scholarly journals Specific pancreatic beta-cell surface antigens recognized by a xenogenic antiserum.

1982 ◽  
Vol 94 (2) ◽  
pp. 472-477 ◽  
Author(s):  
T Dyrberg ◽  
S Baekkeskov ◽  
A Lernmark
Keyword(s):  
Cell Surface ◽  
Beta Cells ◽  
Islet Cell ◽  
Islet Cells ◽  
Protein A ◽  
Cell Suspensions ◽  
Nmri Mouse ◽  
Mouse Islet ◽  
Gold Particles ◽  
Spleen Lymphocytes

An antiserum (R4) from a rabbit immunized with suspensions of C57BL/61 ob/ob mouse islet cells contains antibodies which in a 125I-protein A radioligand assay can be demonstrated to bind to single cell suspensions of normal Naval Medical Research Institute (NMRI) mouse islet cells. The binding of 125I-protein A to islet cells was about four times that of normal rabbit serum (NRS) after incubation at a 1/600 dilution of R4 antiserum quantitatively absorbed to mouse spleen lymphocytes (R4A antiserum) and hepatocytes. Subsequent absorption of the R4A antiserum to islet cells significantly reduced the binding of 125I-protein A to islet cells incubated with the doubly absorbed serum. Immunoprecipitation of radiolabeled islet cell lysates followed by SDS polyacrylamide gel electrophoresis and autoradiography suggested that the R4A antiserum recognized a Mr 40,000 glycoprotein. This glycoprotein was not detected in spleen lymphocytes. Electron microscope detection of gold-protein A complexes suggested that the binding of islet cell surface antibodies was cell specific. islet cell suspensions incubated with R4A antiserum and gold-protein A showed that 86 +/- 3 gold particles were bound per 100 beta-cells (mean +/- SE for six experiments). In contrast, the number of gold particles per 100 endocrine non-beta-cells was 8 +/- 1 which was similar to the number achieved with NRS (3 +/- 1) on all endocrine islet cells. Our observations suggest that the pancreatic islet cells, in particular the beta-cells, express a specific antigen.

Detection of islet cell surface antibodies by an indirect rosette assay, using islet cells and protein A-labeled sheep red blood cells

1984 ◽  
Vol 74 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Shoji Maruyama ◽  
Masahiko Sugiura ◽  
Michio Nakazawa ◽  
Hiroko Tomiyama ◽  
Miyuki Shizawa ◽  
...  
Keyword(s):  
Cell Surface ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Islet Cell ◽  
Islet Cells ◽  
Protein A ◽  
Sheep Red Blood Cells ◽  
Islet Cell Surface Antibodies ◽  
Surface Antibodies

A pre-embedding, protein a-gold immunolabelling technique for cytocentrifuged cell monolayers for lm and tem

1986 ◽  
Vol 44 ◽  
pp. 220-221
Author(s):  
K. Chien ◽  
I.P. Shintaku ◽  
A.F. Sassoon ◽  
R.L. Van de Velde ◽  
R. Heusser
Keyword(s):  
Cell Surface ◽  
Staining Method ◽  
Protein A ◽  
Surface Antigens ◽  
Cell Suspensions ◽  
Cellular Morphology ◽  
Cellular Phenotype ◽  
Cell Surface Antigens ◽  
Cell Monolayers ◽  
Diagnostic Histopathology

Identification of cellular phenotype by cell surface antigens in conjunction with ultrastructural analysis of cellular morphology can be a useful tool in the study of biologic processes as well as in diagnostic histopathology. In this abstract, we describe a simple pre-embedding, protein A-gold staining method which is designed for cell suspensions combining the handling convenience of slide-mounted cell monolayers and the ability to evaluate specimen staining specificity prior to EM embedding.

scholarly journals Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

1986 ◽  
Vol 103 (6) ◽  
pp. 2273-2281 ◽  
Author(s):  
L Orci ◽  
M Ravazzola ◽  
M Amherdt ◽  
O Madsen ◽  
A Perrelet ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Islet Cells ◽  
Protein A ◽  
Secretory Vesicle ◽  
Average Density ◽  
Secretory Vesicles ◽  
Gold Particles ◽  
Single Polypeptide Chain ◽  
Igg Binding ◽  
Single Polypeptide

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.

Quantitative determination of islet cell surface antibodies using 125I-protein A

Diabetes ◽  
1983 ◽  
Vol 32 (5) ◽  
pp. 460-465 ◽  
Author(s):  
A. H. Huen ◽  
M. Haneda ◽  
Z. Freedman ◽  
A. Lernmark ◽  
A. H. Rubenstein
Keyword(s):  
Cell Surface ◽  
Quantitative Determination ◽  
Islet Cell ◽  
Protein A ◽  
Islet Cell Surface Antibodies ◽  
Surface Antibodies

scholarly journals Alloxan cytotoxicity in vitro. Microscope photometric analyses of Trypan Blue uptake by pancreatic islet cells in suspension

