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Immunohistochemical and biochemical studies have demonstrated several different proteins in amyloid deposits that are not intrinsic components of the fibril itself but may play a role in their deposition and fibril formation. We compared the distribution of several amyloid-associated proteins, ie, amyloid P component, apolipoprotein-E, apolipoprotein-J, and vitronectin, in the deposits of several different amyloids, in particular light chain amyloid, with those in the deposits of nonamyloid monoclonal immunoglobulin, which may be considered a form of preamyloid disease. Although 100% of amyloid specimens (7 amyloid A, 15 immunoglobulin light chain, and 1 transthyretin) had amyloid P component and 100% had apolipoprotein-E (2 amyloid A, 10 immunoglobulin light chain, and 1 transthyretin) co-localized with the primary amyloid protein, none of the monoclonal nonamyloid cases (14 light chain deposition disease and 6 light and heavy chain deposition disease) had amyloid P component and only 1 of 11 had apolipoprotein-E. On the other hand, staining for apolipoprotein-J and vitronectin was positive in 100% of cases of amyloid and nonamyloid monoclonal deposits. The association between the presence of apolipoprotein-E and amyloid P component in the fibrillar form of monoclonal light chain deposits and their absence in the nonfibrillar form of deposits suggest a role for these proteins in the process of fibrillogenesis. This lends support for the previously proposed concept that apolipoprotein-E functions as a pathological chaperone by altering the conformation of amyloidogenic proteins.

Purpose: To analyze the deposition of amyloid and its precursors in eyes of patients with familial amyloidosis, Finnish (FAF; Meretoja's syndrome), a hereditary systemic amyloidosis., Methods: Autopsy eyes from three patients with FAF and ten control eyes were studied by Congo red staining and with antibodies to the nonmutated part of gelsolin (GS-2C4), the mutated gelsolin Asn-187 fragment (AGel), and amyloid-P component (AP)., Results: Congo red and antisera to AP and AGel bound to amyloid deposits in the cornea and conjunctiva, the sclera, the perineurium of ciliary nerves, the walls of ciliary vessels, the optic nerve sheaths, the stroma of the ciliary body, and along the choriocapillaris. mAb GS-2C4 bound weakly and focally to most deposits and strongly around the choriocapillaris. It labeled the corneal epithelium and endothelium, keratocytes, scleral fibroblasts, trabecular and lens epithelial cells, the ciliary muscle and epithelium, the iris sphincter and dilator, and stromal cells of the conjunctiva and uveal tract., Conclusions: Local production, especially in the cornea, conjunctiva, sclera, and ciliary muscle, and systemic deposition, particularly in blood vessles and in the sclera, may contribute to amyloid deposits in FAF. To explain the complex pattern of deposition, microenvironmental factors such as lamellar architecture of the cornea and sclera, altered processing of gelsolin, or blood-tissue barriers must be invoked. In addition to corneal lattice dystrophy type II, the observed deposits help to explain glaucoma in patients with FAF.

Amyloid beta (A beta) is a normal proteolytic fragment of a large precursor protein (beta PP) which undergoes altered conformation, leading to fibril formation. Two main beta PP processing pathways have been described, and we are now reporting the characterization of a third beta PP pathway. A membrane-associated 16 kDa component identified in human platelets isolated from normal donors. Based on size, immunoreactivity and amino acid sequence analysis, the fragment is a C-terminal beta PP component which starts at position 642 (APP770 numbering) and contains the intact A beta sequence. The presence of this novel pathway of beta PP processing in resting platelets suggest that it occurs as a normal event.

The discovery of soluble amyloid beta (sA beta) suggests that the role of amyloid in Alzheimer's disease (AD) is similar to the previously studied systemic amyloidoses and alters the notion that membrane damage is the initial event in AD. The disease state is characterized by the abnormal accumulation of a normal degradative peptide, which becomes resistant to further proteolysis due to a conformational change. Mutations in the beta PP gene have been found in a very small percentage of AD cases; hence other factors, both genetic and environmental, need to be identified. Priority needs to be given to detailed studies of the structural differences between sA beta and the A beta in amyloid deposits. This will help uncover the determining factors governing the aggregation of sA beta. These structural alterations may be critical for the possible toxic effects A beta and/or associated proteins (molecular chaperones, e.g., apolipoprotein E) have on brain cell function.

