Abstract: Amyloid beta protein deposition is a universal feature of Alzheimer's disease brain. To investigate the effects of amyloid beta protein in aged primates, intracerebral microinjections of solubilized amyloid beta (A beta (1-40)) and control peptides were made into the frontal cortex of 7 primates under stereotactic guidance. Control injections consisted of vehicle alone, a 37 amino acid non toxic peptide (A37), scrambled peptide (CA4), and reverse peptide (A beta (40-1)). Amyloid beta peptide produced dose-dependent cortical lesions that were significantly larger than those produced by vehicle or by isomolar control peptides (3.28 and 2.20 fold larger respectively) (p = < 0.005). In 5 aged primates, the cortex surrounding the amyloid beta lesions contained argyrophilic, thioflavine S fluorescent, Alz 50 and ubiquitin immunoreactive neurons and perikarya. The number of Alz 50 immunoreactive neurons surrounding the amyloid beta injections was significantly greater (mean 127 +/- 39) than the number found surrounding reverse peptide injections (mean 20 +/- 13) and other control peptides (mean 0.8 +/- 0.3) (p < 0.05). Neuronal and neuritic alterations were not found adjacent to the amyloid beta peptide lesions in young monkeys and control injections produced insignificant Alz 50 neuronal positivity. These findings suggest that amyloid beta peptide is neurotoxic in primate brain and that the cytoskeletal response to amyloid beta protein is specific and age-related.

Abstract: beta-amyloid peptide (A beta) and several A beta-fragments decrease the fluidity of human cortex membranes in a concentration dependent fashion. The effect of A beta on membrane fluidity increases with peptide length, is most pronounced for A beta 1-43 and can be seen at concentrations as low as 100 nmol/l. While the fragment A beta 25-35 is active, scrambled peptide (A beta 35-25) when investigated under similar conditions shows no effects on membrane fluidity. The effect of A beta peptides on fluidity of the phospholipid bilayer is more pronounced in the hydrocarbon core (labeled with the fluorescence probe 1,6-diphenylhexa-1,3,5-triene) than in the region of the hydrophilic heads (labeled with the fluorescence probe 1-[4'-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene). It is suggested that the effect of A beta on neuronal membranes is probably a major initial mechanism in a cascade of events finally leading to neurotoxicity and cell death in Alzheimer's disease.

Abstract: The most common form of hereditary systemic amyloidosis is familial amyloidotic poly neuropathy associated with single amino acid changes in the plasma protein transthyretin.So far, high resolution structures of only three amyloidogenic variants (Met30, Ser84, tle 122) and one non-amyloidogenic variant (Thr109) have been reported complemented by X-ray fiber diffraction studies and image reconstruction from electron micrographs of amyloid fibrilsTo investigate the role of structural factors in this disease, we extended our studies to other transthyretin variants. We report crystallization and structural investigations of three amyloidogenic (Arg10, Ala60, Tyr77) and two non-amyloidogenic variants (Ser6, Met119). The similarity oj these structures to normal transthyretin does not give direct clues to the fibril forming process. Since transthyretin amyloid fibrils contain a major fragment starting at position 49, besides the intact molecule, we calculated the solvent accessibility of residue 48. Indeed, all ...

