SMART MODE:

NORMAL GENOMIC

• Liepnieks JJ, Burt C, Benson MD
• Shoulder-Pad Sign of Amyloidosis: Structure of an Ig Kappa III Protein.
• Scand J Immunol. 2001; 54: 404-8
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• While amyloid infiltration of articular structures is rare, the 'shoulder-pad' sign resulting from periarticular soft tissue amyloid deposition is essentially pathognomonic for immunogloblin (Ig) (AL) amyloidosis. We report the characterization of an amyloid protein (GRA) which produced articular amyloid deposits and the shoulder-pad sign. Amyloid fibrils were isolated from soft tissue shoulder mass of a patient with systemic AL amyloidosis. The fibrils were solubilized in guanidine HCl and proteins separated by Sepharose chromatography. Amino acid sequence of fractionated protein was determined after tryptic digestion. Sequence analysis of the major amyloid protein yielded a kappa III Ig light chain structure. The entire variable region (VL) plus the constant region (CL) to residue 207 was identified; but lesser amounts of CL than VL were present. A number of amino acid residues previously not observed in kappa III VL proteins, plus a two amino acid insert (95A, 95B), were identified. Kappa III VL amyloid proteins are rare and may show an increased predilection for soft tissue deposition. While several unique amino acid residues that were identified in protein GRA may contribute to soft tissue amyloid deposition, no definite pattern is obvious from comparison with other reported kappa III amyloid proteins.

• Cunnane G
• Amyloid precursors and amyloidosis in inflammatory arthritis.
• Curr Opin Rheumatol. 2001; 13: 67-73
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• Recent data demonstrating the multifunctional role of serum amyloid A (SAA) in the pathogenesis of amyloidosis have yielded important insights into this potentially fatal consequence of chronic inflammation. SAA has been shown to participate in chemotaxis, cellular adhesion, cytokine production, and metalloproteinase secretion and is thus integrally involved in the disease process. In addition to its production by the liver as part of the acute phase response, SAA is also expressed by several pathologic tissues such atherosclerotic plaques, rheumatoid synovitis and in the brains of patients with Alzheimer disease. Its constitutive production in normal tissue suggests a role for SAA in host defense and tissue turnover. Many pathways are involved in the regulation of SAA, and as more becomes known about these, potential therapeutic targets may be identified. However, the prevention of secondary amyloidosis is best achieved by early and adequate treatment of patients with chronic inflammatory disorders. Suppression of the acute phase response and normalization of SAA levels are likely to significantly impact on the incidence of amyloidosis in inflammatory arthritis.

• Urieli-Shoval S, Linke RP, Matzner Y
• Expression and function of serum amyloid A, a major acute-phase protein, in normal and disease states.
• Curr Opin Hematol. 2000; 7: 64-9
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• Serum amyloid A (SAA), the precursor protein in inflammation-associated reactive amyloidosis (AA-type), is an acute phase reactant whose level in the blood increases in response to various insults. It is expressed in the liver, but its physiological role is not well understood. Recently, a broader view of SAA expression and function has been emerging. Expression studies show local production of SAA proteins in histologically normal, atherosclerotic, Alzheimer, inflammatory, and tumor tissues. Binding sites in the SAA protein for high density lipoproteins, calcium, laminin, and heparin/heparan-sulfate were described. Adhesion motifs were identified and new functions, affecting cell adhesion, migration, proliferation and aggregation have been described. These findings emphasize the importance of SAA in various physiological and pathological processes, including inflammation, atherosclerosis, thrombosis, AA-amyloidosis, rheumatoid arthritis, and neoplasia. In addition, recent experiments suggest that SAA may play a "housekeeping" role in normal human tissues.

• Kho YJ et al.
• Rapid communication: cloning of bovine serum amyloid A3 cDNA.
• J Anim Sci. 2000; 78: 2756-7

• Ma Z, Mizukoshi T, Khatlani TS, Okuda M, Onishi T
• Molecular cloning and sequencing of equine cDNA encoding serum amyloid A (SAA).
• Vet Immunol Immunopathol. 2000; 77: 321-7
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• The serum amyloid A (SAA) protein is a characteristic and sensitive acute phase reactant in all vertebrates investigated. We molecularly cloned the equine cDNA encoding SAA from the liver of a healthy horse by polymerase chain reaction (PCR). The cloned cDNA is 480 bases in length, and contains an open reading frame (ORF) of 387 nucleotides encoding a precursor SAA protein of 128 amino acids. The precursor of horse SAA seems to have an 18-residue signal peptide and differs from the reported amino acid sequences of the horse SAA by substitution of valine at residue 81. It shows high homology with SAA amino acid sequence of other species such as dog (80.6%), mink (77.5%), human (76.9%) and duck (71.9%). An insertion of eight amino acids at residues between 85 and 92, as compared to human SAA, has also been found in horse SAA. The availability of the equine SAA cDNA will provide a useful reagent for studying its role in diseased horses.

• Sipe JD
• Serum amyloid A: from fibril to function. Current status.
• Amyloid. 2000; 7: 10-2

• Kindy MS, de Beer MC, Yu J, de Beer FC
• Expression of mouse acute-phase (SAA1.1) and constitutive (SAA4) serum amyloid A isotypes: influence on lipoprotein profiles.
• Arterioscler Thromb Vasc Biol. 2000; 20: 1543-50
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• The serum amyloid A (SAA) family of proteins consists of inducible acute-phase members and a constitutive member that are minor apolipoproteins of normal high density lipoprotein (HDL). During inflammation, HDL cholesterol and apolipoprotein A-I (apoA-I) protein are decreased, and these changes are thought to be partly related to the increase in acute-phase SAA proteins that associate with the HDL particle to become the major apolipoprotein species. To determine the specific role of SAA in the alteration of HDL in the absence of a generalized acute-phase response, acute-phase Saa1.1 transgene expression was directed via an inducible mouse metallothionein promoter. Elevated levels of SAA1.1 (28+/-9 mg/dL) comparable to a moderate acute-phase response were achieved over a 5-day period. SAA association with the HDL particles at this concentration did not significantly alter the apoA-I or HDL cholesterol levels or change the lipoprotein profiles in the transgenic mice compared with wild-type mice. In addition, we used adenoviral vectors to increase the SAA expression to levels seen in a major acute-phase response. Injection of adenovirus expressing the mouse SAA1.1 protein resulted in high-level expression (72+/-8 mg/dL) but did not alter apoA-I levels. However, the SAA associated with the HDL particle gave rise to significantly larger HDL particles ( approximately 10%). Adenoviral expression of the constitutive SAA4 protein resulted in an increase in HDL size ( approximately 10%) and an increase in very low density lipoprotein levels (20-fold) and triglyceride levels (1.7-fold). These studies suggest that increases in acute-phase SAA proteins alone are insufficient to alter HDL cholesterol or apoA-I levels during inflammation. A role for constitutive SAA4 in HDL-very low density lipoprotein interactions should be considered.

• Furlaneto CJ, Campa A
• A novel function of serum amyloid A: a potent stimulus for the release of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-8 by human blood neutrophil.
• Biochem Biophys Res Commun. 2000; 268: 405-8
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• High density lipoprotein (HDL) and its main apolipoproteins, AI and serum amyloid A (SAA), present in physiological and acute phase response conditions, respectively, affect the inflammatory process. This study focuses on the effect of AI, SAA, and HDL from healthy (N-HDL) and acute phase individuals (AP-HDL) on the release of TNF-alpha, IL-1beta, and IL-8 by human blood neutrophils. It was observed that SAA (100 microg/mL) causes a dramatic increase (75-400 times) in the basal liberation of the three cytokines assayed. This effect is not triggered by AP-HDL. Although AI (100 microg/ml) increases the release of IL-1beta and IL-8 modestly, N-HDL does not. Both HDLs (0.16-0.32 mg of protein/mL) had an anti-inflammatory action, decreasing the basal and LPS-stimulated cytokine release. Given that the biological role of SAA is still uncertain, the present study adds an important finding potentially pertinent to the biological role of this acute phase protein.

• Wada A, Yamada T, Itoh K, Igari J
• [No significant influence of serum amyloid A1 genotypes on serum lipids or HDL clearance]
• Rinsho Byori. 2000; 48: 174-7
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• Serum amyloid A1(SAA1), the major acute phase isotype of SAA protein family, consists of three common allelic variants(SAA1.1, SAA1.3 and SAA1.5) in the Japanese population. We have recently reported that subjects with the SAA1.5 allele have higher plasma SAA concentrations than those without it, a phenomenon probably due to the delayed catabolism of the isotype SAA1.5. Since SAA is present in high density lipoprotein(HDL), this study assessed whether SAA genotype influenced the serum lipid study by altering HDL metabolism. In a total of 279 healthy adults, no difference was noted in their total cholesterol, HDL-cholesterol or triglyceride concentrations among six genotype groups. Plasma clearance of human apolipoprotein AI(apoAI) was studied in mice by giving HDL reconstituted with each recombinant human SAA1 isotype. The apoAI clearance did not differ among each of the SAA1 isotype-conjugated HDLs. Moreover, the changes in content of SAA in HDL also did not alter the apoAI clearance. These results suggest that SAA1 may not play an active role in plasma HDL metabolism.