Amyloidosis is a generic term for a group of clinically and biochemically diverse diseases that are characterized by the deposition of an insoluble fibrillar protein in the extracellular space. Over 16 biochemically distinct amyloids are known. Despite this diversity, all amyloids have a particular ultrastructural and tinctorial appearance, a beta-pleated sheet structure, and are codeposited with a group of amyloid-associated proteins. The most common amyloidosis is Alzheimer's disease (AD), where A beta is the main component of the amyloid. Recently it has been found that A beta exists as a normal soluble protein (sA beta) in biological fluids. This links AD more closely to some of the systemic amyloidoses, where the amyloid precursor is found in the circulation normally. Numerous mutations have been found in the A beta precursor (beta PP) gene, associated with familial AD. Many mutations are also found in some of the hereditary systemic amyloidoses. For example, over 40 mutations in the transthyretin (TTR) gene are associated with amyloid. However, both A beta and TTR related amyloid deposition can occur with no mutation. The pathogenesis of amyloid is complex, and appears to be associated with genetic and environmental risk factors that can be similar in the systemic and cerebral amyloidoses.

The origin of amyloid beta (A beta) deposited in brain and cerebral blood vessels of patients with Alzheimer's Disease (AD) is not known. In this study, we tested whether soluble A beta (sA beta) can cross the blood-brain barrier (BBB). An in vivo vascular brain perfusion model and capillary depletion technique in guinea-pigs were used to determine cerebral capillary sequestration and blood-brain transport of a synthetic peptide identical with residues 1-40 (SP-40) of A beta. A saturable, specific binding of SP-40 has been demonstrated at the luminal side of the BBB, with the Kd of 25 +/- 2 nM, and Bmax of 188 +/- 11 fmol/min/g of isolated microvessels. A specific transcellular BBB transport of SP-40 into brain parenchyma exhibited the Km of 49 +/- 10 nM, and Vmax of 111 +/- 19 fmol/min/g of capillary depleted brain. We concluded that the BBB has the capability to control the cerebrovascular sequestration and blood-to-brain transport of circulating sA beta. Hence, sA beta can contribute to both cerebrovascular and parenchymal amyloid formation.

Corneal buttons from four patients with lattice corneal dystrophy (LD) type I, thought to be an isolated corneal amyloidosis, and from six patients with LD type II, part of systemic familial amyloidosis, Finnish type (FAF; Meretoja's syndrome), were studied by immunohistochemistry to determine the differential distribution in the amyloid deposits of amyloid P component (AP), mutated gelsolin specific for FAF, and native gelsolin. In both types of LD, antibodies to AP labelled lattice lines and a discontinuous layer of amyloid deposits under Bowman's layer. In LD type II, particularly, they also reacted with streak-like amyloid deposits between corneal almellae, especially in the limbal region. While the anti-FAF antiserum strongly labelled all amyloid deposits in LD type II, it failed to react unequivocally with them in LD type I. Both in LD type I and in two control specimens representing granular dystrophy, the monoclonal antibody (MAb) GS-2C4 to gelsolin faintly labelled some deposits, while in LD type II it reacted non-homogeneously with most amyloid deposits. In all specimens, MAb GS-2C4 labelled corneal epithelial cells and occasional stromal keratocytes and endothelial cells. The results suggest that Meretoja's syndrome, a systemic disease, can be diagnosed even retrospectively from corneal buttons subjected to histopathological study.

Alzheimer's disease is characterized by the deposition of beta-protein (A beta) as amyloid. Recently, it was found that A beta is a normal component of serum and cerebrospinal fluid. Synthetic peptides homologous to A beta form amyloid-like fibrils spontaneously in water or physiological solutions. Using a peptide homologous to A beta 1-40, we find that fibril formation is inhibited by the presence of cerebrospinal fluid.

We amplified DNA encoding the 3' 109 codons of Alzheimer's-disease amyloid precursor protein (APP) inclusive of the beta protein (A beta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13. We also modified this construct to generate recombinant molecules incorporating two recently described APP mutants by site-directed mutagenesis. Both native C109 (deletion construct inclusive of the C-terminal 109 residues of APP) and constructs with a single mutation at codon 642 (T-->G, resulting in a substitution of glycine for valine) or a double mutation at codons 595 (G-->T, substituting asparagine for lysine) and 596 (A-->C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20% of total bacterial protein after induction. The major constituent of expressed C109 protein had an apparent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the full-length construct by size and N-terminal microsequencing. Also present was a 4-5 kDa species that co-purified with C109, constituting only approximately 1% of expressed protein, which was revealed by Western-blot analysis with antibodies specific for A beta epitopes and after biotinylation of purified recombinant C109. This fragment shared N-terminal sequence with, and appeared to arise by proteolysis of, full-length C109 in biosynthetic labelling experiments. C109 spontaneously precipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5%) fibrillar component that was reactive with antibodies to a C-terminal epitope of APP. Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal A beta-inclusive fragments of APP that have been found in transfected cells, brain cortex and cerebral microvessels.