Abstract: Familial amyloidotic polyneuropathy (FAP) is characterized by deposits of amyloid fibers in which the major protein component is transthyretin (TTR). Nearly fifty mutations have been reported for the TTR in hereditary FAP. Protein crystallography of mutant TTRs has shown that the molecular structures of the variant molecules are similar to those found in the wild type. On this basis, the FAP fibers were initially proposed to consist of native-like TTR tetramers. In the current paper, we used x-ray fiber diffraction to study the structure of FAP fibers from biopsy samples of vitreous humor and kidney. The reflections of the vitreous sample showed a cross-beta diffraction pattern. All the meridional reflections were indexed by a one-dimensional, 29 A-period lattice, and the equatorial reflections were indexed by an apparent one-dimensional 67 A-period lattice. The x-ray intensity distribution indicated that the unit structure, which is similar to a TTR monomer, is composed of a pair of beta-sheets consisting of four hydrogen-bonded beta-chains per sheet, with the beta-chains oriented approximately normal to the fiber axis. The axial disposition of these units, with a 29 A-period, constitutes the protofilament; and a tetrameric lateral assembly of the protofilaments containing the core domain of the approximately 20 A-wide beta-sheet structure constitutes the FAP amyloid fiber. An inter-fiber separation of 75 A in these concentrated samples accounts for the apparent one-dimensional lattice perpendicular to the fiber axis. In the delipidated kidney FAP sample, the diffraction pattern indicated a pair of beta-sheets, suggesting that the protofilament structure in kidney is similar to that in vitreous humor. In the non-delipidated sample the successive sharp reflections indexed to a one-dimensional, 48.9 A-lattice, and the electron density projection showed a density elevation at the center of a lipid bilayer. This suggests that lipid may be associated with the monomeric TTR in the kidney FAP protofilament.

Abstract: The basic pathogenesis of numerous neurodegenerative disorders is now thought to be related to abnormal protein conformation. The common theme in all these diseases is the conversion of a normal cellular andor circulating protein into an insoluble, aggregated, β-sheet rich form which is deposited in the brain, sometimes in the form of amyloid. These deposits are toxic and produce neuronal dysfunction and death. The most common of these illnesses is Alzheimer's disease (AD), in which a central event is the conversion of the normal soluble amyloid p (SAP) peptide to amyloid p (AP) within neuritic plaques and cerebral vessels. A unique category of the conformational conditions areprion related diseases (or prionoses), where the etiology is thought to be related to conversion of the normal prion protein, PrPC, into an infectious and pathogenic form, PrPSc. In the case of AD and the prionoses, the conformational change can be influenced by the presence of mutations in various gene products, as well as by chape...

Abstract: Islet amyloid formed from islet amyloid polypeptide (IAPP, amylin) is found in spontaneously diabetic monkeys and cats. Islet amyloidosis is progressive, apparently irreversible and is associated with destruction of insulin-secreting cells. The role of macrophages in the destruction and removal of islet amyloid is unknown. Therefore, the presence and morphology of macrophages were determined by electron and quantitative light microscopy in islets of diabetic and nondiabetic man and monkeys and in transgenic mice expressing the gene for human IAPP. Tissue macrophages were present in all pancreatic sections and tissue distribution was similar in exocrine and endocrine areas. There was no difference in macrophage density in amyloidotic and amyloid-free islets in monkeys and man. Macrophage density was similar in islets of transgenic mice expressing human IAPP which do not contain amyloid in vivo but in which fibrils are formed in vitro following islet isolation compared to islets from mice expressing rat IAP...

Abstract: Amyloid fibril formation is believed to be a nucleation-dependent polymerization process which may be influenced by various other factors with important consequences for the development, prevention or treatment of amyloidosis. We have previously shown that laminin inhibits A beta peptide fibril formation in vitro. Here we present a kinetic study that indicates laminin to be a potent anti-amyloidosis factor, as it not only inhibited A beta 1-40 fibril aggregation, but also inhibited the aggregation of the Dutch A beta 1-40 variant, a peptide with a higher capacity to aggregate than the wild-type A beta 1-40. The inhibitory effect of laminin on amyloid fibril formation was not overcome by the addition of pre-formed A beta fibrils, suggesting that laminin inhibits the fibril elongation process. At the present time, however, we cannot rule out the possibility that laminin also affects the initial nucleation process of A beta fibril formation. On other hand, laminin was not able to counteract the amyloid fibril formation promoted by acetylcholinesterase (AChE), another component of the amyloid deposits found in AD brains. The effect of laminin may be important as an inhibitor of A beta amyloidogenesis in vivo, specifically at the level of cerebral blood vessels.

