Designing conditions for in vitro formation of amyloid protofilaments and fibrils.
(9/3010)
We have been able to convert a small alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 A in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions. (+info)
The degrees of plasma cell clonality and marrow infiltration adversely influence the prognosis of AL amyloidosis patients.
(10/3010)
BACKGROUND AND OBJECTIVE: Primary amyloidosis is a lethal form of plasma cell (PC) dyscrasia characterized by deposits of monoclonal immunoglobulin light chains that cause organ dysfunction. In contrast to multiple myeloma, the amyloid clone is typically indolent and of small size, and marrow PC clonality is not always apparent. This is generally investigated by analyzing the light chain isotype ratio in bone marrow PC. We investigated whether the degree of PC infiltration (PC%) and clonality (PC isotype ratio) affected survival in 56 consecutive patients with primary amyloidosis. DESIGN AND METHODS: PC% was determined by morphologic examination. Immunofluorescence microscopy was used to determine the PC light chain isotype ratio. Statistical analysis was carried out using Cox regression models. RESULTS: The degrees of PC clonality and infiltration were inversely correlated with survival (PC isotype ratio, p = 0.001; PC%, p = 0.008). The two variables were weakly correlated (p = 0.02; r = 0.3). Bone marrow PC isotype ratio demonstrated a powerful independent prognostic value at multivariate analysis when analyzed together with congestive heart failure (the major known negative prognostic factor) and PC%. k/l ratio cut-off values of 0.2 (l patients, p = 0.022) and 16 (k patients, p = 0.03) discriminated two groups with a similar number of patients and significantly different survivals. INTERPRETATION AND CONCLUSIONS: PC clonality and marrow infiltration are important parameters that influence prognosis, presumably because they reflect the amount of pathogenic light chain synthesis. (+info)
Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology.
(11/3010)
Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization. (+info)
Characterization of receptors for calcitonin gene-related peptide and adrenomedullin on the guinea-pig vas deferens.
(12/3010)
1. The receptors which mediate the effects of calcitonin gene-related peptide (CGRP), amylin and adrenomedullin on the guinea-pig vas deferens have been investigated. 2. All three peptides cause concentration dependant inhibitions of the electrically stimulated twitch response (pD2s for CGRP, amylin and adrenomedullin of 7.90+/-0.11, 7.70+/-0.19 and 7.25+/-0.10 respectively). 3. CGRP8-37 (1 microM) and AC187 (10 microM) showed little antagonist activity against adrenomedullin. 4. Adrenomedullin22-52 by itself inhibited the electrically stimulated contractions of the vas deferens and also antagonized the responses to CGRP, amylin and adrenomedullin. 5. [125I]-adrenomedullin labelled a single population of binding sites in vas deferens membranes with a pIC50 of 8.91 and a capacity of 643 fmol mg(-1). Its selectivity profile was adrenomedullin> AC187>CGRP=amylin. It was clearly distinct from a site labelled by [125I]-CGRP (pIC50=8.73, capacity=114 fmol mg(-1), selectivity CGRP>amylin=AC187>adrenomedullin). [125I]-amylin bound to two sites with a total capacity of 882 fmol mg(-1). 6. Although CGRP has been shown to act at a CGRP2 receptor on the vas deferens with low sensitivity to CGRP8-37, this antagonist displaced [125I]-CGRP with high affinity from vas deferens membranes. This affinity was unaltered by increasing the temperature from 4 degrees C to 25 degrees C, suggesting the anomalous behaviour of CGRP8-37 is not due to temperature differences between binding and functional assays. (+info)
Organ-specific (localized) synthesis of Ig light chain amyloid.
(13/3010)
Ig amyloidosis is usually a systemic disease with multisystem involvement. However, in a significant number of cases amyloid deposition is limited to one specific organ. It has not been determined if the Ig light chain (LC) amyloid precursor protein in localized amyloidosis is synthesized by circulating plasma cells with targeting of the amyloid fibril-forming process to one specific organ, or whether the synthesis of Ig LC and fibril formation occurs entirely as a localized process. In the present study local synthesis of an amyloid fibril precursor LC was investigated. Amyloid fibrils were isolated from a ureter that was obstructed by extensive infiltration of the wall with amyloid. Amino acid sequence analysis of the isolated fibril subunit protein proved it to be derived from a lambdaII Ig LC. Plasma cells within the lesion stained positively with labeled anti-lambda Ab and by in situ hybridization using an oligonucleotide probe specific for lambda-LC mRNA. RT-PCR of mRNA extracted from the tumor and direct DNA sequencing gave the nucleotide sequence coding specifically for the lambdaII amyloid subunit protein, thus confirming local synthesis of the LC protein. (+info)
Intracellular amyloidogenesis by human islet amyloid polypeptide induces apoptosis in COS-1 cells.