The amyloid fibrils deposited in Alzheimer's neuritic plaque cores and cerebral blood vessels are mainly composed of aggregated forms of a unique peptide, 39-42 amino acids long, named amyloid beta (A beta). A similar, although soluble, A beta ('sA beta') has been identified in cerebrospinal fluid, plasma and cell supernatants, indicating that it is normally produced by proteolytic processing of its precursor protein, amyloid precursor protein (APP). Using direct binding experiments we have isolated and characterized an 80 kDa circulating protein that specifically interacts with a synthetic peptide identical with A beta. The protein was unmistakably identified as SP-40,40 or ApoJ, a cytolytic inhibitor and lipid carrier, by means of amino acid sequence and immunoreactivity with specific antibodies. Immunoprecipitation with anti-SP-40,40 retrieved soluble A beta from cerebrospinal fluid, indicating that the interaction occurs in vivo.

We synthesized polypeptides corresponding to sequences encoded by normal and mutant alleles in the regions of codon 178 (Asp-->Asn) and codon 200 (Glu-->Lys) of the chromosome 20 amyloid gene that have been linked to familial Creutzfeldt-Jakob disease. Peptide suspensions from both regions spontaneously formed amyloid fibrils with different morphological characteristics and aggregation tendencies. Fibrillar arrays were denser and more profuse in mutant than in normal peptide suspensions and were even more marked when the homologous mutant and normal peptides were mixed together. Preparations from the region of codon 200 were in all cases more fibrillogenic than corresponding peptides from the region of codon 178. These in vitro observations support the hypothesis that amino acid changes from pathogenic single-allele point mutations in Creutzfeldt-Jakob disease may nucleate the in vivo folding behavior of the normal host protein to favor formation of insoluble amyloid fibrils.

Apolipoprotein E (apo E) is associated with Alzheimer's beta-amyloid (A beta) in senile plaques. A beta is now known to be a normal soluble peptide (sA beta) found in the cerebrospinal fluid (CSF) and other biological fluids. We have used synthetic A beta peptides bound to affinity membranes in order to determine whether apo E or any other amyloid associated protein will bind to these membranes, when they are bathed in CSF. Under these conditions apo E, as well as another apolipoprotein, apolipoprotein J (Apo J), bound to the membranes. Using ELISA and ligand binding studies, we found a high avidity binding of A beta peptides to apo E. This suggests that apo E, as well as other related proteins may bind not only amyloid A beta but also sA beta. This interaction may be critical in amyloid formation.

The predominant component of amyloid plaques of Alzheimer's disease is the amyloid beta protein (A beta), a 39-42-amino-acid peptide derived by proteolysis of a family of precursors known as amyloid precursor proteins (APP). In mammalian brain and in cultured mammalian cells, the release of APP amino-terminal fragments into the extracellular medium occurs by a proteolytic cleavage within the A beta domain, thereby precluding amyloidogenesis. Infection of Sf9 insect cells with baculovirus vectors containing APP cDNAs results in high levels of APP expression. The concomitant release of amino-terminal fragments of APP and the production of carboxyl-terminal, cell-associated cleavage products are observed. Here we demonstrate by direct protein microsequencing that the proteolytic processing of APP in the Sf9 cells generates a prominent carboxyl-terminal species that is identical to that produced in human cells, suggesting that the major pathway for proteolytic processing of APP is conserved among metazoans.