• Yu J, Zhu H, Guo JT, de Beer FC, Kindy MS
• Expression of mouse apolipoprotein SAA1.1 in CE/J mice: isoform-specific effects on amyloidogenesis.
• Lab Invest. 2000; 80: 1797-806
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• Amyloid A (AA) amyloid deposition in mice is dependent upon isoform-specific effects of the serum amyloid A (SAA) protein. In type A mice, SAA1.1 and SAA2.1 are the major apolipoprotein-SAA isoforms found on high-density lipoproteins. During inflammation, both isoforms are increased 1000-fold, but only SAA1.1 is selectively deposited into amyloid fibrils. Previous studies showed that the CE/J mouse strain is resistant to amyloid induction. This resistance is not due to a deficiency in SAA synthesis, but is probably related to the unusual SAA isoform present. The CE/J mouse has a single acute-phase SAA protein (SAA2.2), which is a composite of the SAA1.1 and SAA2.1, with an amino terminus similar to the nonamyloidogenic SAA2.1. Recently, genetic experiments suggested that the SAA2.2 isoform might provide protection from amyloid deposition. To determine the amyloidogenic potential of the CE/J mouse, we generated SAA adenoviral vectors to express the various isoforms in vitro and in vivo. Purified recombinant SAA proteins demonstrated that SAA1.1 was fibrillogenic in vitro, whereas SAA2.2 was unable to form fibrils. Incubation of increasing concentrations of the nonamyloidogenic SAA2.2 protein with the amyloidogenic SAA1.1 did not inhibit the fibrillogenic nature of SAA1.1, or alter its ability to form extensive fibrils. Injection of the mouse SAA1.1 or SAA2.2 adenoviral vectors into mice resulted in isoform-specific expression of the SAA proteins. Amyloid induction after viral expression of the SAA1.1 protein resulted in the deposition of amyloid fibrils in the CE/J mouse, whereas SAA2.2 expression had no effect. Similar expression of the SAA2.2 protein in C57BL/6 mice did not alter amyloid deposition. These data demonstrate that the failure of the CE/J mouse to deposit amyloid is due to the structural inability of the SAA2.2 to form amyloid fibrils. This mouse provides a unique system to test the amyloidogenic potential of altered SAA proteins and to determine the important structural features of the protein.

• Artl A, Marsche G, Lestavel S, Sattler W, Malle E
• Role of serum amyloid A during metabolism of acute-phase HDL by macrophages.
• Arterioscler Thromb Vasc Biol. 2000; 20: 763-72
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• The serum amyloid A (SAA) family of proteins is encoded by multiple genes that display allelic variation and a high degree of homology in mammals. Triggered by inflammation after stimulation of hepatocytes by lymphokine-mediated processes, the concentrations of SAA may increase during the acute-phase reaction to levels 1000-fold greater than those found in the noninflammatory state. In addition to its role as an acute-phase reactant, SAA (104 amino acids, 12 kDa) is considered to be the precursor protein of secondary reactive amyloidosis, in which the N-terminal portion is incorporated into the bulk of amyloid fibrils. However, the association with lipoproteins of the high-density range and subsequent modulation of the metabolic properties of its physiological carrier appear to be the principal role of SAA. Because SAA may displace apolipoprotein A-I, the major protein component of native high density lipoprotein (HDL), during the acute-phase reaction, the present study was aimed at (1) investigating binding properties of native and acute-phase (SAA-enriched) HDL by J774 macrophages, (2) elucidating whether the presence of SAA on HDL particles affects selective uptake of HDL-associated cholesteryl esters, and (3) comparing cellular cholesterol efflux mediated by native and acute-phase HDL. Both the total and the specific binding at 4 degrees C of rabbit acute-phase HDL were approximately 2-fold higher than for native HDL. Nonlinear regression analysis revealed K(d) values of 7.0 x 10(-7) mol/L (native HDL) and 3.1 x 10(-7) mol/L (acute-phase HDL), respectively. The corresponding B(max) values were 203 ng of total lipoprotein per milligram of cell protein (native HDL) and 250 ng of total lipoprotein per milligram of cell protein (acute-phase HDL). At 37 degrees C, holoparticle turnover was slightly enhanced for acute-phase HDL, a fact reflected by 2-fold higher degradation rates. In contrast, the presence of SAA on HDL specifically increased (1. 7-fold) the selective uptake of HDL cholesteryl esters from acute-phase HDL by J774 macrophages, a widely used in vitro model to study foam cell formation and cholesterol efflux properties. Although ligand blotting experiments with solubilized J774 membrane proteins failed to identify the scavenger receptor-BI as a binding protein for both native and acute-phase HDL, 2 binding proteins with molecular masses of 100 and 72 kDa, the latter comigrating with CD55 (also termed decay-accelerating factor), were identified. During cholesterol efflux studies, it became apparent that the ability of acute-phase HDL with regard to cellular cholesterol removal was considerably lower than that for native HDL. This was reflected by a 1.7-fold increase in tau/2 values (22 versus 36 hours; native versus acute-phase HDL). Our observations of increased HDL cholesteryl ester uptake and reduced cellular cholesterol efflux (acute-phase versus native HDL) suggest that displacement of apolipoprotein A-I by SAA results in considerable altered metabolic properties of its main physiological carrier. These changes in the apolipoprotein moieties appear (at least in the in vitro system tested) to transform an originally antiatherogenic into a proatherogenic lipoprotein particle.

• Hayat S, Raynes JG
• Acute phase serum amyloid A protein increases high density lipoprotein binding to human peripheral blood mononuclear cells and an endothelial cell line.
• Scand J Immunol. 2000; 51: 141-6
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• Serum Amyloid A (SAA) is an acute-phase protein secreted mainly by hepatocytes and is largely associated with high-density lipoprotein (HDL) in plasma. It has been suggested that SAA alters HDL binding to the cell surface and that this in turn changes HDL-mediated cholesterol delivery to cells. Incorporation of SAA into HDL at concentrations equivalent to those found physiologically in moderate inflammation mediated a 1.5-fold increase in the binding of HDL to adherent peripheral blood mononuclear cells but had no effect on binding of the lipoprotein to the monocyte cell lines, U937 or THP-1. SAA incorporation also increased binding to an endothelial cell line, EA.hy.926. Hepatoma cells (HuH-7) showed no change in specific binding of the SAA-enriched HDL particle compared to normal HDL. These results suggest that a specific receptor for HDL-bound SAA is found on differentiated human macrophages and an endothelial cell line, which may have functional significance in lipid metabolism or other macrophage responses during inflammation.

• Hosoai H et al.
• Expression of serum amyloid A protein in the absence of the acute phase response does not reduce HDL cholesterol or apoA-I levels in human apoA-I transgenic mice.
• J Lipid Res. 1999; 40: 648-53
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• Plasma concentrations of high density lipoprotein (HDL) cholesterol and its major apolipoprotein (apo)A-I are significantly decreased in inflammatory states. Plasma levels of the serum amyloid A (SAA) protein increase markedly during the acute phase response and are elevated in many chronic inflammatory states. Because SAA is associated with HDL and has been shown to be capable of displacing apoA-I from HDL in vitro, it is believed that expression of SAA is the primary cause of the reduced HDL cholesterol and apoA-I in inflammatory states. In order to directly test this hypothesis, we constructed recombinant adenoviruses expressing the murine SAA and human SAA1 genes (the major acute phase SAA proteins in both species). These recombinant adenoviruses were injected intravenously into wild-type and human apoA-I transgenic mice and the effects of SAA expression on HDL cholesterol and apoA-I were compared with mice injected with a control adenovirus. Plasma levels of SAA were comparable to those seen in the acute phase response in mice and humans. However, despite high plasma levels of murine or human SAA, no significant changes in HDL cholesterol or apoA-I levels were observed. SAA was found associated with HDL but did not specifically alter the cholesterol or human apoA-I distribution among lipoproteins. In summary, high plasma levels of SAA in the absence of a generalized acute phase response did not result in reduction of HDL cholesterol or apoA-I in mice, suggesting that there are components of the acute phase response other than SAA expression that may directly influence HDL metabolism.