1977 ◽  
Vol 162 (1) ◽  
pp. 19-24 ◽  
Author(s):  
K Grankvist ◽  
Å Lernmark ◽  
I B Täljedal
Keyword(s):  
Pancreatic Islets ◽  
Beta Cells ◽  
Trypan Blue ◽  
Islet Cell ◽  
Visual Inspection ◽  
Islet Cells ◽  
Pancreatic Islet Cells ◽  
Cell Nuclei ◽  
20 Nm

Suspensions of islet cells were prepared by shaking pancreatic islets from non-inbred ob/ob mice in a Ca2+-free buffer. The cells were incubated with or without 20 mM-alloxan, and subsequently with Trypan Blue. The uptake of Trypan Blue by cell nuclei was analysed by microscope photometry and by counting the frequency of cells appearing stained on visual inspection. Cells classified as stained or unstained by inspection showed no overlap in nuclear absorbance. Suspensions not exposed to alloxan contained 70-80% of unstained cells. Alloxan markedly decreased the frequency of unstained cells, an effect counteracted by 5 or 20 mM-D-glucose. The spectrum of Trypan Blue in islet-cell nuclei was red-shifted by about 20 nm. A similar red-shift was observed on adding the dye to solutions of albumin or histones, but not on mixing the dye with DNA. Binding to basic proteins may explain the concentrative uptake of Trypan Blue in dead cells and contribute to the oncogenic transformation of phagocytotically active cells. Beta-Cells in vitro are killed by alloxan and hence represent a valid model for studying the diabetogenic action of the drug.

scholarly journals Cell-mediated cytotoxic islet cell surface antibodies to human pancreatic beta cells

Diabetologia ◽  
1984 ◽  
Vol 26 (1) ◽  
Author(s):  
T. Maruyama ◽  
I. Takei ◽  
I. Matsuba ◽  
A. Tsuruoka ◽  
M. Taniyama ◽  
...  
Keyword(s):  
Cell Surface ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Islet Cell ◽  
Islet Cell Surface Antibodies ◽  
Surface Antibodies

Reevaluation of Autoantibodies to Islet Cell Membrane in IDDM: Failure to Detect Islet Cell Surface Antibodies Using Human Islet Cells as Substrate

Diabetes ◽  
1992 ◽  
Vol 41 (12) ◽  
pp. 1624-1631 ◽  
Author(s):  
M. Vives ◽  
N. Somoza ◽  
G. Soldevila ◽  
R. Gomis ◽  
A. Lucas ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Cell Surface ◽  
Islet Cell ◽  
Islet Cells ◽  
Human Islet ◽  
Islet Cell Surface Antibodies ◽  
Surface Antibodies

scholarly journals Monoclonal antibodies to different epitopes on a cell-surface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold.

1987 ◽  
Vol 35 (11) ◽  
pp. 1277-1284 ◽  
Author(s):  
R Jemmerson ◽  
M Agre
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Colloidal Gold ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Placental Alkaline Phosphatase ◽  
Gold Particles ◽  
Human Placental Alkaline Phosphatase ◽  
Membrane Components

Two monoclonal antibodies (mAbs) to different epitopes on human placental alkaline phosphatase (PLAP), both of the immunoglobulin G2a heavy-chain class and having similar affinities for PLAP, were compared for their ability to label the enzyme on the HeLa cell surface. In one type of experiment employing [125I]-labeled mAbs, the results demonstrated quantitative differences in binding of the mAbs to the cells. At saturating levels, the number of molecules of mAb E5 bound to the cells was almost eight times the number of mAb B10 molecules bound. In another type of experiment, mAbs were indirectly visualized on the cell surface using protein A tagged with colloidal gold particles in transmission electron microscopy. Only one of the antibodies (E5) displayed a clustered distribution of PLAP that previously had been observed with rabbit polyclonal antibodies and goat anti-rabbit IgG-labeled gold (J Histochem Cytochem 33:1227, 1985). The other antibody (B10) showed less frequent and more scattered labeling; three to four times more gold particles were visualized in each cluster on cells bound by mAb E5 compared to cells bound by B10. These results are consistent with the idea that not all epitopes on a membrane-bound antigen may be equally accessible for antibody binding. Even identical epitopes on different PLAP molecules are not equally hindered by other membrane components, since at least some of the PLAP molecules are labeled by the more sterically hindered mAb B10. Quantification of the number of gold particles employing the more abundantly bound mAb E5 provides an average estimate of seven to eight molecules of PLAP in each cluster. Because of inefficiencies in labeling, however, this value is probably lower than the real number.

IgG subclasses of islet cell surface antibodies and their cytotoxic activity against pancreatic islet cells

1987 ◽  
Vol 3 (4) ◽  
pp. 215-220 ◽  
Author(s):  
Masao Suzuki ◽  
Shoji Kawazu ◽  
Kiyohiko Negishi ◽  
Toshiro Watanabe ◽  
Asanori Hokama ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cytotoxic Activity ◽  
Pancreatic Islet ◽  
Islet Cell ◽  
Islet Cells ◽  
Igg Subclasses ◽  
Pancreatic Islet Cells ◽  
Islet Cell Surface Antibodies ◽  
Surface Antibodies
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