Amyloid beta (A beta), the major constituent of the fibrils composing senile plaques and vascular amyloid deposits in Alzheimer's disease (AD) and related disorders, is a 39-42-residue self-aggregating degradation peptide of a larger multidomain membrane glycoprotein designated amyloid precursor protein (APP). An array of biological functions has been assigned to different APP domains, including growth regulation, neurotoxicity, inhibitory activity of serine proteinases and promotion of cell-cell and cell-matrix interactions. A beta is generated through an as-yet-unknown catabolic pathway that by-passes or inhibits the cleavage of APP within the A beta sequence. We have identified a 16 kDa intermediate APP C-terminal fragment containing A beta in leptomeningeal vessels of aged normal individuals and AD patients by means of its immunoreactivity with a panel of four different anti-(APP C-terminal) antibodies, indicating a different pathway of APP processing. Previous studies have indicated that the APP C-terminal domain is the most likely to be involved in cell-matrix interactions. A 109-amino-acid construct C109 with a sequence analogous to the C-terminal of APP (positions 587-695 of APP695), similar in length and immunoreactivity to the 16 kDa fragment, was found to promote cell adhesion. By use of synthetic peptides, this activity was initially located to the extracellular 28 residues of A beta. Inhibition studies demonstrated that the sequence RHDS (amino acids 5-8 of A beta, corresponding to residues 601-604 of APP695 was responsible for the adhesion-promoting activity. The interaction is dependent on bivalent cations and can be blocked either by the tetrapeptides RHDS and RGDS or by an anti-(beta 1 integrin) antibody. Thus, through integrin-like surface receptors, APP or its derivative proteolytic fragments containing the sequence RHDS may modulate cell-cell or cell-matrix interactions.

The 4-kilodalton (39 to 43 amino acids) amyloid beta protein (beta AP), which is deposited as amyloid in the brains of patients with Alzheimer's diseases, is derived from a large protein, the amyloid beta protein precursor (beta APP). Human mononuclear leukemic (K562) cells expressing a beta AP-bearing, carboxyl-terminal beta APP derivative released significant amounts of a soluble 4-kilodalton beta APP derivative essentially identical to the beta AP deposited in Alzheimer's disease. Human neuroblastoma (M17) cells transfected with constructs expressing full-length beta APP and M17 cells expressing only endogenous beta APP also released soluble 4-kilodalton beta AP, and a similar, if not identical, fragment was readily detected in cerebrospinal fluid from individuals with Alzheimer's disease and normal individuals. Thus cells normally produce and release soluble 4-kilodalton beta AP that is essentially identical to the 4-kilodalton beta AP deposited as insoluble amyloid fibrils in Alzheimer's disease.

We present the immunohistochemical and biochemical identification of an amyloidoma localized to the cerebral white matter in a patient who shows no evidence of systemic or extracranial localized amyloid deposits. Immunohistologic and immunochemical studies, using antibodies against biochemically different amyloid fibrils and amyloid-associated proteins, showed reactivity with antibodies only to lambda light chain and serum amyloid P-component. Amino acid sequence analysis of the purified amyloid fibrils extracted from the brain tumor indicates that the amyloid protein is an unusual immunoglobulin lambda light chain, starting at residue five of the variable domain. These fibrils consist of lambda chain fragments of different lengths (10 to 30 kd) very likely arising by polymerization of the amyloid subunit or sequential proteolytic cleavage of the light chain, or both. After exposure to denaturing agents, the 10-kd subunit retains the characteristics of native amyloid fibrils by electron microscopy.

Background: The origin of the amyloid beta protein (A beta) that is the main constituent of amyloid fibrils occurring in the senile plaques and cerebrovasculature of individuals afflicted with Alzheimer's Disease, Down's Syndrome, Hereditary Cerebral Hemorrhage with Amyloidosis--Dutch Type, and Sporadic Cerebral Amyloid Angiopathy, is central to the pathogenesis of these disorders. Evidence exists to support a neuronal and/or a vascular origin. We have reported that platelets may serve as one possible source of the A beta sequence via an intact, membrane-associated Beta Protein Precursor (beta PP) which is encoded by a platelet transcript (BBRC 173:1292-1298, 1990)., Experimental Design: Immunoaffinity chromatography and western blotting of extracted cellular proteins, polymerase chain reaction amplification of beta PP mRNA, fluorescence activated flow cytometric analysis, and confocal scanning laser microscopy have been employed to characterize the presence and distribution of thrombocytic beta PP., Results: Immunoblot analysis with antibodies specific for the carboxyl-terminal end of beta PP indicates that platelets and the Dami megakaryocyte cell line express membrane-associated species of intact beta PP ranging in molecular weight from 110 to 140 kilodaltons (kd), as well as carboxyl-terminal reactive forms ranging from 16 to 22 kd. Thrombin stimulation of platelets induces the release of five soluble beta PP species, which possess apparent isofocusing points in the range of 4.1-5.5. By contrast, extracts of peripheral blood mononuclear cells enriched by ficoll centrifugation, endothelial cells and a B cell line were not immunoreactive by western blot, even though beta PP transcripts could be amplified by polymerase chain reaction. The distribution of platelet beta PP was localized by flow cytometric analysis and scanning laser microscopy, using fluorescein-labeled antibodies. Study of the subcellular distribution of platelet beta PP indicates that these translation products are accumulated in discrete foci throughout the thrombocyte, possibly corresponding to secretory granules., Conclusions: The size of the carboxyl-terminal forms of beta PP indicate that the A beta sequence is present as a membrane associated constituent in unstimulated platelets, and may represent alternative pathways of beta PP processing. Cleavage or other abnormal processing of platelet-associated beta PP in Alzheimer's disease provides one mechanism whereby cerebral amyloid might derive from the circulation.