• Miida T, Yamada T, Yamadera T, Ozaki K, Inano K, Okada M
• Serum amyloid A protein generates pre beta 1 high-density lipoprotein from alpha-migrating high-density lipoprotein.
• Biochemistry. 1999; 38: 16958-62
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• Serum amyloid A protein (SAA), an acute-phase reactant in reactive amyloidosis, has high affinity for high-density lipoprotein (HDL). When SAA is added to HDL, SAA displaces apolipoprotein A-I (apoA-I) and phospholipid from the HDL particles. These dissociated components may form prebeta1-HDL because free apoA-I can associate with phospholipid to become a lipoprotein having prebeta mobility. To determine whether SAA generates prebeta1-HDL from alpha-migrating HDL, we investigated the effects of recombinant SAA on HDL subfraction concentration using nondenaturing two-dimensional gradient gel electrophoresis. When we added SAA (0.5 mg/mL) to plasma, the prebeta1-HDL concentration increased by 164% (from 4.7% +/- 1.3% to 12.4% +/- 3.2% of apoA-I, p < 0.005). The increase in prebeta1-HDL was proportional to the dose of SAA. When we added SAA to a column of Sepharose beads coupled to the isolated HDL (alpha-migrating HDL), prebeta1-HDL was dissociated from the column together with the SAA-associated HDL. In summary, we demonstrate that SAA generates prebeta1-HDL from alpha-migrating HDL. We speculate that SAA-mediated HDL remodeling may take place in inflammation.

• Kindy MS, de Beer FC
• A mouse model for serum amyloid A amyloidosis.
• Methods Enzymol. 1999; 309: 701-16

• Ohno K, Terado M, Iwata H, Inokuma H, Onishi T
• Expression of recombinant feline serum amyloid A (SAA) protein.
• J Vet Med Sci. 1999; 61: 915-20
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• Feline serum amyloid A (SAA) cDNA clone was isolated from a hepatic mRNA of a mixed-breed cat. The feline SAA cDNA clone contains 333 nucleotides and deduced amino acid sequence shows 87.4%, 73.9%, and 65.8% homology with dog, human and mouse SAA respectively. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified in the feline cDNA clone. Recombinant feline SAA (rfSAA) was expressed at high levels using pGEX bacterial expression system. Cleavage from the fusion moiety, and purification using glutathione-sepharose yielded pure soluble form of rfSAA. Antibodies generated against rfSAA were specific for feline SAA and showed no cross-reactivity with human SAA. Furthermore, antibodies against human SAA did not react with feline SAA. These results indicate that antigenicity of feline SAA is totally different from human SAA.

• Lammers M
• Assays for serum amyloid A (SAA)
• Clin Chem Lab Med. 1999; 37: 927-927

• Yamada T, Wada A, Itoh Y, Itoh K
• Serum amyloid A1 alleles and plasma concentrations of serum amyloid A.
• Amyloid. 1999; 6: 199-204
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• Serum amyloid A1 (SAA1), the predominant isotype of acute phase SAA in plasma and the predominant precursor of fibrillar deposits in reactive amyloidosis, is encoded by a gene, for which six allelic variants have been described. Recent studies proposed that the allele SAA1.3 was positively correlated with the development of reactive amyloidosis in Japanese. This study examined whether the plasma concentration of total SAA is influenced by specific SAA1 alleles. Two hundred and eighty healthy Japanese subjects were examined to determine the allelic distribution of SAA1 and SAA2 genes by the PCR-RFLP method, and to measure the total plasma SAA concentrations. SAA concentrations were significantly higher (p < 0.001) in subjects with the allele SAA1.5 than those without it, suggesting that SAA1.5 may have a distinctive feature in the process of synthesis or catabolism. Subjects with the allele SAA1.3 had lower SAA concentrations, though not statistically significant, than those with SAA1.1. There was not significant correlation of SAA2 alleles with SAA concentrations. These results are discussed in terms of amyloidogenicity.

• Solomon A et al.
• Transgenic mouse model of AA amyloidosis.
• Am J Pathol. 1999; 154: 1267-72
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• AA amyloidosis can be induced in mice experimentally through injection of certain chemical or biological compounds. However, the usefulness of this approach is limited by its dependence on exogenous inflammatory agents that stimulate cytokines to increase the synthesis of precursor serum amyloid A (SAA) protein and the transitory nature of the pathological fibrillar deposits. We now report that transgenic mice carrying the human interleukin 6 gene under the control of the metallothionein-I promoter had markedly increased concentrations of SAA and developed amyloid in the spleen, liver, and kidneys by 3 months of age. At the time of death about 6 months later, organs obtained from these animals had extensive amyloid deposits. This disease process was apparent radiographically using small-animal computer axial tomography and magnetic resonance imaging equipment. The AA nature of the amyloid was evidenced immunohistochemically and was unequivocally established by sequence analysis of protein extracted from the fibrils. The availability of this unique in vivo experimental model of AA amyloidosis provides the means to assess the therapeutic efficacy of agents designed to reduce or prevent the fibrillar deposits found in AA and other types of amyloid-associated disease.

• Sipe J
• Revised nomenclature for serum amyloid A (SAA). Nomenclature Committee of the International Society of Amyloidosis. Part 2.
• Amyloid. 1999; 6: 67-70

• Gruys E
• [Amyloidosis, a current problem]
• Tijdschr Diergeneeskd. 1998; 123: 45-9

• Gruys E, Knol BW, Meyer HP, van Oost BA
• [Amyloidosis in cats: a genetic problem?]
• Tijdschr Diergeneeskd. 1998; 123: 50-1

• Patel H, Fellowes R, Coade S, Woo P
• Human serum amyloid A has cytokine-like properties.
• Scand J Immunol. 1998; 48: 410-8
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• Human serum amyloid A (SAA) proteins are a group of 12-14 kDa apolipoproteins found predominantly in the high-density lipoprotein (HDL) fraction of plasma. Several functions have been proposed for SAA, but its primary physiological function remains elusive. In this report, we used the monocytic cell line THP-1 to investigate whether recombinant SAA1 (rSAA) or the HDL-rSAA protein complex can affect the capacity of these cells to produce inflammatory cytokines in vitro. Incubation of rSAA, plasma HDL (which contains < or = 30 microg/ml of SAA) or HDL-rSAA complex with THP-1 cells induced synthesis of IL-1beta, IL-1ra and sTNFR-II protein and mRNA. The induction of cytokine synthesis was not due to endotoxin contamination since the effect was abrogated by protein denaturation. The rSAA and HDL-rSAA complex did not induce detectable levels of IL-6 or TNFalpha protein or mRNA. In contrast 10 microg/ml LPS stimulated secretion of the inflammatory cytokines, IL-1beta, IL-6 and TNFalpha, as well as IL-1ra and sTNFR-II from THP-1 cells. We confirmed that rSAA has chemoattractant properties in vivo, by subcutaneous injections into mice and examined the histology of the injection site at 72 h, however, the HDL-rSAA complex has a substantially reduced effect.

• Johan K, Westermark G, Engstrom U, Gustavsson A, Hultman P, Westermark P
• Acceleration of amyloid protein A amyloidosis by amyloid-like synthetic fibrils.
• Proc Natl Acad Sci U S A. 1998; 95: 2558-63
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• Amyloid protein A (AA) amyloidosis is a consequence of some long-standing inflammatory conditions, and subsequently, an N-terminal fragment of the acute phase protein serum AA forms beta-sheet fibrils that are deposited in different tissues. It is unknown why only some individuals develop AA amyloidosis. In the mouse model, AA amyloidosis develops after approximately 25 days of inflammatory challenge. This lag phase can be shortened dramatically by administration of a small amount of amyloid extract containing an as yet undefined amyloid-enhancing factor. In the present study, we show that preformed amyloid-like fibrils made from short synthetic peptides corresponding to parts of several different amyloid fibril proteins exert amyloidogenic enhancing activity when given i.v. to mice at the induction of inflammation. We followed i.v. administered, radiolabeled, heterologous, synthetic fibrils to the lung and to the perifollicular area in the spleen and found that new AA-amyloid fibrils developed on these preformed fibrils. Our findings thus show that preformed, synthetic, amyloid-like fibrils have an in vivo nidus activity and that amyloid-enhancing activity may occur, at least in part, through this mechanism. Our findings also show that fibrils of a heterologous chemical nature exert amyloid-enhancing activity.

• Hulten C, Sletten K, Foyn Bruun C, Marhaug G
• The acute phase serum amyloid A protein (SAA) in the horse: isolation and characterization of three isoforms.
• Vet Immunol Immunopathol. 1997; 57: 215-27
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• Serum amyloid A (SAA) from acute phase horse serum was isolated using hydrophobic interaction chromatography, gel filtration and ion exchange chromatography. Three SAA isoforms with different isoelectric points, i.e. SAA pI 8.0, SAA pI 9.0 and SAA pI 9.7, were identified by two-dimensional electrophoresis and further characterized with amino acid sequence analysis. These isoforms were found in similar concentrations in all animals investigated, with SAA pI 9.7 constituting about half of the total SAA content. Partial amino acid sequence analysis verified the previously published heterogeneous SAA sequence. SAA pI 8.0 was found to have isoleucine in Position 16, glutamine in Position 44 and glycine in Position 59. SAA pI 9.0 had leucine, glutamine and alanine in the corresponding positions. In SAA pI 9.7 leucine, lysine and alanine were detected. The three isoforms characterized in this study are all acute phase SAAs. SAA pI 9.0 and 9.7 correspond to amyloid A protein variants previously isolated from amyloid deposits of equine liver, while there are no reports on an amyloid A variant corresponding to SAA pI 8.0.