(14/3010)
Human islet amyloid polypeptide (hIAPP) is co-secreted with insulin from pancreatic islet beta cells. This peptide spontaneously aggregates in the form of fibrils, and amyloid deposits are associated with dead or degenerating beta cells, a hallmark of noninsulin-dependent diabetes mellitus. We demonstrated that COS-1 cells transfected with vectors expressing hIAPP exhibited intracellular amyloid deposits that were associated with cell death (O'Brien, Butler, Kreutter, Kane, Eberhardt, Am J Pathol 1995, 147:609-616). To establish the mechanism of cell death, we transfected COS-1 cells with vectors expressing amyloidogenic hIAPP or nonamyloidogenic rat IAPP and mutant hIAPP constructs and assayed them for markers characteristic of apoptosis and necrosis by fluorescence-activated cell sorting analysis. Amyloidogenic hIAPP-transfected COS cells contained up to threefold more apoptotic cells present at 96 hours after transfection compared with the nonamyloidogenic vector controls. The hIAPP-induced apoptosis was negligible at 24 and 48 hours after transfection and was maximal at 96 hours which parallels the time course of amyloidogenesis. Immunohistochemical staining and confocal microscopy showed that hIAPP is localized with distinct clustering in the endoplasmic reticulum and Golgi apparatus with no discernable extracellular staining. These experiments provide direct evidence that intracellular hIAPP amyloid causes cell death by triggering apoptotic pathways. (+info)
Islet amyloid: a long-recognized but underappreciated pathological feature of type 2 diabetes.
(15/3010)
Islet amyloid has been recognized as a pathological entity in type 2 diabetes since the turn of the century. It has as its unique component the islet beta-cell peptide islet amyloid polypeptide (IAPP), or amylin, which is cosecreted with insulin. In addition to this unique component, islet amyloid contains other proteins, such as apolipoprotein E and the heparan sulfate proteoglycan perlecan, which are typically observed in other forms of generalized and localized amyloid. Islet amyloid is observed at pathological examination in the vast majority of individuals with type 2 diabetes but is rarely observed in humans without disturbances of glucose metabolism. In contrast to IAPP from rodents, human IAPP has been shown to form amyloid fibrils in vitro. Because all human subjects produce and secrete the amyloidogenic form of IAPP, yet not all develop islet amyloid, some other factor(s) must be involved in islet amyloid formation. One hypothesis is that an alteration in beta-cell function resulting in a change in the production, processing, and/or secretion of IAPP is critical to the initial formation of islet amyloid fibrils in human diabetes. This nidus of amyloid fibrils then allows the progressive accumulation of IAPP-containing fibrils and the eventual replacement of beta-cell mass by amyloid and contributes to the development of hyperglycemia. One factor that may be involved in producing the changes in the beta-cell that result in the initiation of amyloid formation is the consumption of increased dietary fat. Dietary fat is known to alter islet beta-cell peptide production, processing, and secretion, and studies in transgenic mice expressing human IAPP support the operation of this mechanism. Further investigation using this and other models should provide insight into the mechanism(s) involved in islet amyloidogenesis and allow the development of therapeutic agents that inhibit or reverse amyloid fibril formation, with the goal being to preserve beta-cell function and improve glucose control in type 2 diabetes. (+info)
Metabolic effects of amylin in lactating goats.
(16/3010)
The objective of this study was to investigate the role of amylin (a pancreatic hormone) in regulating metabolism in support of lactation. Rat amylin was infused (320 pmol.kg LW(-1).h(-1)) for 6 h via an external pudic (mammary) artery into six lactating goats. This dose of amylin led to a sixfold increase in plasma concentrations of amylin relative to baseline. Amylin infusion increased plasma concentrations (jugular) of glucose and NEFA up to 16 and 168%, respectively, relative to saline infusion. In contrast, plasma concentrations of Ca and PO4 during amylin infusion were reduced by 18 and 30%, respectively, relative to saline infusion. Plasma concentrations of IGF-I, insulin, and Mg were not different between the two treatments, although IGF-I concentrations in the amylin-infused group, 1 and 6 h postinfusion, were significantly higher than those in the saline-infused group. Similarly, amylin infusion failed to affect milk yield and major constituents of milk except protein; milk protein content decreased progressively until the end of amylin infusion and remained low thereafter. Amylin also had no effect on minerals in milk (Ca, PO4, Mg, Fe, Sr, S, K, or Na) except Zn, which was significantly decreased from 56.8+/-5.8 micromol/L at 0 h to 44.5+/-2.4 micromol/L at 6 h postinfusion. Mammary blood flow (measured with a transit-time blood flow probe) increased up to 26% during amylin infusion, although this effect lasted only for the first 3 h. In conclusion, amylin increased plasma concentrations of glucose and NEFA, and mammary blood flow, while decreasing plasma concentrations of Ca and PO4. Despite these metabolic changes, amylin infusion did not increase milk yield of lactating goats. (+info)