Search Results - (Author, Cooperation:D. F. Steiner)

2013-01-11

Nature Publishing Group (NPG)

0028-0836

1476-4687

Biology

Chemistry and Pharmacology

Medicine

Natural Sciences in General

Physics

Animals ; Binding Sites ; Calorimetry ; Cattle ; Cell Line ; Crystallography, X-Ray ; Humans ; Insulin/*chemistry/*metabolism ; Leucine/metabolism ; Ligands ; Models, Molecular ; Protein Binding ; Protein Structure, Secondary ; Receptor, Insulin/*chemistry/*metabolism ; Reproducibility of Results

1471-4159

Blackwell Publishing Journal Backfiles 1879-2005

Medicine

Abstract: Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A-8 and a substantial reduction in the levels of Dyn A-17 and Dyn B-13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A-8, Dyn A-17, and Dyn B-13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild-type mice; however, the levels of Dyn peptides in these mice are similar to those of wild-type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up-regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild-type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.

Electronic Resource

1471-4159

Blackwell Publishing Journal Backfiles 1879-2005

Medicine

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.

Electronic Resource

1476-4687

Nature Archives 1869 - 2009

Biology

Chemistry and Pharmacology

Medicine

Natural Sciences in General

Physics

[Auszug] Immunoprecipitation and tryptic peptide analysis of newly synthesised proteins from rat islets have identified an 18,000 molecular weight (MW) protein as proglucagon. Conversion of this precursor was kinetically similar to the conversion of proinsulin and resulted in the formation of both ...

Electronic Resource

1476-4687

Nature Archives 1869 - 2009

Biology

Chemistry and Pharmacology

Medicine

Natural Sciences in General

Physics

[Auszug] We have recently studied the biosynthesis and structure of insulin in a cyclostome, the Atlantic hagfish (Myxine gluti-nosa)Q-w. The cyclostomes represent highly specialised survivors of the earliest vertebrates, the ostracaderms, and thus probably diverged from the gnathostomian vertebrates about ...

Electronic Resource

1476-4687

Nature Archives 1869 - 2009

Biology

Chemistry and Pharmacology

Medicine

Natural Sciences in General

Physics

[Auszug] Fig. 1 Double-antibody radioimmunoassay using bovine proinsulin antiserum and 125I-tyrosylated [59-formyllysine]-bovine proinsulin 31-60 as tracer. o , Synthetic [59-formyllysine]-bovine proinsulin 31-60; A, natural bovine C-peptide; O, natural bovine proinsulin. The peptides were constructed ...

Electronic Resource

1476-4687

Nature Archives 1869 - 2009

Biology

Chemistry and Pharmacology

Medicine

Natural Sciences in General

Physics

[Auszug] WORK with a wide variety of secretory proteins has by now amply substantiated the 'signal hypothesis'. Proteins that are eventually going to be secreted are synthesised on ribosomes attached to the endoplasmic reticulum (ER) and transferred into the lumen of the ER from where they are finally ...

Electronic Resource

1476-4687

Nature Archives 1869 - 2009

Biology

Chemistry and Pharmacology

Medicine

Natural Sciences in General

Physics

[Auszug] Fig. 1 Hypothetical representation of a proinsulin hexamer viewed along the three-fold axis of the hexamer. The central density represents the two zinc atoms which coordinate to the six histidine side chains at position 10 in the B chain. The densities surrounding the zinc atoms and extending ...

Electronic Resource

1432-0428

Islet amyloid polypeptide precursors ; monkey (Macaca nemestrina) ; dog (Canis familiaris) ; amyloidogenic properties

Springer Online Journal Archives 1860-2000

Medicine

Summary The 37-amino acid islet amyloid polypeptide represents the major protein component present in islet amyloid deposits. Although the presence of islet amyloid is a characteristic pathological feature of the islets of humans, monkeys and cats with Type 2 (non-insulin-dependent) diabetes mellitus, it is not found in the islets of diabetic rats, mice or dogs. To further explore the molecular basis for these species differences in amyloid deposition we have used a polymerase chain reaction based method to clone cDNAs encoding the monkey (Macaca nemestrina) and dog (Canis familiaris) islet amyloid polypeptide precursors. The predicted amino acid sequence of the monkey precursor is 96% identical to that of the human protein; differences include one replacement in the signal peptide and three in the islet amyloid polypeptide domain. The sequence of the dog precursor is most closely related to that of the cat protein (85% identity); the sequences of dog and cat islet amyloid polypeptide differ only at two positions and are identical in the region of amino acids 20–29, the region thought to be primarily responsible for amyloidogenesis. Thus, amino acid residues in addition to those at positions 20–29 may facilitate the aggregation of islet amyloid polypeptide. The presence of amyloid deposits in some dog pancreatic endocrine tumours suggests that the dog protein can be amyloidogenic, perhaps due to elevated expression of islet amyloid polypeptide by the tumours relative to normal islets.