Two synthetic peptides with sequences identical with those of fragments of the extracellular domain of the Alzheimer's-disease amyloid precursor protein (APP) were used to raise antibodies. SP28 comprises positions 597-624 of the APP695 isoform, whereas SP41 extends towards the N-terminus (amino acids 584-624) and contains the entire SP28 peptide. Using e.l.i.s.a. and inhibition experiments we identified the two beta-turn-containing segments 602-607 and 617-624 as the epitopes recognized by anti-SP41 and anti-SP28 respectively. Both antibodies immunolabelled amyloid lesions in brains from Alzheimer's-disease patients and patients with related disorders, whereas they were unreactive in control brains. However, when probed on immunoblots, anti-SP28 failed to detect full-length APP from baculovirus-infected Sf9 cells, and anti-SP41 reacted weakly compared with other anti-APP antisera. The data suggest that these antibodies are directed to conformational epitopes not existent in the native molecules but present after alternative APP processing.

Alzheimer's disease and cerebral amyloid angiopathies (CAA) are clinically heterogeneous diseases, but pathogenically related by the deposition of beta A4-amyloid in the brain in the form of neuritic plaques and/or vascular infiltrates. Antibodies directed against the N-terminal region of the predicted sequence of the beta A4 amyloid protein precursor (APP) were used to investigate the cellular distribution of this protein in the brain of normal humans and rats. We found a widespread presence of APP throughout the nervous tissue, including neurons, blood vessels, meningeal membranes, choroid plexus and ependymal cells. The highest APP immunoreactivity in both species was found in neuronal cell bodies and their processes, and around blood vessels. These findings may account for the clinical, pathological and aetiological differences found among the beta A4-amyloidosis.

beta-Trace, a 23.5 kDa glycoprotein of unknown biological functions, is present in all body fluids tested. It is found in higher concentration in human seminal fluid and cerebrospinal fluid (CSF) than in serum. A one-step procedure for the isolation of beta-trace from pooled CSF is described, by affinity chromatography using a specific antibody made against beta-trace. Amino terminal sequence analysis yields the sequence A P E A Q V S V Q P N F Q Q D K F L G with no homology to known proteins, indicating that beta-trace is a novel CSF protein.

In patients with Alzheimer's disease, in patients with Down's syndrome and in aged non-demented individuals, anti-beta-protein antibodies label not only the fibrillary amyloid, but also preamyloid deposits. The latter are made up of amorphous material lacking the tinctorial, optical and ultrastructural properties of amyloid fibrils. To investigate the antigenic profile of preamyloid deposits, we have carried out an immunohistochemical study on specimens of cerebral cortex from 4 Alzheimer patients and two non-demented individuals, using antibodies to the beta-protein (anti-SP28), the C-terminal region of the amyloid precursor protein (APP) (anti-SP20) and an APP extracellular epitope between residues 50 and 100 (anti-preA4). Anti-preA4 and anti-SP28 immunoreactivity was found to be present in preamyloid deposits, whereas anti-SP20 immunoreactivity was not. These findings suggest that an extracellular portion of APP, close to the N-terminus of the molecule, participates with beta-protein in the composition of preamyloid deposits.

Familial amyloidosis, Finnish type (FAF), is an autosomal dominant form of amyloidosis which is related to a point mutation in the gelsolin gene localized on chromosome 9. The mutation corresponds to codon 187 of the secreted form of gelsolin, and is expressed in the amyloid fibril at residue 15. Our original FAF patient was demented, and neuropathological analysis showed Alzheimer type brain lesions associated with both classical and cortical Lewy bodies. Furthermore, antiserum against the gelsolin-derived FAF amyloid reacted strongly with both classical and cortical Lewy bodies of this FAF patient. In preliminary experiments similar results were obtained in cases of Parkinson's disease and diffuse Lewy body disease. These observations may indicate a role for gelsolin in the pathogenesis of Parkinson's disease and related conditions.