• Yamada T, Hirano N, Kuroda T, Okuda Y, Itoh Y
• Generation and characterization of rat monoclonal antibodies against human serum amyloid A.
• Scand J Immunol. 1997; 46: 175-9
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• Monoclonal antibodies against human serum amyloid A (SAA) were generated in rat (which seems not to have mature SAA proteins) by immunizing intact human SAA. Thirteen clones selected by initial screening were analysed based on reactivity with synthetic peptides of SAA and with carboxyl-terminal truncated recombinant SAA. Antibodies were divided into four types, i.e. those recognizing the area around residue 18, 30, 90, and 100, respectively, of SAA. The antibody to the carboxyl terminus (around residue 100) of SAA, when subjected to immunohistochemistry for amyloid deposits in specimens from patients with reactive amyloidosis, always yielded negative reactivity, supporting the general concept that the carboxyl terminus of SAA is absent from human AA deposits.

• Yamada T, Sakurai A
• [Day to day changes in SAA (serum amyloid A) values of healthy subjects and the effect of dexamethasone administration]
• Rinsho Byori. 1997; 45: 696-8
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• Serum amyloid A (SAA) is a sensitive acute phase reactant; its serum level increases up to 1,000 fold of the healthy level (less than 10 micrograms/ml). Recently, the diagnostic usefulness of this protein in comparison with C-reactive protein (CRP) has been discussed in disorders or situations which show slight elevation of these protein levels, especially, in subjects receiving glucocorticoid therapies. To clarify physiological changes of this protein, day to day variations in SAA values of 23 healthy individuals were investigated and it was shown that majority (91%) had dynamic ranges of less than 3.0 micrograms/ml. Effect of dexamethasone administration on SAA and CRP values was studied in 10 subjects who received a dexamethasone suppression test. Changes in SAA values were within the physiological variation, and discrepancy in the changes between SAA and CRP as previously described in subjects with glucocorticoid therapies was not shown.

• Uhlar CM, Black IL, Shields DC, Brack CM, Schreiber G, Whitehead AS
• Wallaby serum amyloid A protein: cDNA cloning, sequence and evolutionary analysis.
• Scand J Immunol. 1996; 43: 271-6
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• A serum amyloid A (SAA) clone was isolated from a Tammar wallaby cDNA library, the most distantly related mammalian species for which an SAA has been described to date. The clone predicts a premolecule of 127 amino acids with good homology to other mammalian SAAs, and consists of an 18 residue leader peptide and a mature protein of 109 amino acids. Evolutionary analysis at both the protein and nucleotide level indicate that the wallaby SAA clone clusters with the acute phase SAAs. However, as the SAA superfamily has undergone concerted evolution it is not possible to determine at this point which acute phase SAA it is most like. The grouping of wallaby SAA inside the acute phase SAA cluster demonstrates that at least some of the duplication events giving rise to multiple acute phase genes occurred prior to the divergence of the eutherian and metatherian mammals.

• Yakar S, Livneh A, Pras M
• [Reactive amyloidosis: advances in understanding its basic mechanisms]
• Harefuah. 1996; 130: 688-93

• Malle E, De Beer FC
• Human serum amyloid A (SAA) protein: a prominent acute-phase reactant for clinical practice.
• Eur J Clin Invest. 1996; 26: 427-35
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• Serum amyloid A (SAA) proteins comprise a family of apolipoproteins synthesized in response to cytokines released by activated monocytes/macrophages. Acute-phase protein concentrations have been advocated as objective biochemical indices of disease activity in a number of different inflammatory processes. Clinical studies in large groups of patients with a variety of disorders confirmed the rapid production and exceptionally wide dynamic range of the SAA response. It is as sensitive a marker for the acute-phase as C-reactive protein (CRP). Recent studies indicate that SAA is the most sensitive non-invasive biochemical marker for allograft rejection. Further studies comparing the measurement of SAA to CRP could reveal other indications for its specific use. These studies are now more feasible given newer assays to measure this acute-phase reactant. Observations that the acutephase response is tightly coupled to lipoprotein abnormalities and the fact that acute-SAA proteins are mainly associated with plasma lipoproteins of the high density range suggested a possible role of this apolipoprotein (apo SAA) in the development of atherosclerosis. The expression of SAA mRNA in human atherosclerotic lesions and the induction of acute-phase SAA by oxidized low-density lipoproteins strengthen the hypothesis that SAA might play a role in vascular injury and atherogenesis.

• Shainkin-Kestenbaum R et al.
• Effect of serum amyloid A, HDL-apolipoprotein, on endothelial cell proliferation. Implication of an enigmatic protein to atherosclerosis.
• Biomed Pept Proteins Nucleic Acids. 1996; 2: 79-84
• Display abstract

• The possible contribution of apo-HDL serum amyloid A (SAA) to the protective effect of HDL against atherosclerosis was studied by evaluating its effect on bovine aortic endothelial cell (BAEC) proliferation. Our results suggest that human SAA, both purified and recombinant, in concentrations relevant to mild acute phase events, significantly inhibit endothelial cell proliferation in a dose-dependent manner (e.g., 50 micrograms/ml causes approximately 88% inhibition; p < 0.001). This inhibition was attenuated by addition of fibroblast growth factor (FGF), which antagonized the SAA-mediated effect. As levels of TNF may be highly elevated during the acute phase response, its effect on BAEC proliferation was evaluated and found, at concentrations of > 1 pg/ml, to be substantially inhibitory Co-incubation of cells with both SAA and TNF was inhibitory, albeit neither additive nor synergistic. FGF antagonized the effect of both proteins. Amyloidic deposit (AA, i.e. SAA 1-76), derived from pathological proteolysis of SAA, practically retains the inhibitory activity (e.g. 50 micrograms/ml causes approximately 66% inhibition; p < 0.001) but apparently lacks the regulatory site towards FGF. In contrast to the above inhibitory effect, synthetic SAA-related peptide corresponding to the sequence 29-33 of SAA enhances BAEC proliferation (50 micrograms/ml causes approximately 64% increase; p < 0.001). The present data, coupled with our previous observations in which SAA was found to induce endothelial PGI2 formation and to inhibit overproduction of PGI2 by TNF and LPS as well as platelet aggregation, may suggest that SAA contributes to the protective effect of HDL against atherosclerosis. This, by means of its modulatory effect on endothelial cell and platelet activation, primarily in the presence of other regulatory proteins. SAA-derived peptides may, potentially, be used as therapeutic agents in the treatment of atherosclerosis and cardiovascular diseases.

• Guo JT, Aldrich CE, Mason WS, Pugh JC
• Characterization of serum amyloid A protein mRNA expression and secondary amyloidosis in the domestic duck.
• Proc Natl Acad Sci U S A. 1996; 93: 14548-53
• Display abstract

• Secondary amyloidosis is a common disease of water fowl and is characterized by the deposition of extracellular fibrils of amyloid A (AA) protein in the liver and certain other organs. Neither the normal role of serum amyloid A (SAA), a major acute phase response protein, nor the causes of secondary amyloidosis are well understood. To investigate a possible genetic contribution to disease susceptibility, we cloned and sequenced SAA cDNA derived from livers of domestic ducks. This revealed that the three C-terminal amino acids of SAA are removed during conversion to insoluble AA fibrils. Analysis of SAA cDNA sequences from several animals identified a distinct genetic dimorphism that may be relevant to susceptibility to secondary amyloid disease. The duck genome contained a single copy of the SAA gene that was expressed in liver and lung tissue of ducklings, even in the absence of induction of acute phase response. Genetic analysis of heterozygotes indicated that only one SAA allele is expressed in livers of adult birds. Immunofluorescence staining of livers from adult ducks displaying early symptoms of amyloidosis revealed what appear to be amyloid deposits within hepatocytes that are expressing unusually high amounts of SAA protein. This observation suggests that intracellular deposition of AA may represent an early event during development of secondary amyloidosis in older birds.

• Yamada T, Miida T, Itoh Y, Kawai T, Benson MD
• Characterization of serum amyloid A4 as a plasma apolipoprotein.
• Clin Chim Acta. 1996; 251: 105-12

• de Beer MC et al.
• Structure of the mouse Saa4 gene and its linkage to the serum amyloid A gene family.
• Genomics. 1996; 34: 139-42
• Display abstract

• The serum amyloid A (SAA) proteins are a polymorphic family of apolipoproteins associated with high-density lipoproteins (HDL). Three distinct subfamilies have been identified: (i) a cytokine-induced acute phase subfamily that is hepatically produced and can become the major apolipoprotein on HDL (SAA1, SAA2); (ii) a peripherally produced acute phase SAA3 that is only a minor HDL apolipoprotein; and (iii) a constitutive subfamily (SAA4) that is a minor normal HDL apolipoprotein comprising more than 90% of the SAA during homeostasis. Here we define the structure of the Saa4 gene. Similar to other Saa family members, it has four exons and three introns. It is 4588 bp long from the transcription start site to the end of the 3'-untranslated region and is approximately 20% larger than other Saa genes. We have located Saa4 11 kb upstream from Saa3 and 5 kb downstream from Saa1, with the pseudogene approximately 1 kb from the 5' end of Saa4. Saa4 has the same orientation as most other Saa family members, with only Saa2 having an opposing orientation. These data promote our understanding of the evolution of the Saa family. They enhance our ability to develop the mouse as a transgenic and gene deletion model to advance the understanding of the function of these apolipoproteins.

