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 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 Comparative study of glycophorin A derived O-glycans from human Cad, Sd(a+) and Sd(a-) erythrocytes

1985 ◽  
Vol 232 (3) ◽  
pp. 813-818 ◽  
Author(s):  
D Blanchard ◽  
C Capon ◽  
Y Leroy ◽  
J P Cartron ◽  
B Fournet
Keyword(s):  
Blood Group ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Red Cells ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Methylation Analysis ◽  
Dolichos Biflorus ◽  
Major Acid

Glycophorin A was purified from the erythrocyte membranes of blood group Cad, Sd(a+) and Sd(a-) donors and the oligosaccharide alditols, obtained after alkaline borohydride degradation, separated by h.p.l.c. on an alkylamine silica gel column, were characterized by sugar analysis. Structure determination of the major acid components by methylation analysis, g.l.c.-m.s. and 1H-n.m.r. indicated that the three blood group Cad red cells under study (samples Cad., Bui. and Des.) carry the same pentasaccharide GalNAc(beta 1-4)[NeuAc(alpha 2-3)]Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc -ol(Cad determinant) but in different amounts. This pentasaccharide, however, was absent from glycophorin A of Sd(a+) and Sd (a-) donors, suggesting that the Sda determinant is not associated with glycophorins. It was calculated that glycophorin A from the original Cad donor (Cad.) carries about 12 O-glycosidically linked pentasaccharide chains per molecule whereas only 2-3 of these chains were present in the samples from the two other unrelated Cad individuals (Bui. and Des.) It is well known from quantitative agglutination studies that the proportion of red cells which can be agglutinated by the Dolichos biflorus lectin varies from one Cad blood sample to another. Some are completely agglutinated (Cad. donor) whereas others are only partially agglutinated (Bui. and Des. donors) suggesting that some red cells might not carry the Cad determinants. From the results presented above and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis studies it is suggested that Cad red cells from Bui. and Des. do not carry a mixture of glycophorin A molecules with or without the Cad pentasaccharides but a spectrum of glycoprotein molecules with varying amounts of Cad determinants.

GCDFP-70 protein in cyst fluid identified as albumin and used to classify cysts in women with breast gross cystic disease

1991 ◽  
Vol 37 (4) ◽  
pp. 547-551 ◽  
Author(s):  
Milagros Balbín ◽  
Francisco Vizoso ◽  
Luis M Sánchez ◽  
Rafael Venta ◽  
Alvaro Ruibal ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Simple Procedure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cyst Fluid ◽  
Type I ◽  
Type Ii ◽  
Cystic Disease ◽  
Functional Changes ◽  
Cyst Fluids

Abstract We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to study cyst fluids from women with breast gross cystic disease. The subjects could be classified into two categories according to the concentrations of protein GCDFP-70 in the cyst fluid: those with Type I cysts had a very low content of this protein; those with Type II cysts had very high concentrations. Analysis of the amino acid sequence of GCDFP-70 from both cyst fluid types confirmed that this protein is human plasma albumin. The average concentration of albumin found in Type I cyst fluids was 0.32 g/L and that corresponding to Type II was 10.16 g/L. Thus, albumin quantification from cyst fluids or analysis by either polyacrylamide or agarose gel electrophoresis provides a simple procedure for classifying these fluids, yielding results that correlate well with previous classifications based on other measurements such as sodium, potassium, and chloride concentrations. This albumin-based quantification method may improve the classification of breast cysts and might be useful in further studies on functional changes in the cysts and their relationship to breast cancer.

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.

150 PROTEOME OF BOVINE CUMULUS CELLS AS RELATED TO OOCYTE MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION

2017 ◽  
Vol 29 (1) ◽  
pp. 183
Author(s):  
I. C. Velez ◽  
M. M. Ramirez ◽  
A. I. Chica ◽  
R. Urrego ◽  
A. A. Moura ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Silico ◽  
Triosephosphate Isomerase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Type I ◽  
Type Ii

