Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach

1989 ◽  
Vol 185 (2-3) ◽  
pp. 205-216 ◽  
Author(s):  
Mo�se Bendayan
Keyword(s):  
High Resolution ◽  
B Cell ◽  
Protein A ◽  
Ultrastructural Localization ◽  
C Peptide ◽  
Pancreatic B Cell ◽  
Antigenic Sites

Glucose intolerance in thyrotoxicosis roles of insulin, glucagon and somatostatin

1987 ◽  
Vol 114 (2) ◽  
pp. 228-234 ◽  
Author(s):  
Karen S. L. Lam ◽  
Rose T. T. Yeung ◽  
Patricia W. M. Ho ◽  
S. K. Lam
Keyword(s):  
B Cell ◽  
Glucose Intolerance ◽  
Plasma Glucose ◽  
Insulin Response ◽  
Oral Glucose ◽  
Insulinogenic Index ◽  
C Peptide ◽  
Pancreatic B Cell ◽  
Significant Difference ◽  
Glucose Ingestion

Abstract. The responses in plasma glucose, insulin, C-peptide, glucagon and somatostatin to an oral glucose load were studied in 10 thyrotoxic patients and 10 matched euthyroid controls. The thyrotoxic patients had higher mean fasting plasma glucose (P < 0.05) and responded to oral glucose with an earlier peak at 30 min which was higher than the corresponding glucose level in the controls (P < 0.05). Impaired glucose tolerance was found in 3 patients. Fasting insulin and C-peptide levels were normal in the thyrotoxic patients when corrected for the higher glucose levels. Following glucose ingestion, there was no significant difference between the areas under the insulin or C-peptide curves in patients and controls, but Seltzer's insulinogenic index was reduced in the patients (P < 0.01) suggesting an impaired pancreatic B-cell response to oral glucose. Mean basal glucagon was normal in the thyrotoxic patients. However, while in the controls plasma glucagon became suppressed following glucose ingestion (P < 0.0001), no significant suppression was found in the patients. In the thyrotoxic patients, mean basal somatostatin was normal, but the area under the somatostatin curve following glucose ingestion was significantly increased (P < 0.02). Our findings suggest that décreased glucagon suppression and impaired insulin response after glucose ingestion are involved in glucose intolerance in thyrotoxicosis. Enhanced somatostatin responses to oral glucose in thyrotoxicosis may have contributed to the observed impairment in pancreatic B-cell responsiveness.

Ultrastructural localization of serotonin and polypeptide YY (PYY) in endocrine cells of the human rectum.

1986 ◽  
Vol 34 (6) ◽  
pp. 719-726 ◽  
Author(s):  
A I Lukinius ◽  
J L Ericsson ◽  
M K Lundqvist ◽  
E M Wilander
Keyword(s):  
Colloidal Gold ◽  
Endocrine Cells ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Enterochromaffin Cells ◽  
Cell Type ◽  
Low Concentration ◽  
Antigenic Sites ◽  
Human Rectum ◽  
Ultrathin Sections

This study was performed with the aim of ultrastructurally localizing serotonin and polypeptide YY (PYY) in the endocrine cells of the human rectum. Existing basic methods for immunolocalization of antigenic sites in ultrathin sections were tested and modified to allow reproducible results with distinct localization of marker (colloidal gold probes coupled either to IgG or protein A). Probes signifying presence of serotonin were distinctly localized over all heteromorphous granules in argentaffin cells and, in addition, over some of the more monomorphous, rounded granules in a second cell type whose granules all were covered by probes showing localization of the PYY antigen. The results suggest that serotonin in endocrine cells of the gut is not confined to the enterochromaffin type but may also be present in trace amounts in non-enterochromaffin endocrine cells storing peptide hormones. Since probes marking sites of PYY were deposited over some heteromorphous granules in enterochromaffin cells, the evidence obtained also suggests that PYY may occur in low concentration in these cells. The distribution of probes in the sections indicated that antigenic sites were confined to granules in the cells.

scholarly journals Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.