Lewy bodies (LB) are intraneuronal, cytoplasmic inclusion bodies. They are invariably present in Parkinson's and diffuse LB diseases. Their composition by direct biochemical methods is unknown, although LBs are immunoreactive with a number of antibodies, including anti-ubiquitin and anti-neurofilament antibodies. Familial amyloidosis, Finnish type (FAF), is an autosomal-dominant form of systemic amyloidosis. The authors have isolated and partially sequenced the amyloid. The protein has significant sequence identity with gelsolin, an actin-modulating protein. Rabbit polyclonal antibodies raised to the FAF amyloid not only immunostain the amyloid but also LBs in the cortex and substantia nigra of Parkinson's and diffuse LB disease brains. Immunoreactivity is absorbed by the purified amyloid but is unaffected by ubiquitin. This provides a link between the LB and one of the human amyloidoses, FAF.

Gerstmann-Sträussler-Scheinker (GSS) disease is a familial neurological disorder pathologically characterized by amyloid deposition in the cerebrum and cerebellum. The GSS amyloid is immunoreactive to antisera raised against the hamster prion protein (PrP) 27-30. This is a proteinase K-resistant glycoprotein of 27-30 kd that is derived from an abnormal isoform of a neuronal glycoprotein of 33-35 kd designated PrPSc and is a molecular marker of amyloid fibrils isolated from animals with scrapie and humans with related disorders. We have purified and characterized proteins extracted from amyloid plaque cores isolated from two patients of the Indiana kindred of GSS disease. We found that the major component of GSS amyloid is an 11 kd degradation product of PrP, whose N-terminus corresponds to the glycine residue at position 58 of the amino acid sequence deduced from the human PrP cDNA. In addition, amyloid fractions contained larger PrP fragments with apparently intact N-termini and amyloid P component. These findings suggest that the disease process leads to proteolytic cleavage of PrP, generating an amyloidogenic peptide that polymerizes into insoluble fibrils. The N-terminal cleavage of PrP in GSS disease occurs at a tryptophan-glycine peptide bond identical to that cleaved by proteinase K in vitro to generate PrP 27-30 from hamster PrPSc at codon 90. Since no mutations of the structural PrP gene have been found in the Indiana family of GSS disease, it is conceivable that factors other than the primary structure of PrP play a crucial role in the process of amyloid formation and the development of clinical neurologic dysfunction.

The complete amino acid sequence of the Fab fragment of protein KAU, a human monoclonal cold agglutinin (IgMk) with anti-I activity, was determined. The light chain (L-chain) consists of 215 residues; the variable (V)L region belongs to the Hum/Kv325/kIIIb sub-subgroup that is preferentially selected in human IgM autoimmune response. The joining (J) region is encoded by the Jk4 gene, and the constant region (C)L domain expresses the km3 allotypic marker. The Fd fragment contains 232 amino acids, and 120 of them comprise the variable domain. The VH region corresponds to the VHIV subgroup and is closely related to the VHIV 2.1 gene isolated from genomic DNA expressed in peripheral blood of a healthy Caucasian. The complementary-determining region 1 has a unique amino acid (Asp) at position 31, and the complementary-determining region 3 codified by the diversity segment (D) gene, shows poor homology with other known D sequences. The joining segment with two unusual substitutions at the D-J junction is encoded by the JH4 gene. Thus, cold agglutinin KAU is an IgM, VkIIIb-Jk4-km3; VHIV-JH4-C mu.

Using antibodies directed against N-terminal and C-terminal epitopes we have immunologically detected APP species in the membrane and saline-soluble fractions of unstimulated platelets, and in the conditioned medium of thrombin-stimulated platelets. These studies demonstrate an intact 140 kD membrane-associated form of APP that is released on degranulation. Evidence that platelets synthesize at least one form of APP (APP751) was obtained by enzymatic amplification of specific mRNA using Polymerase Chain Reaction (PCR) and direct sequence analysis of PCR product. Processing of APP for release may occur via successive C-terminal truncations, and/or by the release and proteolysis of an intact membrane associated form. An intact form of APP in platelets provides a circulating substrate upon which proteases from many tissues may act to produce beta protein (AB) during pathologic conditions.