Abstract: Quantitative immunohistochemical analysis of islet amyloid polypeptide (IAPP) in normal, impaired glucose tolerant, and diabetic cats.

Abstract: There is an inconsistency between the ultrastructural organization of AA amyloid fibrils that have been isolated, which are composed of a slowly twisting set of two or more protofibrils, and those seen in situ, which are tubular entities with a tight helical substructure. In this study, the ultrastructure of fibrils isolated from experimental murine AA amyloid were observed at high resolution and compared with those seen in situ in the hope of clarifying the reason for this inconsistency. The fibrils in situ were composed of a microfibril-like 8-9 nm wide core covered by a layer of heparan sulfate proteoglycan (HSPG) to which 1 nm wide filaments, immunohistochemically identified as AA protein, were externally associated. Following isolation with the standard distilled water washing procedure, the HSPG layer and AA protein filaments detached from their core and dispersed into the water. The remaining denuded, variously loosened cores lost their typical appearance. In distilled water the detached 1 nm wide AA protein filaments became quite conspicuous and coiled themselves into 3 nm wide tight helices which in turn assembled into the characteristic slowly twisting sets of two parallel protofibrils similar to that previously reported as "isolated amyloid fibrils". The results emphasize that great caution must be taken in extrapolating amyloid fibril structure from isolated preparations to in situ tissue conditions.

Abstract: Proteolytic cleavage of the amyloid precursor protein (A beta PP) results in the generation of the amyloidogenic fragment known as amyloid beta peptide (A beta). Deposition of A beta in the brain parenchyma and cerebrovasculature is a feature of Alzheimer's disease (AD). To date, the process whereby A beta is generated and deposited remains unclear. We have previously established that activated platelets from AD patients retain more A beta PP on their surface than control platelets. We report here that an endothelial cell-derived enzyme can cleave this surface platelet A beta PP. Human blood brain barrier endothelial cells from brains of AD patients were assayed for potential A beta PP-cleaving enzymes using synthetic peptide substrates encompassing the A beta N-terminus cleavage site. A protease activity capable of cleaving A beta PP on the surface of AD platelets was noted. The A beta PP cleavage is partially inhibited by EDTA, by ZincOV, as well as by a specific inhibitor of the Zn metalloprotease E.C.3.4.24.15. Furthermore, the protease is recognized by an antibody directed against it, using immunohistochemistry, Western blot analysis and flow cytometry. The protease is not secreted, but rather resides intracellularly as well as on the surface of the endothelial cells. The data suggest that E.C.3.4.24.15 synthesized by brain endothelial cells may process the platelet-derived A beta PP, yielding fragments which could contribute to cerebrovascular A beta deposits.

Abstract: A Hungarian family with familial amyloid polyneuropathy (FAP) was studied. The disease presented in two individuals with carpal tunnel syndrome in the fourth and fifth decades of life. The proband subsequently developed vitreous opacities requiring vitrectomy and now has evidence of cardiomyopathy. Single strand conformation polymorphism analysis and direct DNA sequencing revealed a variant AGC (serine) codon at amino acid position 84 of the amyloid precursor protein, transthyretin (TTR). The same single amino acid substitution in TTR was detected in an Indiana kindred with Swiss/German origin. Six individuals of the 11 tested being at risk for FAP proved to have the mutation in the present Hungarian kindred. This is the first description of this TTR gene mutation in Europe. Despite TTR gene haplotype analysis which suggests that the Hungarian and Indiana kindreds may have a common origin, no genealogical link has been identified between the families living in Indiana and Hungary.

Abstract: A French family with hereditary renal amyloidosis (HRA) was studied. The disease presented in 7 of the 8 affected individuals with proteinuria or the nephrotic syndrome. The age of onset was in the fifth decade of life. There is currently no sign of extrarenal involvement in any affected individual. However, the nephropathy in this family is progressive and led to terminal renal failure in 4 patients. Immunohistochemistry studies of glomerular amyloid deposits suggested that the amyloid protein was the fibrinogen A α chain. Direct DNA sequencing revealed a G 4993 T transversion and subsequently Arg 554 Leu mutation in the fibrinogen Aa chain. This is the first description of this fibrinogen Aα chain mutation in Europe. This family is of French descent and cannot be related to the previously reported Peruvian/Mexican and African-American kindreds.

