A new “marker” protein for astrocytes

1986 ◽  
Vol 6 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Rajkumari Singh ◽  
Bhagirath Singh ◽  
S. K. Malhotra
Keyword(s):  
Monoclonal Antibody ◽  
Glial Fibrillary Acidic Protein ◽  
Immunofluorescence Microscopy ◽  
Brain Homogenate ◽  
Marker Protein ◽  
Sds Page ◽  
Double Label ◽  
Cryostat Sections ◽  
The Brain ◽  
New Protein

A monoclonal antibody (Mab J1-31) has been produced by using human brain homogenate as immunogen in mouse. Double-label immunofluorescence microscopy on cryostat sections of human, rabbit and rat brain, reveals staining of cells that are also stained with antiserum to glial fibrillary acidic protein (GFAP, a commonly used marker protein for astrocytes). However, there is no decrease in staining due to Mab J1-31 in sections incubated in antiserum to GFAP prior to incubation with the J1-31 ascites fluid. Immunoprecipitation of aqueous and detergent extracts of brain homogenate gives a single band at 30K by SDS PAGE followed by autoradiography. Immunoelectron microscopy shows that the J1-31 antigen is associated with the cytoskeleton. Thus, the Mab J1-31 recognizes a new protein present in GFAP positive cells (astrocytes) in the brain.

A new protein (J1-31 antigen, 30 kD) is expressed by astrocytes, Müller glia and ependyma

1987 ◽  
Vol 7 (6) ◽  
pp. 491-502 ◽  
Author(s):  
R. Predy ◽  
D. Singh ◽  
R. Bhatnagar ◽  
R. Singh ◽  
S. K. Malhotra
Keyword(s):  
Direct Evidence ◽  
Immunofluorescence Microscopy ◽  
Brain Homogenate ◽  
Ependymal Cells ◽  
Cell Type ◽  
Intracellular Protein ◽  
Reducing Conditions ◽  
Core Proteins ◽  
A Cell ◽  
New Protein

Monoclonal antibody (MAb) J1–31 raised using human brain homogenate as immunogen in mice can be used as a cell type marker for certain types of CNS macroglia, namely astrocytes, Müller cells and tanycytes as well as ciliated ependymal cells. Except for the ciliated ependymal cells, these types of macroglia express glial fibrillary acidic protein (GFAP). J1–31 antigen is an intracellular protein which has a MW of 30 kD under reducing conditions for gel electrophoresis (Singh et al., 1986). This protein is distinct from GFAP (MW 50 kD) and vimentin (MW 55 kD), the two core proteins of 10 nm IFs known to be expressed in the above types ofmacroglia. This conclusion is based on several criteria including temporal differences in the onset of expression of GFAP and J1–31 antigen during development of the rat cerebellum. Also, there is no detectable (by immunofluorescence microscopy) expression of J1–31 antigen in the prenatal CNS or outside the CNS where vimentin has been reported to be abundant. The most direct evidence that J 1–31 antigen and GFAP are distinct proteins comes from studies on the mature ciliated ependymal cells which do not express GFAP and yet show intense immunostaining for J1–31 antigen.

scholarly journals Identification of a Golgi-associated protein that undergoes mitosis dependent phosphorylation and relocation.

1990 ◽  
Vol 110 (5) ◽  
pp. 1513-1523 ◽  
Author(s):  
I McMorrow ◽  
W E Souter ◽  
G Plopper ◽  
B Burke
Keyword(s):  
Monoclonal Antibody ◽  
Nuclear Envelope ◽  
Immunofluorescence Microscopy ◽  
Nuclear Lamina ◽  
Early Prophase ◽  
Perinuclear Region ◽  
Nuclear Envelope Breakdown ◽  
Peripheral Membrane Protein ◽  
Double Label ◽  
Late Telophase

By means of a monoclonal antibody (BH3), we have identified a 57-kD protein (p57) that in interphase is restricted largely to the perinuclear region of the cell. Double label immunofluorescence microscopy suggests localization of p57 to the Golgi complex and associated membranous structures. Protease protection experiments and chemical extractability indicate that p57 is a peripheral membrane protein exposed to the cytoplasm. p57 displays unique behavior during mitosis. At the end of G2 or in early prophase, p57 leaves the perinuclear region and accumulates very rapidly within the nucleus, at a time when the nuclear envelope is still intact and before nuclear lamina disassembly. This relocation of p57 coincides with its hyperphosphorylation on serine and threonine residues. After nuclear envelope breakdown p57 becomes uniformly distributed throughout the mitotic cytoplasm until in late telophase when it returns to its perinuclear location and is once again excluded from the nucleus. The behavior of p57 during mitosis suggests that it may play a role in the cellular reorganization evident during mitotic prophase.