1983 ◽  
Vol 31 (1) ◽  
pp. 101-109 ◽  
Author(s):  
M Bendayan ◽  
M Zollinger
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Liver Mitochondria ◽  
Secretory Proteins ◽  
Additional Advantage ◽  
Sodium Metaperiodate ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Structural Preservation

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.

scholarly journals Association of secreted insulin with particular domains of the pancreatic B-cell plasma membrane: the actin-rich microvilli.

1992 ◽  
Vol 40 (3) ◽  
pp. 327-331 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Plasma Membrane ◽  
B Cell ◽  
Binding Sites ◽  
Glucose Transporter ◽  
Protein A ◽  
Secretory Activity ◽  
Insulin Binding ◽  
Functional Roles ◽  
Pancreatic B Cell ◽  
Cell Plasma

Association of insulin with the plasma membrane of the pancreatic B-cell was revealed using the high-resolution protein A-gold immunocytochemical approach. The labeling was found to be heterogeneously distributed, the plasma membrane of the actin-rich microvilli being preferentially labeled. Morphometrical evaluation of labeling intensities confirmed the microvilli location of the insulin binding sites. This membrane domain, which also displays the glucose transporter, therefore appears to play important functional roles in the secretory activity of the B-cell. That the autocrine influence insulin exerts on its own secretion is receptor mediated through these insulin binding sites remains, however, to be determined.

scholarly journals Protein G-gold complex: comparative evaluation with protein A-gold for high-resolution immunocytochemistry.

1988 ◽  
Vol 36 (6) ◽  
pp. 597-607 ◽  
Author(s):  
M Bendayan ◽  
S Garzon
Keyword(s):  
Monoclonal Antibodies ◽  
High Resolution ◽  
Guinea Pig ◽  
Comparative Evaluation ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Gold Complex ◽  
Protein G ◽  
Specific Antibodies ◽  
Antigenic Sites

We combined the protein G-gold complex with several polyclonal and monoclonal antibodies for localization of various antigenic sites. The labelings were compared with those obtained using the protein A-gold complex. The results from either the immunodot experiment or immunoelectron microscopy have demonstrated that, for rabbit and guinea pig antibodies, both protein G-gold and protein A-gold complexes label several different specific antibodies with similar efficiency. However, with antibodies raised in goats or in mice, and particularly with mouse monoclonal antibodies, protein G-gold yielded intense and specific labeling, whereas protein A-gold yielded intense and specific labeling, whereas protein A-gold was very variable; it either gave weaker signals or failed to reveal any specific site or, as with one monoclonal, both protein G and protein A gave similar results. The higher affinity and versatility of protein G over protein A, established by the immunochemical approach, was confirmed by immunocytochemistry. Because of its enhanced reactivity with monoclonal antibodies and its broader affinity for polyclonal antibodies, protein G-gold complex appears to be a better and more versatile probe for high-resolution immunocytochemistry.

scholarly journals Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell.

1984 ◽  
Vol 98 (1) ◽  
pp. 222-228 ◽  
Author(s):  
L Orci ◽  
M Ravazzola ◽  
M Amherdt ◽  
C Yanaihara ◽  
N Yanaihara ◽  
...  
Keyword(s):  
B Cell ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Core Material ◽  
Insulin Degradation ◽  
C Peptide ◽  
Lysosomal Compartment ◽  
Pancreatic B Cell ◽  
Ready Access ◽  
Gain Access

We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded. Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C-peptide, immunoreactivity was detectable. The absence of C-peptide (proinsulin) immunoreactivity in multigranular bodies, i.e., in early morphological stages of lysosomal digestion, was compatible with the ready access and breakdown of C-peptide and/or proinsulin by lysosomal degrading enzymes, while the insulin crystallized in secretory granule cores remained relatively protected. However, in the final stage of lysosomal digestion, i.e., in residual bodies where the secretory granule core material is no longer present, insulin immunoreactivity became undetectable. Lysosomal digestion thus appears to be a normal pathway for insulin degradation in the pancreatic B-cell.

scholarly journals Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

1978 ◽  
Vol 26 (12) ◽  
pp. 1074-1081 ◽  
Author(s):  
J Roth ◽  
M Bendayan ◽  
L Orci
Keyword(s):  
Endocrine Pancreas ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Gold Complex ◽  
Secretory Proteins ◽  
Staphylococcal Protein A ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Step Procedure ◽  
General Method

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.

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