Islet amyloid polypeptide/amylin messenger RNA and protein expression in human insulinomas in relation to amyloid formation.
(1/3010)
OBJECTIVE: Islet amyloid polypeptide (IAPP), also named amylin, is the predominant protein component of amyloid deposits in human islet beta cell tumours of the pancreas (insulinomas). IAPP is co-produced with insulin by islet beta cells. We investigated IAPP expression in relation to insulin expression and to amyloid formation in eleven insulinomas. DESIGN AND METHODS: RNA and protein extracts were prepared from the same pieces of tumour tissue, and from specimens of two normal human pancreata. IAPP and insulin mRNA and peptide content were quantified using Northern blot analysis and radioimmunoassay (RIA) respectively. Molecular forms of IAPP immunoreactivity were analysed by reversed-phase high-performance liquid chromatography (HPLC). The presence of islet hormones and of amyloid was assessed by (immuno)histochemical staining of paraffin sections. Plasma levels of IAPP and insulin prior to tumour resection were determined by RIA. RESULTS: IAPP and insulin mRNA and peptide content varied widely between the tumour specimens, and there was considerable intratumour heterogeneity of peptide content. HPLC analysis indicated correct proteolytic processing of the IAPP precursor protein. Amyloid deposits were detected only in the three tumours with the highest IAPP content. In contrast to insulin, plasma levels of IAPP were not elevated in the insulinoma patients. CONCLUSIONS: The spectrum of hormone production by insulinomas cannot be inferred from only a few tissue sections due to intratumour heterogeneity. Expression of the IAPP and insulin genes is not coupled in insulinomas, which produce properly processed mature IAPP. In addition to IAPP overproduction, additional factors such as intracellular accumulation of IAPP are involved in amyloidogenesis in insulinomas. (+info)
Prion domain initiation of amyloid formation in vitro from native Ure2p.
(2/3010)
The [URE3] non-Mendelian genetic element of Saccharomyces cerevisiae is an infectious protein (prion) form of Ure2p, a regulator of nitrogen catabolism. Here, synthetic Ure2p1-65 were shown to polymerize to form filaments 40 to 45 angstroms in diameter with more than 60 percent beta sheet. Ure2p1-65 specifically induced full-length native Ure2p to copolymerize under conditions where native Ure2p alone did not polymerize. Like Ure2p in extracts of [URE3] strains, these 180- to 220-angstrom-diameter filaments were protease resistant. The Ure2p1-65-Ure2p cofilaments could seed polymerization of native Ure2p to form thicker, less regular filaments. All filaments stained with Congo Red to produce the green birefringence typical of amyloid. This self-propagating amyloid formation can explain the properties of [URE3]. (+info)
Interaction of amylin with calcitonin gene-related peptide receptors in the microvasculature of the hamster cheek pouch in vivo.
(3/3010)
1. This study used intravital microscopy to investigate the receptors stimulated by amylin which shares around 50% sequence homology with the vasodilator calcitonin gene-related peptide (CGRP) in the hamster cheek pouch microvasculature in vivo. 2. Receptor agonists dilated arterioles (diameters 20-40 microm). The -log of the concentrations (+/- s.e.mean; n = 8) causing 50% increase in arteriole diameter were: human betaCGRP (10.8 +/- 0.3), human alphaCGRP (10.8 +/- 0.4), rat alphaCGRP (10.4 +/- 0.3). Rat amylin and the CGRP2 receptor selective agonist [Cys(ACM2,7]-human alphaCGRP were 100 fold less potent (estimates were 8.5 +/- 0.4 and 8.2 +/- 0.3 respectively). 3. The GCRP1 receptor antagonist, CGRP8-37 (300 nmol kg(-1); i.v.) reversibly inhibited the increase in diameter evoked by human alphaCGRP (0.3 nM) from 178 +/- 22% to 59 +/- 12% (n = 8; P < 0.05) and by rat amylin (100 nM) from 138 +/- 23% to 68 +/- 24% (n = 6; P < 0.05). CGRP8-37 did not inhibit vasodilation evoked by substance P (10 nM; n = 4: P > 0.05). 4. The amylin receptor antagonist, amylin8-37 (300 nmol kg(-1); i.v.) did not significantly inhibit the increase in diameter evoked by human alphaCGRP (0.3 nM) which was 112 +/- 26% in the absence, and 90 +/- 29% in the presence of antagonist (n = 4; P < 0.05); nor that evoked by rat amylin (100 nM) which was 146 +/- 23% in the absence and 144 +/- 32% in the presence of antagonist (n = 4; P > 0.05). 5. The agonist profile for vasodilatation and the inhibition of this dilatation by CGRP8-37, although not the amylin8-37 indicates that amylin causes vasodilatation through interaction with CGRP1 receptors in the hamster cheek pouch. (+info)
The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles.