• Yamada T, Kakihara T, Kamishima T, Fukuda T, Kawai T
• Both acute phase and constitutive serum amyloid A are present in atherosclerotic lesions.
• Pathol Int. 1996; 46: 797-800
• Display abstract

• The polymorphic protein, serum amyloid A (SAA), consists of acute phase isotypes and a constitutive isotype. Both are associated mostly with high density lipoproteins (HDL) in the circulation. In the present study, both SAA isotypes were detected by immunohistochemistry and immunoblotting using monoclonal antibodies in atherosclerotic lesions. As the distribution of SAA was identical with that of apolipoprotein B and SAA is known to be associated also with low density lipoproteins (LDL), SAA may also be delivered to the artery wall by LDL.

• Preciado-Patt L, Pras M, Fridkin M
• Binding of human serum amyloid A (hSAA) and its high-density lipoprotein3 complex (hSAA-HDL3) to human neutrophils. Possible implication to the function of a protein of an unknown physiological role.
• Int J Pept Protein Res. 1996; 48: 503-13
• Display abstract

• Serum amyloid A (SAA) is an acute-phase serum protein which exists in the body in a complex with high-density lipoprotein (HDL3). It is involved in chronic inflammation and neoplastic diseases in an as yet unknown manner. Toward an understanding of the possible physiological role of SAA we initiated a study of its association with blood proinflammatory cells with which it may interact functionally in vivo. In the following we describe the binding characteristics of recombinant human SAA to human neutrophils (polymorphonuclear leukocytes; PMNLs) and their plasma membranes. Scatchard analysis of rSAA binding and displacement curves revealed Kd in the nanomolar range. The C-terminal domain of the protein, i.e. amino acid residues 77-104, which might reside in serum following SAA degradation and amyloid A formation, was found to inhibit efficiently the binding of the whole protein to neutrophils. The interaction of SAA, and of its related peptides while complexed in HDL3, with human PMNs was also studied. The results suggest that SAA may be involved, in an as yet unknown manner, in the neutrophil-associated inflammatory mechanism.

• Gonnerman WA, Elliott-Bryant R, Carreras I, Sipe JD, Cathcart ES
• Linkage of protection against amyloid fibril formation in the mouse to a single, autosomal dominant gene.
• J Exp Med. 1995; 181: 2249-52
• Display abstract

• Inbred strains of mice provide a model for studies of the pathogenesis of amyloid A (AA) amyloidosis. All susceptible strains of mice described to date codominantly express two serum amyloid A (apoSAA) isoforms, apoSAA1 and apoSAA2, of which only apoSAA2 serves as a precursor for amyloid fibrils. In previous studies, we have shown that the CE/J strain, which produces a single, novel apoSAA isoform, apoSAACE/J, is amyloid resistant. In the present study amyloid-resistant CE/J females were mated with amyloid-susceptible CBA/J males to produce F1 hybrid offspring which were then backcrossed to the parental CBA/J mouse strain. Amyloid susceptibility was determined in 30 backcrossed mice 72 h after injection of murine amyloid enhancing factor and silver nitrate. ApoSAA isoforms in plasma were separated by isoelectric focusing gel electrophoresis and visualized after immunoblotting with anti-AA antiserum. Amyloid A fibrils in spleen homogenates were denatured by formic acid and AA protein was quantified by ELISA using anti-mouse apoSAA antibodies. Values < 5 apoSAA equivalent units were considered negative. 13 mice expressed an apoSAA1 and apoSAA2 doublet characteristic of CBA/J mice, whereas 17 mice, expressed the apoSAACE/J isoform codominantly with apoSAA1 and apoSAA2. The correlation of amyloid resistance to expression of the apoSAACE/J isoform was absolute (17/17 were negative; mean score 2.6 +/- 0.17 [standard error of the mean] apoSAA equivalent units) and the correlation between amyloid susceptibility and the expression of apoSAA2/apoSAA1 was also striking (12/13 were amyloid positive; mean score 47.9 +/- 9.0 [standard error of the mean] apoSAA equivalent units (P < 0.001). This is not significantly different from the 50% segregation of apoSAA phenotypes expected for linkage to a single gene. These results indicate that a single gene governs apoSAACE/J expression and thus confers protection against amyloid deposition even in the presence of apoSAA1 and apoSAA2 isoforms and show for the first time that resistance to AA amyloidosis is a dominant trait governed by a single gene.

• Droz D, Nochy D
• [Amyloid substance and amyloidosis]
• Ann Pathol. 1995; 15: 11-20

• Alsemgeest SP, Kalsbeek HC, Wensing T, Koeman JP, van Ederen AM, Gruys E
• Concentrations of serum amyloid-A (SAA) and haptoglobin (HP) as parameters of inflammatory diseases in cattle.
• Vet Q. 1994; 16: 21-3
• Display abstract

• The concentrations of the acute-phase proteins, serum amyloid-A (SAA) and haptoglobin (Hp), were determined in the plasma of healthy cows (n = 25) and cows with spontaneous acute (n = 6), subacute (n = 37), or chronic (n = 7) inflammatory diseases. The plasma concentration of SAA alone, Hp alone, and the Hp/SAA ratio, differed significantly (p < 0.001) between healthy animals and animals with inflammatory diseases. Plasma Hp concentrations in the group of cows with acute inflammatory diseases were significantly (p < 0.01) different from those in the group with chronic inflammatory diseases. Moreover, the Hp/SAA ratio in chronic inflammatory diseases was significantly different from this ratio in acute (p < 0.01) and subacute (p < 0.05) inflammatory diseases. It is therefore concluded that the plasma concentrations of SAA and Hp and the Hp/SAA ratio are useful parameters to distinguish healthy animals from animals with inflammation and can be helpful in distinguishing between acute and chronic of inflammatory diseases.

• Niewold TA, Murphy C, Gruys E
• Hamster hepatic amyloid A (AA) protein is derived from a novel member of the SAA gene family.
• Scand J Immunol. 1994; 39: 225-7
• Display abstract

• The nearly complete amino acid sequence of casein-induced hepatic hamster AA is described. Hamster AA appeared to be C-terminally ragged as found in other species. Furthermore, the N-terminus was (partially) truncated. Hamster hepatic AA sequence showed high homology with, but was not identical to hamster SAA-isotype sequences deduced earlier from mRNA in hepatic and extra-hepatic tissue. Therefore, hamster hepatic AA is derived from a novel member of the SAA gene family. However, the origin of hepatic AA-amyloid remains unclear.

• Syversen PV, Juul J, Marhaug G, Husby G, Sletten K
• The primary structure of serum amyloid A protein in the sheep: comparison with serum amyloid A in other species.
• Scand J Immunol. 1994; 39: 88-94
• Display abstract

• Serum amyloid A (SAA) protein was isolated from acute phase sheep sera by ultracentrifugation, gel filtration and ion-exchange chromatography. The purified protein was characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, amino acid composition and Edman degradation. Protein SAA sheep consists of 112 amino acid residues and has a blocked N-terminus. The amino acid sequence showed a high degree of homology with SAA proteins from other species, especially at positions 32 to 54, indicating that this particular part of the protein is important for its function. When compared to human protein SAA, nine inserted amino acids could be demonstrated, located in regions 69 to 77. Similar observations have been seen in cow, horse, dog, cat, and mink protein SAA. Heterogeneities were found in positions 28, 55, 63, 64, 66, 75, 77, 78, 80 and 89. Positions 63, 64, 66, 75, 77, 78 and 80 revealed the existence of a minor gene product of protein SAA sheep. The minor variant of protein SAA sheep is identical in these positions with the corresponding positions in protein SAA cow. By comparing the amino acid sequences of the different SAA proteins, two separate branches in the evolutionary pattern of protein SAA appear. One of the branches includes the species with the insertion which represents also one of the more heterogeneous part of the protein.

• de Beer MC, Kindy MS, Lane WS, de Beer FC
• Mouse serum amyloid A protein (SAA5) structure and expression.
• J Biol Chem. 1994; 269: 4661-7
• Display abstract

• A novel member of the mouse serum amyloid A protein family, SAA5, has been identified as a normal apolipoprotein component of non-acute-phase high density lipoprotein (HDL). The structure of SAA5 was derived from a clone isolated from a normal Balb/c liver cDNA library. The clone predicts a pre-SAA5 molecule of 130 residues from which an 18-residue leader peptide is cleaved. The mature molecule has an octapeptide insert spanning from position 70 to 77. Similar inserts are found in human C-SAA and, paradoxically, in acute-phase SAA molecules of a number of other species. There is 48% amino acid identity between apo-SAA5 and the other mouse SAA proteins and 57% identity between the human C-SAA and apo-SAA5. The SAA5 mRNA is three times larger than previously identified SAA mRNAs. Although SAA5 is constitutively expressed in the liver, it has a rapid albeit muted response to inflammatory stimuli. The increase of SAA5 mRNA is due to increased transcription rather than mRNA stabilization. Plasma SAA5 levels during the acute phase are biphasic, either because of translational control or displacement from HDL and rapid clearance. We propose that constitutive SAAs (SAA5) on normal HDL contribute to its normal physiological role, whereas the dramatically inducible family members (SAA1, SAA2, SAA3) equip this particle for an altered functional role during inflammation.

