1. 10% SDS-PAGE Immunoblot for Epitope H (O-GlcNAcH)
Expression of the O-linked N-Acetylglucosamine containing epitope H (O-GlcNAcH) in lamb glial cells in vitro and in vivo  (#112) D. Arvanitis 1, Y. Drosos 2, A. Zibis 1, S. Havaki 3, L. Arvanitis 4 1Dept. of Anatomy, University of Thessaly, Medical School, Larissa, Greece 2Dept. of Oncology, St. Jude Children’s Research Hospital, Memphis TN, USA 3Dept. of Histology-Embryology, National and Kapodistrian University of Athens, Medical School, Athens, Greece 4Dept. of Pathology, Rush, University, Medical Center, Chicago, Ill, USA
Abstract
INTRODUCTION: Epitope H contains an O-linked N-Acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal IgM antibody Η (mabH). O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, the latter in cells under stress. The post-translational modification of serine and threonine residues of polypeptides by the addition of the sugar moiety N-Acetylglucosamine (O-GlcNAc) occurs in many nuclear, cytoplasmic and mitochondrial proteins, which are involved in cell processes such as cancer, diabetes mellitus, cardiovascular and neurodegenerative diseases.
PURPOSE: In the present work, we examined the expression of the O-GlcNAcH in cultures of lamb oligodendrocytes - astrocytes, in sections of lamb brains and in lamb brain polypeptides, using the mabH and anti-GFAP in immunostaιn for the formalin fixed paraffin embedded sections and the western-blot immunoblot. RESULTS: The expression of the O-GlcNAcH is as follows: 1) Oligodendrocytes and astrocytes in culture showed strong cytoplasmic and process staning  2) Double immunofluoresence of the basis pontis from two cases showed staining only of the astrocytes. 3) The indirect immunoperoxidase staining of 30 cases revealed: a) Strong and widespread  cytoplasmic and process staining of the external glial limitans astrocytes of the cerebral hemispheres, b) very few stained astrocytes in the cerebral cortex and basal ganglia, c) a minority of stained fibrous astrocytes within the cerebral and cerebellar white matter, d) strong and widespread staining of the astrocytes within the granular  layer of the cerebellar cortex ,  e) a good number of stained astrocytes in the fastigial nucleus, ( only vermis sections were examined) and f) a minority  of  cytoplasmicly stained ependymal cells with heterogeneous distribution. A western blot- immunoblot from one case including samples from the cerebral and cerebellar cortex, the cerebellar white matter and the ependymal lining showed polypeptides with common KD of 160, 135, 120, and 74 in all samples. In addition both the cerebellar white matter and cortex showed two polypeptides of 44.8 and  44.1 KD  and the cerebral cortex a polypeptide of 44.3 KD. The ependyma in addition to four polypeptides showed four more with 151, 57.3, 52.6 and KD. 1a 1b 2a 2b 3a 3b
Epitope H
GFAP 4 5 6 7a 7b 8a 8b
10% SDS-PAGE Immunoblot for Epitope H (O-GlcNAcH)
Lane 1: Lamb cerebellar cortex; 2: Cerebral cortex;
3: Cerebellar white matter;
4: Cerebral ependymal lining
4: External glia limitans cerebral astrocytes intensely stained for epitope H. Mag: 200x
Double immunofluorescence: Epitope H (Rhodamine). GFAP (Fluoresceine). 1a, 1b: culture of oligodendrocytes - astrocytes. Epitope H appears in both, while GFAP only in the one astrocyte. Mag: 200x. 2a,2b: one astrocyte with broader processes stained for epitope H and GFAP. Mag: 1000x. 3a, 3b: Basis pontis with stained astrocytes for H. GFAP shows that the epitope H-stained cells are astrocytes only. Mag: 100x 9a 9b 10 11
5: Caudate nucleus with few stained astrocytes (large arrow). Unstained oligodendrocyte (small arrow) and neuron (arrowhead). Mag: 200x. 6: Weakly stained white matter cerebral astrocytes for epitope H. Mag: 200x. 7a: Area of caudate nucleus with abundant stained astrocytes with GFAP. Mag: 100x. 7b: Consecutive section showing an occasional stained astrocyte for H (arrow). Mag: 100x. 8a: Ependymal and subependymal area showing stained ependymal cells (arrow), astrocytes and abundant astrocytic processes (open asterisk) with the GFAP. Mag: 200x. 8b: Epitope H immunostaining. Consecutive section showing stained (small arrow) and unstained (arrowhead) ependymal cells, unstained astrocytic processes (open asterisk) and a stained astrocyte (large arrow). Same vessel: closed asterisk. Mag: 200x.
Cerebellar Epitope H immunostaining. 9a: Cerebellar cortex showing intensely stained protoplasmic astrocytes in the granular layer and unstained Bergmann glia. Mag: 100x. 9b: Higher magnification of 9a showing the stained protoplasmic astrocytes. Mag: 630x. 10: Part of cerebellar cortex and white matter with stained astrocytes in both areas. Fibrous astrocytes in white matter with weak stain. Mag: 200x. 11: Fastigial nucleus with stained astrocytes (small arrows), unstained neurons (large arrows), unstained oligodendrocytes (arrowheads). Mag: 200x.
CONCLUSIONS: There is heterogeneous expression of the O-GlcNAcH in the subpopulations of the astrocytes and in the ependymal cells.