Familial amyloidosis, Finnish type (FAF), is an inherited form of systemic amyloidosis clinically characterized by cranial neuropathy and lattice corneal dystrophy. We have demonstrated that the protein subunit isolated from amyloid fibrils shows considerable sequence identity with gelsolin, an actin-binding protein. We have purified the amyloid subunit from a second case and further analysed different fractions from the previous one. Sequence analysis shows that, in both cases, the amyloid subunit starts at position 173 of the mature molecule; it has a heterogeneous N-terminus and contains one amino acid substitution, namely asparagine for aspartic acid, at position 15 (gelsolin residue 187), that is due to a guanine-to-adenine transversion corresponding to nucleotide-654 of human plasma gelsolin cDNA. The substitution maps in a fragment with actin-binding activity and is located in a repetitive motif highly conserved among species. Thus FAF is the first human disease known to be caused by an internal abnormal degradation of a gelsolin variant. We designate this variant of gelsolin-associated amyloidosis 'Agel Asn-187'.

Familial amyloidosis, Finnish type (FAF), is an autosomal dominant form of familial amyloid polyneuropathy. The novel amyloid fibril protein found in these patients is a degradation fragment of gelsolin, an actin-binding protein. We found a mutation (adenine for guanine) at nucleotide 654 of the gelsolin gene in genomic DNA isolated from five FAF patients. This site is polymorphic since the normal allele was also present in all the patients tested. This mutation was not found in two unaffected family members and 11 normal controls. The A for G transition causes an amino acid substitution (asparagine for aspartic acid) that was found at position 15 of the amyloid protein. The mutation and consequent amino acid substitution may lead to the development of FAF.

The complete sequence of the toxin TsTX-VI from the venom of the scorpion Tityus serrulatus Lutz and Mello is presented. The sequence has been determined by automated Edman analysis of the reduced and carboxymethylated protein as well as of the resulting peptides, obtained from S. aureus protease and tryptic digestions. TsTX-VI is composed of 62 residues and has a calculated molecular weight of 6717. Homology studies with other scorpion toxins show that TsTX-VI is more similar to the Old World than to the North American scorpion toxins. The hydropathic index indicates that TsTX-VI is more hydrophobic than Ts-gamma. Toxicity studies carried out in mice demonstrate that i.v. injection of TsTX-VI is unable to evoke the usual symptoms induced by the typical neurotoxins of this venom, but only a generalized allergic reaction. These properties are important in clarifying the relationship between primary structure and biological function of scorpion toxins.

Immunohistochemical studies of six patients with familial amyloidosis, Finnish type, showed that their amyloid deposits did not react with polyclonal antibodies against the amyloid proteins of other, established forms of systemic or cerebral amyloidosis. However, strong immunoreactivity was observed with rabbit antiserum raised against a low molecular weight purified amyloid subunit isolated from one of the patients. This immunoreactivity was abolished by absorption with the low molecular weight amyloid fraction. The amino terminal sequence of the amyloid protein subunit was homologous to gelsolin, an actin-modulating protein, and the amyloid deposits in tissues reacted with a monoclonal antibody against gelsolin. These studies show that the amyloid protein in familial amyloidosis, Finnish type, is not related to previously identified forms of amyloid, including prealbumin (transthyretin) variants, but represents a novel amyloidogenic protein related to gelsolin, a plasma and cytoplasmic protein.

Polyclonal antibodies to synthetic peptides homologous to amino acid residues 45-62, 597-624, and 676-695 of the predicted sequence of Alzheimer's amyloid precursor protein (APP) were used to investigate the site of origin of APP, and the relationship between APP and amyloid protein in Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D). Cortical sections as well as homogenates of isolated leptomeningeal and cortical microvessels from three patients with AD, two patients with HCHWA-D, and two nondemented controls were probed. In vessel extracts of both groups of patients and the controls, APP was detected as a set of proteins with electrophoretic mobility of 105 to 135 kilodaltons. In cortical sections of all subjects, APP immunoreactivity was found in leptomeningeal and cortical vessel walls. In patients with AD and HCHWA-D, APP and amyloid fibrils coexisted in the same vessels. Moreover, APP immunoreactivity was found in association with 50% of senile plaques in AD brains, but was not evidenced in parenchymal amyloid deposits in patients with HCHWA-D. These data suggest that the vascular system is a source of APP and that the processing of APP into insoluble fibrils in AD and HCHWA-D may take place in situ.