Electronic Resource

1432-0428

Insulin gene ; expression ; precursor processing ; glucokinase ; glucose transport ; signal transduction

Springer Online Journal Archives 1860-2000

Medicine

Summary Considerable progress has been made in our understanding of islet-cell function and its relationship to regulation of whole body glucose metabolism. At the genetic level, the regulatory regions in islet-specific genes are being characterised. Transcription factors that interact with these regions have been cloned and these will be instructive in elucidating how islet-specific genes are regulated during development and regeneration. Identification of the enzymes responsible for proteolytic conversion of proinsulin to insulin represents a major advance in understanding prohormone processing. Cleavage of proinsulin is mediated by at least two prohormone convertases (PC3/PC1 and PC2). Their activity is regulated by an acidic gradient between the Golgi and secretory granules and by calcium ions. It is not yet clear how insulin or the PC's are specifically diverted into the regulated secretory pathway. Regulation at this step may be defective in some diabetic patients resulting in relatively elevated circulating proinsulin levels. Specific features of GLUT 2 and glucokinase (GK), proteins that regulate Beta-cell glucose transport and phosphorylation, indicate that these may be key components of the glucose sensor. GLUT 2 is necessary to reconstitute glucose-sensitive insulin secretion in pituitary tumour cells expressing a proinsulin cDNA. Furthermore, the expression of GLUT 2 in Beta cells, but not in hepatocytes, is decreased in diabetes mellitus. However, under normal circumstances GK is probably rate limiting for Beta-cell glucose utilisation. Thus, it is likely that both GLUT 2 and GK determine the set point for glucose-stimulated insulin secretion. Elucidation of distal effectors that regulate insulin secretion is also crucial to our understanding of Beta-cell function. Elevations in cytosolic Ca2+ are essential for stimulus secretion coupling. A novel second messenger, cyclic ADP ribose, has been implicated as a regulator of glucose-stimulated Ca2+ release from the Beta-cell endoplasmic reticulum. These and other recent advances provide optimism for the ultimate development of an artificial Beta cell capable of making insulin and releasing it in response to glucose.

Electronic Resource

1432-0428

Pancreatic islet cells ; cell suspensions ; islet cell surface antibodies ; cell surface immunofluorescence ; Protein A radioassay ; cell surface antigens ; autoimmunity ; diabetes mellitus

Springer Online Journal Archives 1860-2000

Medicine

Summary Rabbits were immunised with suspensions of viable, insulin-producing islet cells prepared from collagenase-isolated rat or ob/ob mouse pancreatic islets. Antibodies reactive with the surface of dispersed rat islet cells were present in both the rabbit anti-rat and the rabbit anti -ob/ob mouse islet sera as revealed by indirect immunofluorescence or by a radioligandassay using 125I-Protein A as a measure of cell bound IgG. In a competition assay the binding of 125I-Protein A was displaced in a concentration dependent manner by non-radioactive Protein A. Maximal displacement was found at concentrations of Protein A higher than 0.1 μg. added to 105 islet cells. Although not always detected by immunofluorescence there was a several-fold increase above normal rabbit serum of 125I-Protein A-binding to rat hepatocytes and spleen lymphocytes incubated with the islet cell antisera. Conversely, rabbit antisera against rat spleen lymphocytes or against a rat liver plasma membrane preparation reacted with rat islet cells. The rabbit anti-rat islet cell antiserum was absorbed to both spleen lymphocytes and hepatocytes until there was no binding of 125I-Protein A to either cell type. Islet specific antibodies were still present since this doubly absorbed antiserum induced cell surface immunofluorescence as well as 125I-Protein A-binding to rat islet cells. It is concluded that apart from common antigenic determinants immunisation with viable islet cells induces formation of antibodies directed against specific islet cell surface components.

Electronic Resource

1432-0428

Insulin ; mutant ; familial ; gene ; high performance liquid chromatography

Springer Online Journal Archives 1860-2000

Medicine

Summary We describe a family from Japan displaying the mutant insulin syndrome with hyperinsulinaemia and an increased insulin: C-peptide molar ratio. Serum insulin isolated from several family members showed reduced in vitro biological activity, and analysis by high performance liquid chromatography revealed a peak co-eluting with human insulin and a second species of increased hydrophobicity co-migrating with the previously reported Insulin Wakayama. The insulin genes from the propositus were cloned and sequenced, revealing one normal allele; the second allele, encoding a leucine for valine amino acid substitution at position 3 of the insulin A chain, was similar to that previously described for Insulin Wakayama. Synthesized [LeuA3] insulin showed 0.14% of receptor binding activity on rat adipocytes and a 10-fold prolonged half-life in a somatostatin-infused dog compared with human insulin. The finding of the same mutant gene in two unrelated Japanese families suggests that Insulin Wakayama may be discovered in additional Japanese families with hyperinsulinaemia and/or diabetes.

Electronic Resource

1432-0428

Islet amyloid polypeptide ; Type 2 (non-insulindependent) diabetes mellitus ; polymerase chain reaction ; direct sequencing ; maturity onset diabetes mellitus of the young (MODY) ; Pima Indian

Springer Online Journal Archives 1860-2000

Medicine

Summary Islet amyloid polypeptide is the major protein component of the islet amyloid of patients with Type 2 (non-insulin-dependent) diabetes mellitus. Since the synthesis of a structurally abnormal or mutant protein may contribute to the formation of amyloid deposits, we have examined the possibility that a mutant form of islet amyloid polypeptide or its precursor contributes to the formation of islet amyloid in Type 2 diabetic patients. We have sequenced the islet amyloid polypeptide precursor coding regions of the gene of 25 patients with Type 2 diabetes. Genomic DNA fragments corresponding to exon 2 and 3 of the islet amyloid polypeptide gene were amplified from patients' peripheral blood leucocyte DNAs using the polymerase chain reaction and specific oligonucleotide primer sets, and then directly sequenced. The nucleotide sequences of the amplified regions of both alleles of the islet amyloid polypeptide gene of these 25 patients were identical to one another and to the sequence of an islet amyloid polypeptide allele isolated from a human fetal liver genomic library. These findings suggest that a primary structural abnormality of islet amyloid polypeptide or its precursor is unlikely to play a significant role in the formation of islet amyloid in Type 2 diabetic patients.

Electronic Resource

1058-8388

Life and Medical Sciences ; Cell & Developmental Biology

Wiley InterScience Backfile Collection 1832-2000

Medicine

Electronic Resource