Temperature-dependent binding of monoclonal antibodies to C hordein

We describe a method for the specific radioiodination of pinocytic vesicles (PVs) based upon the simultaneous endocytosis of lactoperoxidase (LPO) and glucose oxidase (GO). Initial experiments indicated that LPO was interiorized by the macrophage cell line J774 by fluid phase pinocytosis and without detectable binding to the plasma membrane (PM). Interiorization varied linearly with enzyme concentration and exposure time, was temperature dependent, and was undetectable at 4 degrees C. Employing EM cytochemistry, LPO activity was restricted to PVs after a 3- to 5-min pulse at 37 degrees C. These results formed the basis of the method for iodinating the luminal surface of PVs: 5-min exposure to both LPO and GO at 37 degrees C followed by washes and iodination (addition of 125I and glucose) at 4 degrees C. Enzyme-dependent incorporation of iodide into the polypeptides of both PV membrane and contents occurred. Several lines of evidence indicated that there was selective labeling of PV as opposed to PM. Iodination did not occur if the pinocytic uptake of LPO ad GO was inhibited by low temperature. EM autoradiography showed a cytoplasmic localization of grains, whereas a clear PM association was evident with surface labeling. LPO was iodinated only after PV labeling and was present within organelles demonstrating latency. After PV iodination, > 75% of several labeled membrane antigens could be immunoprecipitated by monoclonal antibodies only after cell lysis. In contrast, all labeled antigens were accessible to antibody on intact cells after surface labeling. The polypeptide compositions of PM and PV membrane were compared by SDS polyacrylamide gel electrophoresis and by quantitative immune precipitation using a panel of anti-J774 monoclonal antibodies. The electrophoretic profiles of iodinated proteins (15-20 bands) were strikingly similar in NP-40 lysates of both PV and PM iodinated cells. In addition, eight membrane antigens examined by immune precipitation, including the trypsin-resistant immunoglobulin (Fc) receptor and the H-2Dd histocompatibility antigen, were found to be iodinated to the same relative extents by both labeling procedures. We conclude that PV membrane is formed from a representative sample of PM polypeptide components.

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(111) Monocrystalline Nickel Films

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095819 ◽

1972 ◽

Vol 30 ◽

pp. 512-513

Author(s):

T.E. Pratt ◽

R.W. Vook

Keyword(s):

Film Thickness ◽

Deposition Rate ◽

Room Temperature ◽

Narrow Range ◽

High Vacuum ◽

Residual Pressure ◽

Temperature Dependent ◽

Deposition Parameters ◽

Transmission Electron ◽

Substrate Temperatures

(111) oriented thin monocrystalline Ni films have been prepared by vacuum evaporation and examined by transmission electron microscopy and electron diffraction. In high vacuum, at room temperature, a layer of NaCl was first evaporated onto a freshly air-cleaved muscovite substrate clamped to a copper block with attached heater and thermocouple. Then, at various substrate temperatures, with other parameters held within a narrow range, Ni was evaporated from a tungsten filament. It had been shown previously that similar procedures would yield monocrystalline films of CU, Ag, and Au.For the films examined with respect to temperature dependent effects, typical deposition parameters were: Ni film thickness, 500-800 A; Ni deposition rate, 10 A/sec.; residual pressure, 10-6 torr; NaCl film thickness, 250 A; and NaCl deposition rate, 10 A/sec. Some additional evaporations involved higher deposition rates and lower film thicknesses.Monocrystalline films were obtained with substrate temperatures above 500° C. Below 450° C, the films were polycrystalline with a strong (111) preferred orientation.

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Ultrastructural analysis of unique glycoconjugates in the rat olfactory system

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124859 ◽

1992 ◽

Vol 50 (1) ◽

pp. 890-891

Author(s):

James E. Crandall ◽

Linda C. Hassinger ◽

Gerald A. Schwarting

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Monoclonal Antibodies ◽

Olfactory System ◽

Olfactory Bulbs ◽

Temporal And Spatial Patterns ◽

Carbohydrate Epitopes ◽

Olfactory Systems ◽

Temporal And Spatial ◽

Cell Surface Glycoconjugates

Cell surface glycoconjugates are considered to play important roles in cell-cell interactions in the developing central nervous system. We have previously described a group of monoclonal antibodies that recognize defined carbohydrate epitopes and reveal unique temporal and spatial patterns of immunoreactivity in the developing main and accessory olfactory systems in rats. Antibody CC2 reacts with complex α-galactosyl and α-fucosyl glycoproteins and glycolipids. Antibody CC1 reacts with terminal N-acetyl galactosamine residues of globoside-like glycolipids. Antibody 1B2 reacts with β-galactosyl glycolipids and glycoproteins. Our light microscopic data suggest that these antigens may be located on the surfaces of axons of the vomeronasal and olfactory nerves as well as on some of their target neurons in the main and accessory olfactory bulbs.

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Structural and Chemical Studies of Pathological Fibers in Human Neurofibrillary Degeneration

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012031x ◽

1985 ◽

Vol 43 ◽

pp. 726-729

Author(s):

K.S. Kosik ◽

L.K. Duffy ◽

S. Bakalis ◽

C. Abraham ◽

D.J. Selkoe

Keyword(s):

Monoclonal Antibodies ◽

Alzheimer Disease ◽

Human Brain ◽

Neurofibrillary Tangles ◽

Morphological Analysis ◽

High Specificity ◽

Cytoskeletal Proteins ◽

Neuritic Plaque ◽

Protein Chemistry ◽

Chemical Studies

The major structural lesions of the human brain during aging and in Alzheimer disease (AD) are the neurofibrillary tangles (NFT) and the senile (neuritic) plaque. Although these fibrous alterations have been recognized by light microscopists for almost a century, detailed biochemical and morphological analysis of the lesions has been undertaken only recently. Because the intraneuronal deposits in the NFT and the plaque neurites and the extraneuronal amyloid cores of the plaques have a filamentous ultrastructure, the neuronal cytoskeleton has played a prominent role in most pathogenetic hypotheses.The approach of our laboratory toward elucidating the origin of plaques and tangles in AD has been two-fold: the use of analytical protein chemistry to purify and then characterize the pathological fibers comprising the tangles and plaques, and the use of certain monoclonal antibodies to neuronal cytoskeletal proteins that, despite high specificity, cross-react with NFT and thus implicate epitopes of these proteins as constituents of the tangles.

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