Familial amyloidosis, Finnish type, is clinically characterized by cranial neuropathy and lattice corneal dystrophy. It is an autosomal dominant form of systemic amyloidosis with small deposits of congophilic material occurring in most tissues, particularly in association with blood vessel walls and basement membranes. Amyloid fibrils were extracted from the kidney of patient VUO, and rabbit antiserum raised against the 12 kDa purified amyloid subunit displayed strong immunohistochemical reactivity with the amyloid deposits. The amino terminal sequence of this 12 kDa amyloid protein (ATEVPVSWESFNNGD) showed homology with gelsolin (or actin depolymerizing factor), a 93 kDa plasma protein. The amyloid peptide is a degradation product, starting at position 173, of the gelsolin molecule.

Hydropathic anticomplementarity of amino acids indicates that peptides derived from complementary DNA strands may form amphiphilic structures and bind one another. By using this concept, we have found that the antisense peptide Ser-Tyr-Asp-Leu complementary to the segment Gln-Ile-Val-Ala-Gly (residues 55-59) in cystatin C (an inhibitor of cysteine proteases) is located at positions 611-614 of the beta chain of human C4, the fourth component of complement. Here we describe and characterize the specific interaction between cystatin C and C4 by ligand affinity chromatography and ELISA. Interaction between the two native proteins was mimicked on replacement of one of them with the corresponding sense-antisense peptide coupled to a carrier protein, and the binding was inhibited by these synthetic peptides in solution. Through the interaction with C4, cystatin C may play a regulatory role in complement activation that might be of particular importance at tissue sites where both proteins are produced by macrophages.

The human cystatin C gene was cloned using a synthetic oligonucleotide predicted from a portion of its amino-acid sequence. The nucleotide sequence of the restriction fragment hybridizing with the oligonucleotide confirms the existence of one exon encoding amino acids 56-93 of human cystatin C and its relationship to kininogens. However the deduced amino-acid sequence differs in one position from the sequence of the cystatin C fragment deposited as amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of icelandic origin.

Two synthetic peptides corresponding to the reported 28-residue sequence of Alzheimer's Disease beta-protein (SP28) and to residues 12-28 (SP17) were used to form fibrils in vitro. Synthetic fibrils bound Congo Red and closely resembled amyloid fibrils isolated from leptomeninges and senile plaques of Alzheimer's brain by electron microscopy. A polyclonal antiserum to SP28 specifically decorated both synthetic and native amyloid by colloidal gold immunoelectron microscopy. Amyloid fibrils isolated from tissue were insoluble on SDS-Polyacrylamide gels, and tended to aggregate while synthetic amyloid fibrils were completely solubilized, releasing only monomers of SP28 and SP17. Anti-SP28 immunostained cerebrovascular and plaque core amyloid, but not neurofibrillary tangles, in tissue section. Western blot analysis showed that anti-SP28 reacted with a 4 kDa band released from amyloid core-enriched preparations and leptomeninges. By contrast, a 16 kDa band corresponding to the tetramer of beta-protein was not recognized. These data suggest that as little as a 17 residue sequence of beta-protein may be required to form fibrils and that the complete sequence of the 4 kDa beta-protein may be important in determining insolubility and the formation of intermediate size polymers.

The amyloid fibrils deposited in cerebral vessel walls and senile plaques in Alzheimer's disease are polymeric forms of a 4 kDa fragment produced by proteolysis of a putative precursor protein (APP). Using antibodies to several fragments of the deduced precursor, we were able to demonstrate the presence of APP in senile plaques, brain extracts and cerebrospinal fluid. Membrane-associated APP is detected as a group of 105-135 kDa proteins while soluble APP is predominantly 105 kDa, does not react with an anti C-terminal antibody, and is 10 kDa shorter than the membrane-bound APP. Amino terminal sequence of the tissue 105 kDa protein indicates that APP begins at residue 18 of the cDNA sequence. These findings imply that i) two forms of APP are detected: membrane-bound and secreted, and ii) APP can be processed in situ.

Hereditary Cerebral Hemorrhage With Amyloidosis is an autosomal dominant form of amyloidosis restricted to the cerebral vasculature. We have previously demonstrated that the amyloid protein subunit is similar to Cystatin C (or gamma-trace), an inhibitor of lysosomal cysteine proteinases, and homologous to kininogens. High pressure liquid chromatography tryptic fingerprint analysis was developed to distinguish Cystatin C from the amyloid protein. Moreover, we isolated and sequenced tryptic peptides in which the differences were detected. The data prove that the amyloid protein is 10 residues shorter than Cystatin C and has one amino acid substitution at residue 58.