scholarly journals Immunohistochemical Characteristics of Ischemic Process in Hippocampal Formation in Posterity of Female Rats after Labor Induction

2021 ◽  
Vol 6 (3) ◽  
pp. 114-119
Author(s):  
I. Yu. Mamay ◽  
◽  
O. A. Hryhorieva ◽  
V. I. Dariy
Keyword(s):  
Monoclonal Antibody ◽  
Glial Fibrillary Acidic Protein ◽  
Dentate Gyrus ◽  
Hippocampal Formation ◽  
Acidic Protein ◽  
Female Rats ◽  
Relative Area ◽  
Santa Cruz ◽  
The Brain ◽  
Stimulation Of

The response to perinatal hypoxia, developing in stimulating generic activity, is the activation of microglia, which induces the development of local inflammation of the brain and leads to the death of neurons. The formation of the hippocampus supports important physiological and behavioral functions, including spatial learning and memory, and is part of the brain, which is especially vulnerable to changes in blood glucose and oxygen. Thus, the study of the features of the development of hippocampal formation in the postnatal period after stimulating generic activity is relevant. The purpose of the study was to study the features of Glial fibrillary acidic protein and NeuN expression in the hippocampal formation in posterity of female rats after PgE2 injection for labor induction. Materials and methods. Pregnant females of the experimental group on the twenty-second day of pregnancy were injected intravaginally with PGE2 in the form of a gel to stimulate the generic activity. Birth occurred on the twenty-third day after conception. Birth in the intact group of rats occurred on the 23-24th day after conception. The large hemispheres of the rat brain were fixed in a 10% neutral formal solution, dehydrated in an ascending alcohol battery. For immunohistochemical studies, paraffin sections were used with 3 microns with a thickness of 3 microns. Glial fibrillary acidic protein Mouse Monoclonal Antibody (Santa Cruz Biotechnology, Inc.) is used to detect astrocytes (Santa Cruz Biotechnology, Inc.). NeuN Mouse Monoclonal Antibody (Santa Cruz Biotechnology, Inc.) was used to identify neurons on the 1st, 7th, 14th, 45th days after birth in histological cuts of hippocampus and gear, using a program for analyzing and processing images Image J, studied the relative area occupied by Glial fibrillary acidic protein + NeuN + cells. Data is processed by variation statistics. The results are reliable at p <0.05. All animal experiments were performed according to international principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” (Strasbourg, 18.03.86) and the Law of Ukraine No. 1759-VI (15.12.2009) On the Protection of Animals from Cruelty. Results and discussion. We identified that by the changes in the hippocampus and the toothed gyrus of the rats after induction of labor, increased Glial fibrillary acidic protein expression on the first day after birth and reducing the NeuN expression on the 14th and 45th day of life in experimental animals compared to the control group were included. Conclusion. The density of the distribution of Glial fibrillary acidic protein + cells in the hippocampus and the dentate gyrus of rats changes wavily throughout the first month and a half of postnatal life. In the offspring of rats, after stimulation of labor on the first day of life, a reliable increase in the relative area occupied by Glial fibrillary acidic protein + astrocytes is determined compared to the control (49.3±2.6% and 36.8±5.9%, respectively). In the hippocampus and the dentate gyrus of rats after stimulation of labor during the first 45 days after birth, the gradual decrease in the relative area occupied by NeuN + neurons is determined, in contrast to the control animals, in which this index practically does not change. On the 14th and 45th days, the index of the relative area occupied by NeuN + neurons in experimental rats is reliably lower than in control (p <0.05).

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

scholarly journals Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D

2004 ◽  
Vol 380 (3) ◽  
pp. 749-756 ◽  
Author(s):  
Yong-Xin SUN ◽  
Kazuhito TSUBOI ◽  
Yasuo OKAMOTO ◽  
Takeharu TONAI ◽  
Makoto MURAKAMI ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Brain Homogenate ◽  
Wide Distribution ◽  
Rat Tissues ◽  
Lysophospholipase D ◽  
Pla2 Enzyme ◽  
First Time ◽  
Hydrolysis Of ◽  
The Brain

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2α were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.

