽  
H. Murer ◽  
B. Kaissling
Keyword(s):  
Electron Microscopy ◽  
Parathyroid Hormone ◽  
Horseradish Peroxidase ◽  
Brush Border Membrane ◽  
Adaptor Protein ◽  
Immunogold Electron Microscopy ◽  
Type Ii ◽  
Lysosomal Degradation ◽  
New Synthesis ◽  
Proximal Tubular

Physiological/pathophysiological alterations in proximal tubular Pireabsorption are associated with an altered brush-border membrane (BBM) expression of type II Na-Pi cotransporter molecules. Reduction is achieved by an internalization and lysosomal degradation and an increase in Pi reabsorption by new synthesis and BBM insertion of type II Na-Pi cotransporters. In the present study, we investigated by immunohistochemistry and immunogold electron microscopy the routing of internalized rat type II Na-Picotransporters (NaPi-2). In kidney of rats on a chronic low-Pi diet, NaPi-2 is mainly localized in the BBM, in cisterns of the Golgi apparatus and sparsely also in large endocytotic vacuoles and lysosomes. Fifteen minutes after the injection of the 1–34 analog of parathyroid hormone (PTH), the amount of NaPi-2 was decreased in the BBM and increased in endocytotic vesicles. NaPi-2 molecules colocalized with horseradish peroxidase injected prior to the injection of PTH. Vesicles labeled for NaPi-2 were occasionally also labeled for clathrin or the adaptor protein AP2. We conclude that NaPi-2 molecules enter the subapical compartment from where NaPi-2-containing vesicles are segregated off and directed to the lysosomes. A clathrin-mediated pathway may contribute to the PTH-induced internalization of NaPi-2.

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