(4/3010)
NIDDM is characterized by islet amyloid deposits and decreased beta-cell mass. Islet amyloid is derived from the locally expressed protein islet amyloid polypeptide (IAPP). While it is now widely accepted that abnormal aggregation of IAPP has a role in beta-cell death in NIDDM, the mechanism remains unknown. We hypothesized that small IAPP aggregates, rather than mature large amyloid deposits, are cytotoxic. Consistent with this hypothesis, freshly dissolved human (h)-IAPP was cytotoxic when added to dispersed mouse and human islet cells, provoking the formation of abnormal vesicle-like membrane structures in association with vacuolization and cell death. Human islet cell death occurred by both apoptosis and necrosis, predominantly between 24 and 48 h after exposure to h-IAPP. In contrast, the addition to dispersed islet cells of matured h-IAPP containing large amyloid deposits of organized fibrils was seldom associated with vesicle-like structures or features of cell death, even though the cells were often encased in the larger amyloid deposits. Based on these observations, we hypothesized that h-IAPP cytotoxicity is mediated by membrane damage induced by early h-IAPP aggregates. Consistent with this hypothesis, application of freshly dissolved h-IAPP to voltage-clamped planar bilayer membranes (a cell-free in vitro system) also caused membrane instability manifested as a marked increase in conductance, increased membrane electrical noise, and accelerated membrane breakage, effects that were absent using matured h-IAPP or rat IAPP solutions. Light-scattering techniques showed that membrane toxicity corresponded to h-IAPP aggregates containing approximately 25-6,000 IAPP molecules, an intermediate-sized amyloid particle that we term intermediate-sized toxic amyloid particles (ISTAPs). We conclude that freshly dissolved h-IAPP is cytotoxic and that this cytotoxicity is mediated through an interaction of ISTAPs with cellular membranes. Once ISTAPs mature into amyloid deposits comprising >10(6) molecules, the capacity of h-IAPP to cause membrane instability and islet cell death is significantly reduced or abolished. These data may have implications for the mechanism of cell death in other diseases characterized by local amyloid formation (such as Alzheimer's disease). (+info)
Specific gene expression in pancreatic beta-cells: cloning and characterization of differentially expressed genes.
(5/3010)
Identification and characterization of genes expressed preferentially in pancreatic beta-cells will clarify the mechanisms involved in the specialized properties of these cells, as well as providing new markers of the development of type 1 diabetes. Despite major efforts, relatively few beta-cell-specific genes have been characterized. We applied representational difference analysis to identify genes expressed selectively in the pancreatic beta-cell line betaTC1 compared with the pancreatic alpha-cell line alphaTC1 and isolated 26 clones expressed at higher levels in the beta-cells than in the alpha-cells. DNA sequencing revealed that 14 corresponded to known genes (that is, present in GenBank). Only four of those genes had been shown previously to be expressed at higher levels in beta-cells (insulin, islet amyloid polypeptide, neuronatin, and protein kinase A regulatory subunit [RIalpha]). The known genes include transcription factors (STAT6) and mediators of signal transduction (guanylate cyclase). The remaining 12 genes are absent from the GenBank database or are present as expressed sequence tag (EST) sequences (4 clones). Some of the genes are expressed in a highly specific pattern-expression in betaTC1 and islet cells and in relatively few of the non-beta-cell types examined; others are expressed in most cell types tested. The identification of these differentially expressed genes may aid in attaining a clearer understanding of the mechanisms involved in beta-cell function and of the possible immunogens involved in development of type 1 diabetes. (+info)
Colchicine inhibition of the first phase of amyloid synthesis in experimental animals.
(6/3010)
Colchicine was found to inhibit the first phase of casein-induced synthesis of murine amyloid. When mice were treated with colchicine during the first 7 days of an amyloid induction regimen or when colchicine was given to the donor mice in a transfer model, the amyloidogenic stimulus of casein was blocked completely. Amyloid synthesis was however, not interrupted by the administration of colchicine during the last 7 days of the casein regimen nor by colchicine treatment of recipient mice in a transfer model. (+info)
Ancestral origins and worldwide distribution of the PRNP 200K mutation causing familial Creutzfeldt-Jakob disease.
(7/3010)
Creutzfeldt-Jakob disease (CJD) belongs to a group of prion diseases that may be infectious, sporadic, or hereditary. The 200K point mutation in the PRNP gene is the most frequent cause of hereditary CJD, accounting for >70% of families with CJD worldwide. Prevalence of the 200K variant of familial CJD is especially high in Slovakia, Chile, and Italy, and among populations of Libyan and Tunisian Jews. To study ancestral origins of the 200K mutation-associated chromosomes, we selected microsatellite markers flanking the PRNP gene on chromosome 20p12-pter and an intragenic single-nucleotide polymorphism at the PRNP codon 129. Haplotypes were constructed for 62 CJD families originating from 11 world populations. The results show that Libyan, Tunisian, Italian, Chilean, and Spanish families share a major haplotype, suggesting that the 200K mutation may have originated from a single mutational event, perhaps in Spain, and spread to all these populations with Sephardic migrants expelled from Spain in the Middle Ages. Slovakian families and a family of Polish origin show another unique haplotype. The haplotypes in families from Germany, Sicily, Austria, and Japan are different from the Mediterranean or eastern European haplotypes. On the basis of this study, we conclude that founder effect and independent mutational events are responsible for the current geographic distribution of hereditary CJD associated with the 200K mutation. (+info)
Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains.
(8/3010)
The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant kappa4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V(L) from a benign light chain. The amyloidogenic V(L)s were significantly less stable than the benign V(L). Furthermore, only the amyloidogenic V(L)s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V(L) could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V(L) that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V(L) beta-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation. (+info)