Abstract: Isolated atrial amyloid (IAA) frequently affects elderly human hearts in which only the atria are involved by the deposits. Biochemical analysis has indicated that the major subunit protein of IAA is alpha-human atrial natriuretic peptide (alpha-ANP), which is synthesized by the atrial muscle cells. To define the exact location of the formation of IAA fibrils, right atria from 25 patients undergoing cardiac surgery have been examined by an immunohistochemistry and immunoelectron microscopy with anti-alpha-ANP, apolipoprotein E, amyloid P component, transthyretin, and cathepsin B antisera. Of 25 patients, 19 were involved with IAA deposits which reacted with anti-alpha-ANP, apolipoprotein E, amyloid P component antisera but not with anti-transthyretin antiserum. In 8 of them, amyloid fibrils were seen not only in the interstitium of the atrial myocardium but also in the dilated transverse tubules of the cardiomyocytes. In some cardiomyocytes, amyloid fibrils were also demonstrated within the organelles such as coated and uncoated secretory vesicles or lysosomes. These findings lead to the inescapable conclusion that the polymerization of amyloid fibrils in IAA occurs within the cytoplasm of cardiomyocytes under some conditions.

Abstract: Autonomic neuropathy is a well-known and prognostically important feature of systemic amyloidosis. In other conditions, autonomic function is commonly assessed by cardiovascular reflex tests, described by Ewing, but the feasibility of these tests has not been investigated in patients with systemic amyloidosis. We studied autonomic function in amyloidotic patients using cardiovascular tests and assessed their feasibilityPatients with AA, AL and ATTR amyloidosis participated In all patients, cardiovascular refex testing (mental arithmetic stress test and head-up tilting, besides the Ewingtests) was performedOf the 46 patients included, only 28 patients could perform all 4 Ewing-tests. In particular, patients with AA amyloidosis secondmy to rheumatoid arthritis could not perform standing up and the isometric handgrip test. However, when the mental stress test replaced the handgrip test and head-up tilting replaced standing up, in 45 of the 46 patients, autonomic function could be assessed with cardiovascular...

Abstract: The incidence of serum amyloid A2 alleles (SAA2 alpha and SAA 2 beta) in the Japanese population was analyzed by PCR-RFLP analysis. The SAA2 alpha allele was predominant (approximately 90%) in both healthy controls and adult patients with rheumatoid arthritis. There was no significant association of allele patterns with AA amyloidosis in the patient group. These results are discussed in comparison with those of previous reports from other areas.

Abstract: An African-American kindred with renal amyloidosis is described Four members in two generations developed nephropathy in the sixth to eighth decade of life Kidney biopsy and subcutaneous fat aspirate biopsy of one patient revealed amyloid deposits DNA analysis showed that patients were heterozygous for a mutation in the fibrinogen Aa chain gene with a guanine to thymine transversion at the second base of codon 554, predicting a leucinefor arginine substitution Peptide sequence analysis 05 isolated plasma fibrinogen showed normal peptide as well as variant peptide with leucine replacing arginine at position 554, as predicted by the DNA sequence The ratio between normal and variant peptides was approximately 1:1 in one patient and 3:2 in another Although this African-American kindred has the exact same mutation as a previously described Peruvian-Mexican kindred, the onset age in this kindred is much later than in the Peruvian-Mexican kindred This finding may indicate the existence of additional facto