• Marhaug G, Dowton SB
• Serum amyloid A: an acute phase apolipoprotein and precursor of AA amyloid.
• Baillieres Clin Rheumatol. 1994; 8: 553-73
• Display abstract

• Serum amyloid A is an acute phase protein complexed to HDL as an apoprotein. The molecular weight is 11.4-12.5 kDa in different species and the protein has from 104 to 112 amino acids, without or with an insertion of eight amino acids at position 72. The protein is very well conserved throughout evolution, indicating an important biological function. The N-terminal part of the molecule is hydrophobic and probably responsible for the lipid binding properties. The most conserved part is from position 38 to 52 and this part is therefore believed to be responsible for the until now unknown biological function. The protein is coded on chromosome 11p in man, and chromosome 7 in mice, and found in all mammals until now investigated, and also in the Peking duck. In the rat a truncated SAA mRNA has been demonstrated, but no equivalent serum protein has been reported. Acute phase SAA is first of all produced in hepatocytes after induction by cytokines, but extrahepatic expression of both acute phase and constitutive SAA proteins have been demonstrated. Several cytokines, first of all IL-1, IL-6 and TNF are involved in the induction of SAA synthesis, but the mutual importance of these cytokines seems to be cell-type specific and to vary in various experimental settings. The role of corticosteroids in SAA induction is somewhat confusing. In most in vitro studies corticosteroids show an enhancing or synergistic effect with cytokines on SAA production in cultured cell. However, in clinical studies and in vivo studies in animals an inhibitory effect of corticosteroids is evident, probably due to the all over anti-inflammatory effect of the drug. Until now no drug has been found that selectively inhibits SAA production by hepatocytes. Effective anti-inflammatory or antibacterial treatment is the only tool for reducing SAA concentration in serum and reducing the risk of developing secondary amyloidosis. The function of SAA is still unclear. Interesting theories, based on current knowledge of the lipid binding properties of the protein and the relation to macrophages, in the transportation of cholesterol from damaged tissues has been advanced. A putative role in cholesterol metabolism is supported by the findings of SAA as an inhibitor of LCAT. The potential that SAA is a modifying protein in inflammation influencing the function of neutrophils and platelets is interesting and more directly related to the inflammatory process itself.(ABSTRACT TRUNCATED AT 400 WORDS)

• Yakar S et al.
• Direct evidence for SAA deposition in tissues during murine amyloidogenesis.
• Scand J Immunol. 1994; 40: 653-8
• Display abstract

• To study the mechanism of amyloid deposition, the nature of amyloid proteins formed in experimental murine amyloidosis, was examined. Spleen specimens, 15-60 mg, were homogenized and extracted using aqueous acidic acetonitrile, in a recently developed procedure, making it possible to obtain amyloid proteins from minute amounts of tissue. The extracted material, 1.5-4 mg, was analysed by Western blotting and ELISA using antibodies recognizing differentially proteins AA and SAA. Two immunoreactive proteins of 8 and 12 KDa were isolated and subjected to amino acid analysis and N-terminal sequence determination. The results of immunochemical and chemical examination showed that the 8 and 12 KDa proteins represented proteins AA and SAA, respectively. The data obtained provide new direct evidence for SAA in tissues during murine amyloidogenesis.

• Sellar GC, Whitehead AS
• Localization of four human serum amyloid A (SAA) protein superfamily genes to chromosome 11p: characterization of a fifth SAA-related gene sequence.
• Genomics. 1993; 16: 774-6
• Display abstract

• The serum amyloid A proteins (SAAs) are heterogeneous differentially expressed apolipoproteins of M(r) 12-19 kDa. Four SAA loci have been described. Two of the loci (SAA1 and SAA2) encode acute-phase SAAs (A-SAAs), which exhibit a dramatic transient increase in serum concentration in response to inflammatory stimuli; a third locus (SAA3) defines a pseudogene; and a fourth locus (SAA4) encodes a constitutively expressed SAA (C-SAA). Using locus-specific polymerase chain reaction, we have definitively assigned all four well-characterized SAA loci, SAA1, -2, -3, and -4, to chromosome 11p. In addition, we have more fully characterized an ill-defined sequence previously misidentified by others as SAA4, which also maps to chromosome 11p and may represent a fifth SAA locus.

• Rygg M, Nordstoga K, Husby G, Marhaug G
• Expression of serum amyloid A genes in mink during induction of inflammation and amyloidosis.
• Biochim Biophys Acta. 1993; 1216: 402-8
• Display abstract

• Serum amyloid A (SAA) is an acute phase protein and the precursor of amyloid protein A (AA) in deposits of secondary amyloidosis. Several isotypes exist in mink, but previous studies suggest that mink AA is derived from only one. To assess the effect of repeated episodes of inflammation and induction of amyloidosis, qualitative and quantitative changes in hepatic and extrahepatic SAA mRNA were studied. Young female mink received subcutaneous lipopolysaccharide injections for amyloid induction. Studies were performed using RNA probes and oligonucleotide probes specific for each of two SAA mRNA species. Northern blot hybridization showed that hepatic SAA1 and SAA2 mRNA levels increased dramatically after inflammatory stimulation, and were subsequently maintained at elevated levels, showing considerable interindividual variation, but only a slight decrease during repeated inflammatory stimuli and the early stages of amyloid deposition. No preferential accumulation of mRNA specifying a particular isotype was found during the experiment. Differential expression of mink SAA mRNA during repeated inflammatory stimulation does not seem to explain why only SAA2-derived AA is found in amyloid deposits. Extrahepatic SAA mRNA seemed to be independently regulated and may thus represent another, yet not characterized, SAA isotype.

• Syversen PV, Sletten K, Marhaug G, Husby G, Lium B
• The revised amino acid sequence of serum amyloid A (SAA) protein in mink.
• Scand J Immunol. 1993; 37: 696-7
• Display abstract

• The revised amino acid sequence of mink protein SAA was shown to be composed of 111 amino acid residues. The protein has an insertion of eight amino acid residues compared with that reported earlier. Microheterogeneities were observed in positions 6, 10, 24, 27, 67 and 71. The amino acid sequence is in accordance with the SAA mink cDNA sequences, except for the phenylalanine in position 6. The data indicate a third SAA gene in mink.

• de Beer MC, de Beer FC, Beach CM, Gonnerman WA, Carreras I, Sipe JD
• Syrian and Armenian hamsters differ in serum amyloid A gene expression. Identification of novel Syrian hamster serum amyloid A subtypes.
• J Immunol. 1993; 150: 5361-70
• Display abstract

• Amyloid A (AA) amyloidosis is widespread throughout the animal kingdom. Several factors including: 1) precursor production; 2) precursor structure; 3) precursor degradation; and 4) precursor/product interaction with the pentraxin serum amyloid P have been implicated in amyloidogenesis, but the exact sequence of events leading to AA fibril formation and deposition remains unclear. Most models of experimental amyloidosis, including golden Syrian hamsters (Mesocricetus auratus), involve massive and repeated inflammatory stimulation; however, the model of spontaneous amyloidosis with aging in female, but not male, Syrian hamsters permits analysis of amyloidogenic factors in the absence of inflammation. Another genus, the Armenian hamster (Cricetulus migratorius), differs from Syrian hamsters both in gender-specific serum amyloid P expression and susceptibility to AA amyloidosis. In this study, we describe novel SAA molecules in the Syrian hamster in the presence and absence of inflammation. We demonstrate that, based on isoelectric separation, the Syrian hamster SAA proteins can be separated into two broad subfamilies. Plasma SAA concentration in female Syrian hamsters increases spontaneously with age, and fragments of a basic SAA isotype expressed both hepatically and extrahepatically are selectively deposited as AA fibrils. After inflammatory stimulation, the patterns of SAA gene expression in Syrian and Armenian hamsters differ. In Syrian hamsters, both hepatic SAA mRNA and the high density lipoprotein apoSAA content increase approximately 1000-fold; in Armenian hamsters, hepatic SAA mRNA is limited in quantity and different in structure; and although plasma SAA proteins increase three- to fivefold, apoSAA is not detectable in high density lipoprotein. The results suggest that regulation and site of precursor production as well as precursor structure influence AA amyloidogenesis in these two hamster genera.