Distribution of hyaluronan in the epiphysial growth plate: turnover by CD44-expressing osteoprogenitor cells

1994 ◽  
Vol 107 (10) ◽  
pp. 2669-2677 ◽  
Author(s):  
P. Pavasant ◽  
T.M. Shizari ◽  
C.B. Underhill
Keyword(s):  
Monoclonal Antibody ◽  
Growth Plate ◽  
Cultured Cells ◽  
Human Cell Line ◽  
Histochemical Staining ◽  
Long Bone ◽  
Thin Sections ◽  
Osteoprogenitor Cells ◽  
Double Label ◽  
Mouse Tibia

In the present study, we have examined the distribution of both hyaluronan and its receptor, CD44, during the process of endochondral ossification in the mouse tibia. Histochemical staining revealed that a large amount of hyaluronan was present in the lacunae located in the zone of hypertrophy, but it was greatly reduced or absent from the zone of erosion. In addition, hyaluronan was present in the cytoplasm of osteoprogenitor cells located in the zone of erosion. These cells also expressed CD44 on their surfaces, as revealed by double-label immunohistochemistry. These results suggested that the osteoprogenitor cells may use CD44 to bind and internalize hyaluronan, and subsequently degrade it with lysosomal enzymes. To test this possibility, we examined the human cell line, MG-63, which closely resembles osteoprogenitor cells. These cells produced several different forms of CD44, as determined by western blotting (85, 116 and 150 kDa). In addition, the binding of isotopically labeled hyaluronan to detergent extracts of these cells was blocked by a monoclonal antibody to CD44. Similarly, the degradation of hyaluronan by these cultured cells was also inhibited by a monoclonal antibody to CD44. To determine if these cells could remove hyaluronan from the growth plate, the cells were cultured directly on top of thin sections of the epiphysial region of long bone. After 16 hours, the sections were stained for hyaluronan. The MG-63 cells removed significant amounts of hyaluronan present in the zone of hypertrophy, and this effect was blocked by an excess of soluble hyaluronan and by a monoclonal antibody to CD44.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of antibodies as probes for structural and biochemical studies of tektins from ciliary and flagellar microtubules

1987 ◽  
Vol 88 (4) ◽  
pp. 453-466
Author(s):  
R.W. Linck ◽  
M.J. Goggin ◽  
J.M. Norrander ◽  
W. Steffen
Keyword(s):  
Immunofluorescence Microscopy ◽  
Strongylocentrotus Purpuratus ◽  
Body Region ◽  
Two Dimensional ◽  
Structural Localization ◽  
Lytechinus Pictus ◽  
Sds Page ◽  
Nuclear Structures ◽  
Sea Urchin Sperm

Rabbit antibodies raised and purified against three tektins, proteins of flagellar doublet microtubules from sea-urchin sperm (Lytechinus pictus and Strongylocentrotus purpuratus), were used to study tektin biochemistry and their structural localization. Doublet microtubules were fractionated into tektin filaments and separated by SDS-PAGE into three major tektin polypeptide bands (Mr = 47, 51 and 55 (X 10(3)), which were used to immunize rabbits. Antibodies against each tektin (anti-tektins) were affinity-purified and then characterized by two-dimensional isoelectric focusing/SDS-PAGE immunoblotting and by immunofluorescence microscopy. In two-dimensional immunoblots of 0.5% Sarkosyl-resistant fractions of flagellar microtubules, the antibody against the 55 X 10(3) Mr tektin (anti-55) stained one major polypeptide of 55 X 10(3) Mr and pI 6.9, anti-51 stained two polypeptides of 51 X 10(3) Mr and pI approximately 6.15, and anti-47 stained one major polypeptide of 47 X 10(3) Mr and pI 6.15. The anti-tektins also stained several minor neighbouring polypeptides, which may be isoelectric variants, novel tektins or unrelated proteins. Furthermore, anti-47 crossreacted with the major 55 X 10(3) Mr polypeptide. By immunofluorescence microscopy all three anti-tektins stained methanol-fixed echinoderm sperm flagella and embryonic cilia. In addition, anti-47 and anti-55 stained unfixed, demembranated axonemes. Besides staining axonemes, all anti-tektins labelled the basal body region, and anti-51 labelled the sperm head envelope. These results indicate that the tektins are a complex family of proteins that are components of axonemal microtubules and possibly other cytoplasmic and nuclear structures.

scholarly journals Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

1996 ◽  
Vol 29 (5) ◽  
pp. 483-489
Author(s):  
Lilian Terezinha de Queiroz Leite ◽  
Mauricio Resende ◽  
Wanderley de Souza ◽  
Elizabeth R.S. Camargos ◽  
Matilde Cota Koury
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Silver Staining ◽  
Colloidal Gold ◽  
Leptospira Interrogans ◽  
Outer Envelope ◽  
Lethal Challenge ◽  
Sds Page

Monoclonal antibodies (MABs) ivere produced against an etbylenediaminetetraacetate (EDTA) extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b). The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

Sign in / Sign up

Export Citation Format

Share Document