Abstract: Different isoforms and derived polypeptides of the Alzheimer's disease amyloid protein precursor (AβPP) have been expressed in the yeast Pichia pastor is. The expression characteristics of the different AβPP polypeptides were studied by post-embedding immunogold electron microscopy with various AβPP antibodies. The site of intracellular expression could be readily identified with specific antibodies. Full length AβPP was expressed in association with the nuclear membrane and the endoplasmic reticulum. Secretory derivatives of AβPP were localized in membrane-bound secretory vesicles. A construct encoding two copies of βA4[1-42] linked head-to-tail (βA4duplex) accumulated as irregular dense cytoplasmic and intranuclear inclusions which reacted with all βA4 antibodies tested. A βA4-C-terminal construct accumulated into membranous structures in the cytoplasm and nucleus and reacted with most antibodies to βA4 and the cytoplasmic domain of AβPP. The two shorter constructs containing the fiA4 sequence formed si...

Abstract: Objective. The diagnosis of AA amyloidosis could not be made in eight patients with pediatric rheumatic diseases as later verlfied employing the more sensitive combination of Congo red and additional immunocytochemistry (CRIC). The objective of this paper is to estimate the benefit of CRIC by reevaluating the historical charts with respect to the question as to which of the diagnostic and therapeutic measures would have been altered if the correct diagnosis had been known at the time of the primary biopsyMethods. All subsequent biopsies of eight children with historically missed AA amyloidosis in their primavy biopsies were retrieved, together with the historical data including the Congo red stains of the biopsies. The biopsies were reexamined blindly for the presence of amyloid and the results were compared with the historical data concerning diagnostic and therapeutic measuresResults. Using CRIC, AA amyloidosis could be identified an average of approximately three years earlier as cornpared to the histo...

Abstract: An extracted Bence Jones lambda protein from a Japanese patient with myeloma-associated Fanconi syndrome was found to contain 5 components, including the dimer and the monomer of the entire light-chain, the dimer and the monomer of the constant domain, and monomer of the variable domain. The entire amino acid sequence of this lambda chain was completed. The protein, containing 5 components, was injected intraperitoneally in C3H mice, 20 mg for 13 days and 200 mg for 3 days. Both groups of CJT mice showed a renal proximal tubular deposition of variable domain fragment of the Bence Jones protein by immunoperoxidase staining. Other control Bence Jones proteins of the lambda type and serum albumin were negative in terms of deposition in the epithelial tubular cells.It is also shown that the proximal tubules of the biopsied kidney from the patient had a deposition of variable domain fragment of Bence Jones protein. Thus, the myeloma-associated Fanconi syndrome could be included in the spectrum of light-chain a...

Abstract: An investigation was undertaken on paraformaldehyde-fixed, Lowicryl resin-embedded renal biopsies from patients with AA amyloidosis to study the association of two small chondroitin sulphate/dermatan sulphate proteoglycans, decorin and biglycan, with amyloid fibrils using an ultrastructural immunogold technique. Biglycan was present in glomerular endothelial cells in both normal kidney and in amyloidosis, but little biglycan or decorin was present in the normal mesangial matrix. By contrast, conspicuous amounts of both biglycan and decorin were seen to be associated with amyloid fibrils in the glomerular matrix in cases of renal AA amyloidosis. The results further emphasise the close association between amyloid and extracellular matrix components which are now considered to be an integral part of the amyloid fibrils.

Abstract: Chronic low-level lead exposure is toxic to the developing nervous system The amyloid β precursor protein (AβPP) plays a pivotal role in this developmental process, both as a neurotrophic/neuroprotective factor and as a mediator of cell adhesion in this study, we have used an in vitro system to examine the interaction between chronic low-level lead and the expression and function of AβPP Chronic exposure of the HN9 mouse hippocampal cell line to lead chloride (10−14 M to 10−6M) for 96 hours resulted in a 50% increase in the levels of the particulate form of the protein with a parallel decrease in the soluble form (AβPPs) This effect of lead was reversible following the removal of the toxin This increase in membrane-bound AβPP was also paralleled by an increase in cell adhesivity to afibronectin substrate in addition, AβPPs also acted to attenuate lead toxicity Cells which secreted high levels of the protein were resistant to lead toxicity when compared with control cells suggesting that the protein