• Steel DM, Sellar GC, Uhlar CM, Simon S, DeBeer FC, Whitehead AS
• A constitutively expressed serum amyloid A protein gene (SAA4) is closely linked to, and shares structural similarities with, an acute-phase serum amyloid A protein gene (SAA2).
• Genomics. 1993; 16: 447-54
• Display abstract

• The acute-phase reactant serum amyloid A (SAA) is a polymorphic apolipoprotein encoded by a family of highly homologous and closely linked genes: SAA1, SAA2, and SAA3. We have isolated a human genomic cosmid clone containing the gene encoding a fourth, constitutively expressed member of the human SAA superfamily, C-SAA, together with an SAA2*2 (SAA2 beta) gene. The gene encoding C-SAA shares the same 5' to 3' orientation as SAA2*2 and has the characteristic four-exon structure of the other members of the SAA superfamily. The exons of the gene encoding C-SAA share only limited sequence identity with those of SAA1, SAA2, and SAA3; they specify an mRNA, represented by the CS-1 cDNA reported previously by us, which is expressed at low levels (relative to the acute-phase SAAs) in normal and acute-phase liver. The gene encoding C-SAA is located 9 kb downstream of SAA2*2 and therefore occupies the locus that has been identified as containing the SAA4 gene.

• Rygg M, Husby G, Marhaug G
• Differential expression of rabbit serum amyloid A genes in response to various inflammatory agents.
• Scand J Immunol. 1993; 38: 417-22
• Display abstract

• Serum amyloid A (SAA) is an acute-phase plasma protein which increases up to 1000-fold after an acute-phase stimulus. Several SAA genes and corresponding protein isotypes exist in individual species. Liver is the main source of production, but extra-hepatic SAA expression has been described. In this study inflammation was induced in rabbits with lipopolysaccharide, turpentine, or casein. Transcription of SAA mRNA was studied using Northern blot analysis with probes specific for three different rabbit SAA isotypes and analysed by scanning densitometry. In the stimulated liver slight variation in SAA mRNA transcription level was seen after stimulation with different inflammatory agents. After lipopolysaccharide-stimulation SAA gene expression was also seen in most of the extra-hepatic organs. After turpentine stimulation SAA mRNA was seen in the liver, the ovary, and the small intestines, and after casein stimulation only in the liver and the ovary. SAA1 and SAA2 were induced exclusively in the liver, while SAA3 was induced mainly in the extra-hepatic organs. This indicates that the SAA genes probably are independently regulated both in relation to stimulus, gene- and tissue-specificity.

• Syversen PV, Juul J, Rygg M, Sletten K, Husby G, Marhaug G
• The primary structure of rabbit serum amyloid A protein isolated from acute phase serum.
• Scand J Immunol. 1993; 37: 447-51
• Display abstract

• Serum amyloid A (SAA) protein, a sensitive acute phase protein and the precursor of protein AA in secondary amyloid, was purified from pooled acute phase rabbit serum using two different methods: isolation of protein SAA directly by octyl-Sepharose chromatography of total serum, and dissociation and isolation of apoSAA from acute phase high density lipoprotein (HDL). The protein SAA fraction obtained was further purified using gel filtration and ion exchange chromatography. Rabbit protein SAA has 104 amino acid residues, like human SAA, and has a partially blocked N terminus. The highly conserved region from position 33 to position 63 found in SAA from all species studied was confirmed also in rabbit SAA. No microheterogeneities were observed. The amino acid sequence showed extensive N-terminal homology with the rabbit amyloid A protein, except for the microheterogeneity in position 12 in protein AA. It also showed identical amino acid sequence with that deduced from the rabbit cDNA clone pSAA 55. Complete homologies were found with clone SAA 2, except for positions 22 and 78, clone SA8-1, except for positions 22 and 79 and clone SA7-3, except for position 22. This pSAA 55/SA7-3/SA8-1/SAA2-like protein was the only SAA isotype found both in total serum and in the HDL fraction. Isotypes corresponding to other SAA-like genes could not be found in this pool of acute phase rabbit sera.

• Cohen AS
• Primary (AL) amyloidosis.
• Ren Fail. 1993; 15: 429-33

• Linke RP
• Therapy of amyloid diseases.
• Ren Fail. 1993; 15: 395-400
• Display abstract

• Deposition of amyloid in the organism can lead to severe clinical symptoms and syndromes which are referred to as amyloidosis. However, amyloidosis is not a pathogenetically single disease entity. Various amyloid diseases are known which can clearly be distinguished by identifying the protein from which the amyloid is derived. Since the amyloid syndromes are pathogenetically diverse, each of the different amyloid diseases needs to be treated differently, and a type-specific amyloid therapy is mandatory. Unfortunately, an efficient therapy is not yet available for most amyloid syndromes. It is the purpose of this review to show how the different amyloid syndromes are distinguished definitively from each other and what has been successful in the effort to establish an efficient therapy of the various amyloid deposits and different amyloidoses.

• Whitehead AS et al.
• Identification of novel members of the serum amyloid A protein superfamily as constitutive apolipoproteins of high density lipoprotein.
• J Biol Chem. 1992; 267: 3862-7
• Display abstract

• A novel serum amyloid A protein (SAA) has been identified as a normal apolipoprotein component of non-acute phase high density lipoprotein. This novel SAA has been designated "constitutive" SAA (C-SAA) to distinguish it from "acute phase" SAA (A-SAA). C-SAA was partially sequenced, and immunochemical analyses indicated that it constitutes a distinct subclass of apolipoproteins within the SAA superfamily. A C-SAA cDNA clone was isolated from a human liver library and sequenced. The clone predicts a pre-C-SAA molecule of 130 residues from which an 18-residue leader peptide is cleaved. The 112-residue mature molecule is 8 residues longer than human A-SAA; the size difference is due to the presence of an octapeptide between positions 70 and 77 that is not found in the corresponding region of human A-SAA. Paradoxically, octapeptides of similar composition are found at similar positions in the A-SAAs of a number of other species. The C-SAA octapeptide specifies the first two residues of a NSS tripeptide, the only potential N-linked glycosylation site in the molecule. Studies indicate that approximately 50% of these sites are glycosylated, thereby giving rise to two size classes, 14 and 19 kDa, of C-SAA in vivo. Human acute phase liver contains little C-SAA mRNA relative to the levels of A-SAA mRNA, and the treatment of PLC/PRF/5 hepatoma cells with monocyte-conditioned medium does not induce C-SAA mRNA concentrations to detectable levels, in contrast to the massive induction of A-SAA mRNA observed. C-SAA is therefore not a major acute phase reactant.

• Smith JW, McDonald TL
• Use of ethanol-eluted hydrophobic interaction chromatography in the purification of serum amyloid A.
• Protein Expr Purif. 1991; 2: 158-61
• Display abstract

• A two-step procedure for the purification of the acute-phase reactant serum amyloid A from serum is described. A hydrophobic interaction chromatography medium, octyl-Sepharose CL4B, eluted with increasing concentrations of EtOH was used as the first step in the purification. The concentrate from this step was applied to a gel filtration column of Sephacryl S-200 and eluted with 10% formic acid. The overall recovery of purified serum amyloid A from serum was 56%. This represents the first time that serum amyloid A has been purified without the use of high concentrations of guanidine or urea. The method presented could easily be scaled up to allow the purification of large quantities of serum amyloid A or readily adapted to the purification of other serum apolipoproteins.

• Sellar GC et al.
• Dog serum amyloid A protein. Identification of multiple isoforms defined by cDNA and protein analyses.
• J Biol Chem. 1991; 266: 3505-10
• Display abstract

• Five distinct serum amyloid A (SAA) cDNA clones have been isolated from a library constructed using hepatic mRNA isolated from an individual beagle dog with canine pain syndrome. This implies the existence of at least three SAA genes in the dog genome. One clone predicts a truncated "amyloid A-like" SAA molecule and offers a possible alternative mechanism for the pathogenesis of secondary amyloidosis. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified by each of the dog cDNA clones. The existence of this peptide in all acute phase dog SAA proteins was confirmed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of acute phase high density lipoprotein and provides supporting evidence for gene conversion as a mechanism for maintaining the homogeneity of the SAA gene family within a species. Analysis of hepatic RNA following induction of an acute phase response shows a dramatic increase in SAA mRNA concentration; the SAA transcripts show a transient increase in size early in inflammation due to an increase in polyadenylation.

• Saile R, Fruchart JC
• [Serum amyloid A apolipoprotein (apo SAA). Implications in inflammation and in lipoprotein modifications]
• Ann Biol Clin (Paris). 1990; 48: 77-85
• Display abstract

• The measurement of serum amyloid A apolipoprotein (apo SAA) during acute phase inflammation offers a high interest because of its specificity, sensitivity and early increase of its levels, compared to other acute phase proteins. Furthermore apo SAA is transported in serum in association with lipoproteins, in particular with their denser subpopulation, HDL3 thus inducing their modification. The decrease in Lp AI:AII concentrations in inflammatory diseases is the consequence of the decrease in HDL3. In general the HDL3 composition was changed with a displacement of apo AI by SAA. Another interest to this protein is its relationship with amyloidosis. Apo SAA is the presumed precursor of amyloid A protein, which can be deposited in various tissues, leading to secondary amyloidosis.