The present study was conducted to study the effect of cumulus-oocyte complex (COC) morphology on subsequent in vitro embryo development and to assess the proteome of their corresponding cumulus cells (CC). Cow ovaries were obtained at an abattoir and COC aspirated from 3–8 mm follicles. The COC were defined as type I (TI): homogeneous ooplasma and ≥4 layers of compact CC; type II (TII): granular ooplasma and ≥4 layers of slight expanded CC. Fifty COC had ~500,000 CC. Cumulus cells were frozen in ammonium bicarbonate and immediately lyophilized for proteome analysis. Other selected COC were matured in vitro in TCM-199-supplemented media for 24 h. After maturation, CC were collected (T24) and processed as described above. The remaining COC were fertilized with sperm from a fertile bull and zygotes, cultured in vitro until Day 7. Ten blastocysts per group were stained (Hoechst 33342) and blastomeres, counted for assessment of embryo quality. The CC proteins were obtained from the following groups: immature type I (TIT0) and type II (TIIT0), and in vitro matured type I (TIT24) and type II (TIIT24). For protein extraction, we used sonication (30 min, 4°C), freezing and unfreezing in liquid nitrogen, and maceration. The CC proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by ESI-MS/MS. Differences in cleavage, embryo rates, and blastomere numbers were analysed by t-test. Protein expression difference was set at 2.5-fold (P < 0.05). In silico protein interactions were investigated using STRING v. 10.0. There were no differences in cleavage (88 ± 4 v. 89 ± 8%) and embryo rates (36 ± 7 v. 33 ± 8%) between COC of TI (n = 220) and TII (n = 161), respectively. Blastomeres were also similar in TI (101) and TII (104) groups. Major proteins expressed in all CC were α-enolase, β-actin, oestradiol 17-β-dehydrogenase 1, glutathione S-transferase, glyceraldehyde 3-phosphate dehydrogenase, heat shock protein β-1, histone H2B type 1-N, histone H4, mitochondrial malate dehydrogenase 2, protein disulfide isomarase A6, triosephosphate isomerase, tubulin α-1C chain, and vimentin. Glyceraldehyde 3-phosphate dehydrogenase appeared to be more expressed in TIT0, whereas tubulin α-1C chain and vimentin had greater expression in TIIT24. As evidenced by in silico analysis, most CC proteins interact among themselves, participating in complex networks involving intracellular signalling and other events. In conclusion, there are no difference in embryo development when using compact and early-expanded COC, indicating that both types can be selected for IVP. Protein profile of cumulus cell may serve as a marker for in vitro embryo competence.

scholarly journals Exclusion of Three Candidate Genes as Determinants of Congenital Dyserythropoietic Anemia Type II (CDA-II)

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 4197-4200 ◽  
Author(s):  
Achille Iolascon ◽  
Emanuele Miraglia del Giudice ◽  
Silverio Perrotta ◽  
Matteo Granatiero ◽  
Leopoldo Zelante ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Linkage Study ◽  
Biochemical Data ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Anion Transporter ◽  
Strong Homology ◽  
Cda Ii

Abstract Congenital dyserythropoietic anemia type II (CDA-II) is the most common form of inherited dyserythropoiesis. Previous studies have shown that the anion transporter (band 3) is narrower and it migrates faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); this aspect was related to insufficient glycosylation. Biochemical data support the hypothesis that this disease is due to a deficiency of N-acetylglucosaminyltransferase II (GnT II) or α-Mannosidase II (α-Man II), which represent the key to glycosylation. In addition, a third candidate gene is α-Man IIx, which shows a strong homology with α-Man II. The knowledge of the chromosomal localization of these putative genes allowed us to perform a linkage study using three sets of microsatellite markers flanking the candidate genes. Six families with two or more affected children were enrolled in this study. The data obtained exclude linkage to all three candidate genes. In consideration of the biochemical data (reduction of enzymatic activity) of the same enzymes, our results suggest the hypothesis that a defect in an unknown transcriptional factor is involved in CDA-II.

Changes in Myosin Expression in Denervated Laryngeal Muscle

1997 ◽  
Vol 106 (12) ◽  
pp. 1076-1081 ◽  
Author(s):  
John M. Delgaudio ◽  
James J. Sciote
Keyword(s):  
Fiber Type ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Type I ◽  
Type Ii ◽  
Laryngeal Muscle ◽  
Fiber Type Composition ◽  
Neural Input ◽  
Type Composition ◽  
Posterior Cricoarytenoid