Abstract: Dialysis related amyloidosis (DRA) is a major complication of long term hemodialysis therapy. It is well recognized that scintigraphic study using radioisotope-labeled beta 2-microglobulin (beta 2M) as a tracer is a sensitive and specific technique to diagnose DRA non-invasively. The aim of this study is to clarify the mechanism of 131I-beta 2M accumulation around the amyloid tissue. Three dialysis patients with carpal tunnel syndromes were examined for consecutive 131I-beta 2M scintigraphies every 24 hours for 3 days till the carpal tunnel synovectomy. Removed synovial tissues were processed for histological study. The scintigraphic study demonstrated tracer accumulations in the joints involved with DRA and the intensity increased in a time dependent fashion. Microscopic observations revealed many inflammatory cells presenting CD68-monocytes/macrophages antigen infiltrated into the synovial tissues. 131I-beta 2M was evident in the cytoplasm of the infiltrating cells, while no radioactivity was detected above background in the amyloid tissues. In conclusion, the tracer accumulations observed in the 131I-beta 2M scintigraphic studies were the consequence of circulating beta 2M assimilated by the infiltrating monocytes/macrophages. Thus, the undetermined elimination pathway of circulating beta 2M in the dialysis patients was identified as the storage pool in those inflammatory cells. The inflammatory change may play a crucial role in the local progression of DRA through the accumulation of circulating beta 2M around the established amyloid tissues.

Abstract: A transthyretin (TTR)-adsorption column has been developed for the removal of variant TTR from the plasma of patients with familial amyloid polyneuropathy (FAP). The adsorbent is an ion-exchange resin made of porous beads of polyvinyl alcohol gel covalently bound with dimethylaminoethanol. This column was used for three patients with type I FAP. It reduced the concentrations of both normal and variant TTR in the plasma to about half of their pre-adsorption levels. Except for thyroxine, retinol-binding protein and IgM, other proteins in serum were not significantly decreased and there were no adverse effects in long term clinical usage of this TTR-adsorption column. In this trial, we did not obtain concrete evidence that TTR-adsorption therapy can stop or delay the progression of the disease in a FAP patient. However, if we are able to apply this technique more frequently and effectively, TTR-adsorption therapy using our column might be useful for the treatment of FAP patients.

Abstract: A sixty-three year old French man presented with isolated late-onset amyloid cardiomyopathy proven by endomyocardial biopsy. There was no known family history of amyloidosis. Immunohistochemistry of cardiac deposits suggested that amyloi fibrils were derived from transthyretin. DNA sequencing revealed a point mutation in exon 2 of the transthyretin gene responsible for a novel amyloidogenic variant Asp42.

Abstract: The metabolism of amyloid precursor protein (A beta PP) in chick neurons cultured in serum-free medium is described. A beta PP immunoreactivity, detected with the 22C11 antibody, was seen at approximately 135 and approximately 120 kDa. A beta PPs (approximately 120 kDa) was released from the cells and could be detected in the culture medium without the need of a purification step. The content of A beta PPs increased with time after medium change, but was not affected by either carbachol (100 microM), glutamate (50 microM), veratrine (20 microM), oleic acid (200 microM), A23187 (5 microM), phorbol 12, 13-dibutyrate (PDBu, 1 microM), staurosporine (1 microM), Go 6976 (1 microM) or okadaic acid (50 nM) although the combination of PDBu and okadaic acid reduced the secretion. Addition of the muscarinic receptor agonist carbachol to the neurons increased the rate of phosphoinositide breakdown. In Western blot experiments using antibodies to the alpha, beta II and epsilon isoforms of protein kinase C and conditions whereby robust signals could be seen with rat brain lysates, no immunoreactive bands that could be inhibited by appropriate positive control peptides were seen. It is concluded A beta PPs production by chick neurons in culture is mainly constitutive in nature.