The effects of chronic denervation on the myosin heavy chain (MyHC) content and muscle fiber type composition of rat laryngeal muscles are described. The posterior cricoarytenoid (PCA) and thyroarytenoid (TA) muscles were removed 3 weeks, 3 months, and 6 months after recurrent laryngeal nerve sectioning. Myofibrillar adenosine triphosphatase staining of cryostat sections was performed, and fiber type percentages were determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate MyHC isoforms, and densitometry was subsequently used for quantitative analysis. Unoperated animals served as controls. In the PCA muscle, denervation resulted in a progressive reduction in type I MyHC (the slow-contracting isoform) to an almost complete loss at 6 months, with a concomitant increase in type II MyHCs (fast-contracting isoforms, excluding type IIL). Type IIL MyHC (laryngeal-specific isoform) remained relatively constant up to 6 months after denervation. The myosin expression in the TA muscle, which contained only type II MyHCs, remained relatively constant with denervation. Changes in fiber type composition of the muscles described from tissue staining correlated with MyHC content. These findings in laryngeal muscle confirm the dependence of type I MyHC expression upon neural input, as has been found previously in limb skeletal muscles. Since the expression of all MyHCs except the IIL was modified after denervation in the PCA muscle, it is possible that the IIL isoform is maintained by factors that differ from those in the other skeletal myosins.

scholarly journals Autophagy facilitates type I collagen synthesis in periodontal ligament cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Nakamura ◽  
Motozo Yamashita ◽  
Kuniko Ikegami ◽  
Mio Suzuki ◽  
Manabu Yanagita ◽  
...  
Keyword(s):  
Periodontal Ligament ◽  
Collagen Synthesis ◽  
Type I Collagen ◽  
Periodontal Diseases ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Physiological Role ◽  
Type I ◽  
Periodontal Tissue ◽  
Pdl Cells

AbstractAutophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

Congenital Dyserythropoietic Anemia

PEDIATRICS ◽  
1973 ◽  
Vol 51 (5) ◽  
pp. 957-958
Author(s):  
G. Bennett Humphrey ◽  
Bahaod-Din Mojab ◽  
Ingomar Mutz
Keyword(s):  
Carbohydrate Metabolism ◽  
Morphological Characteristics ◽  
Type I ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
The 1950S ◽  
Deja Vu ◽  
Déjà Vu ◽  
Excellent Article

Reading the excellent article by Drs. Murphy and Oski, "Congenital Dyserythropoietic Anemia (CDA)",1 which further defines type II, produced a sense of deja vu. In the 1950s, nonspherocytic, hemolytic anemias (HNHA) were categorized as type I and II based on the in vitro autohemolysis test.2 This group of anemias has subsequently been demonstrated to be due to a series of enzymatic abnormalities in carbohydrate metabolism.3 In CDA, the morphological characteristics which define types I, II, and III probably reflect nuclear rather than cytoplasmic abnormalities.

Shortening velocity and ATPase activity of rat skeletal muscle fibers: effects of endurance exercise training

1994 ◽  
Vol 266 (6) ◽  
pp. C1699-C1713 ◽  
Author(s):  
J. M. Schluter ◽  
R. H. Fitts
Keyword(s):  
Atpase Activity ◽  
Endurance Exercise ◽  
Citrate Synthase ◽  
Fiber Type ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Treadmill Running ◽  
Type I ◽  
Marker Enzyme ◽  
Shortening Velocity

Mechanical properties were measured in single skinned fibers from rat hindlimb muscle to test the hypothesis that the fast type IIb fiber exhibits a higher maximal shortening velocity (Vo) than the fast type IIa fiber and that the difference is directly attributable to a higher myofibrillar adenosinetriphosphatase (ATPase) activity in the type IIb fiber. Additional measurements were made to test the hypotheses that regular endurance exercise increases and decreases the Vo of the type I and IIa fiber, respectively, and that the altered Vo is associated with a corresponding change in the fiber ATPase activity. Rats were exercised by 8-12 wk of treadmill running for 2 h/day, 5 day/wk, up a 15% grade at a speed of 27 m/min. Fiber Vo was determined by the slack test, and the ATPase was measured fluorometrically in the same fiber. The myosin isozyme profile of each fiber was subsequently determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mean +/- SE Vo (7.9 +/- 0.22 fiber lengths/s) of the type IIb fiber was significantly greater than the type IIa fiber (4.4 +/- 0.21 fiber lengths/s), and the higher Vo was associated with a higher ATPase activity (927 +/- 70 vs. 760 +/- 60 microM.min-1.mm-3). The exercise program induced cardiac hypertrophy and an approximately twofold increase in the mitochondrial marker enzyme citrate synthase. Exercise had no effect on fiber diameter or peak tension per cross-sectional area in any fiber type, but, importantly, it significantly increased (23%) both the Vo and the ATPase activity of the slow type I fiber of the soleus.(ABSTRACT TRUNCATED AT 250 WORDS)

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