• Sletten K, Husebekk A, Husby G
• The primary structure of equine serum amyloid A (SAA) protein.
• Scand J Immunol. 1989; 30: 117-22
• Display abstract

• The complete amino acid sequence of equine serum amyloid A (SAA) was elucidated. The protein consists of 110 amino acid residues and contains an 8-amino acid residue insertion tentatively located between positions 69 and 70, as compared with human SAA. Microheterogeneities were detected at positions 16, 44, and 59, compatible with the existence of more than one SAA gene in the horse. This corresponds to the situation in man and mouse. Pronounced homology with SAA from man and several animal species was observed, thus confirming the conserved structure of this acute phase reactant and apoprotein of high-density lipoprotein (HDL).

• Webb CF, Tucker PW, Dowton SB
• Expression and sequence analyses of serum amyloid A in the Syrian hamster.
• Biochemistry. 1989; 28: 4785-90
• Display abstract

• Reactive amyloidosis occurs during chronic inflammation and involves deposition of amyloid A (AA) fibrils in many organs. Amyloid A is derived by proteolysis from serum amyloid A component (SAA), a major acute-phase reactant in many species. Since spontaneous amyloidosis occurs commonly in Syrian hamsters, we have studied the structure and expression of SAA genes during inflammation in these animals. Two cDNA clones and one genomic clone were sequenced, suggesting that Syrian hamster SAA is encoded by at least two genes. Hepatic mRNA analyses showed that SAA was inducible in many hamster organs during acute inflammation. These studies also demonstrated that SAA mRNA for one isotype is maximally expressed at a site of local tissue damage.

• Naiki H, Higuchi K, Hosokawa M, Takeda T
• Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1.
• Anal Biochem. 1989; 177: 244-9
• Display abstract

• We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.

• Kluve-Beckerman B, Dwulet FE, Benson MD
• Human serum amyloid A. Three hepatic mRNAs and the corresponding proteins in one person.
• J Clin Invest. 1988; 82: 1670-5
• Display abstract

• Serum amyloid A protein (SAA) is a major acute-phase protein in humans and most other mammals. In addition, it is the serum precursor of the major protein constituent of reactive amyloid fibrils. Sequence analyses have identified a number of polymorphic forms of human SAA and amyloid A protein (AA), but the question of the number of genes encoding SAA in the human has not been addressed. In addition, there are insufficient data to predict whether one form of SAA predisposes to amyloid fibril formation. In the present study three separate SAA proteins have been isolated from the plasma of one individual and completely sequenced. While two of the SAA forms (SAA2 alpha and SAA2 beta) differ from each other only at position 71, they differ from the most abundant form (SAA1) at seven and eight other positions, respectively. Nucleotide sequencing of cDNAs from a liver library of this individual identified all three mRNs coding for these proteins and proved that: (a) the often-reported absence of arginine at the amino terminus of SAA proteins must result from proteolytic processing of the protein; (b) the polymorphism involving histidine and arginine at position 71 is present at the DNA level and therefore is not due to an event at the translational level; (c) there are at least two genes coding for human SAA. Comparison of these data to published sequences of SAA and AA proteins may help in identifying genetically determined forms of SAA which predispose to reactive amyloid fibril formation.

• Husebekk A, Husby G, Sletten K, Skogen B, Nordstoga K
• Characterization of bovine amyloid proteins SAA and AA.
• Scand J Immunol. 1988; 27: 739-43
• Display abstract

• The bovine serum amyloid A (SAA) and tissue amyloid A (AA) proteins were isolated and characterized. SAA was isolated from acute phase high density lipoprotein (HDL) of a cow suffering from acute mastitis, and was identified by amino acid sequence analysis. No AA-like protein was found in complex with HDL in serum. Amyloid fibrils isolated from a bovine kidney contained a 9 kDa AA protein and a considerable amount of a 14 kDa protein. Amino acid sequence analysis showed that the largest protein probably represents undegraded SAA. This is an interesting observation which confirms previous works indicating that SAA can be incorporated in the amyloid fibrils without a prior degradation to AA. The partial amino acid sequences of bovine SAA and AA were strikingly homologous to the sequences of corresponding proteins in man and other species.

• Ali-Khan Z, Sipe JD, Du T, Riml H
• Echinococcus multilocularis: relationship between persistent inflammation, serum amyloid A protein response and amyloidosis in four mouse strains.
• Exp Parasitol. 1988; 67: 334-45
• Display abstract

• LPS-hyporesponsive (C3H/HeJ) and LPS-sensitive (C57BL/6, CBA/J, C3H/HeSn) strains of mice were infected intraperitoneally with 50 alveolar hydatid cysts (AHC) to assess the effect of protracted severe inflammation on serum amyloid A protein (SAA) concentrations, splenic amyloid deposition, and pre- and postamyloidotic alterations in the splenic architecture. In general, the SAA concentrations in all the four mouse strains showed a moderate but steady increase throughout the course of infection. Splenic amyloid deposition commenced between 6 to 8 weeks postinfection (p.i.) when the SAA concentrations were relatively low and increased progressively until 12 weeks p.i. when 52 to 78% of the splenic parenchyma was obliterated. CBA mice which harbored the largest AHC throughout the 12-week course of infection showed the poorest SAA and amyloid responses; the situation was reversed in the C3H/HeSn strain. Histologically, most of the splenic follicles, during the stage of maximum amyloid deposition, appeared hypocellular. Their T-cell-dependent periarterial sinuses were either totally depleted of cells or contained plasma cells or myeloid cells. These results show that (a) there is no direct correlation between the intensity of inflammation, SAA concentrations, or amounts of amyloid deposition in either of the four mouse strains and (b) amyloidosis secondary to AHC infection differs from other experimental mouse models of amyloidosis in the magnitude of SAA elevation during the preamyloid phase.

• Husby G et al.
• Serum amyloid A (SAA)--the precursor of protein AA in secondary amyloidosis.
• Adv Exp Med Biol. 1988; 243: 185-92

• Dwulet FE, Benson MD
• Primary structure of amyloid fibril protein AA in azocasein-induced amyloidosis of CBA/J mice.
• J Lab Clin Med. 1987; 110: 322-9
• Display abstract

• Secondary amyloidosis was induced in CBA/J mice by subcutaneous injections of azocasein after priming with amyloid-enhancing factor. Amyloid fibrils were isolated from spleens and the subunit amyloid A (AA) protein purified by gel filtration on a column of Sepharose CL6B. The AA protein was fragmented with trypsin, cyanogen bromide, and Staphylococcus protease, and the peptides were purified by reverse-phase high-performance liquid chromatography. This protein is composed of 73 amino acid residues arranged in a single polypeptide chain with homogeneous amino and carboxyl terminals. Sequence homology with protein AA from other species is quite high with near identity for residues 31 through 54. This sequence is identical to the partial structures for CBA/J mouse AA and serum amyloid A (SAA) previously reported. It is also an exact match to a predicted 73-residue segment from one form of mouse SAA complementary DNA.

• Syversen V, Sletten K, Marhaug G, Husby G, Lium B
• The amino acid sequence of serum amyloid A (SAA) protein in mink.
• Scand J Immunol. 1987; 26: 763-7
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• The amino acid sequence of serum amyloid A (SAA) protein from mink was established by characterization of peptides derived from digestion of the protein with trypsin and from cleavage with BNPS-skatole. In three positions, two amino acid residues were found, showing that the protein is polymorphic. In position 10 both valine and isoleucine were found, while only valine was observed in protein AA. Prominent sequence homologies with protein SAA and protein AA from other species were seen, particularly corresponding to the segment between positions 31 and 54, but also in the C-terminal part of protein SAA, which is not shared by protein AA.

• Husebekk A, Husby G, Sletten K, Marhaug G, Nordstoga K
• Characterization of amyloid protein AA and its serum precursor SAA in the horse.
• Scand J Immunol. 1986; 23: 703-9
• Display abstract

• Amyloid was extracted from the liver of a horse that had developed amyloidosis after being used for several years for the production of antibodies to bacterial antigens. The amyloid fibrils were shown to be of the AA type. Two AA proteins with molecular weights of 9000 and 11,000 and with identical partial N-terminal amino acid sequences were identified. Marked structural homology with AA from other species including man was seen, although clear species-related antigenic specificity was observed. SAA isolated from an acute phase (septic abortion) horse serum was identical to AA with respect to antigenicity and the 10 first N-terminal amino acid residues that have been studied up to now. The bulk of SAA was present in the high-density lipoprotein complex in serum. Also SAA was heterogeneous with respect to size, most molecules having a molecular weight of 11,000, and a minority 9000.

• Stearman RS, Lowell CA, Peltzman CG, Morrow JF
• The sequence and structure of a new serum amyloid A gene.
• Nucleic Acids Res. 1986; 14: 797-809
• Display abstract

• The acute phase response is characterized by changes in the serum concentrations of many proteins. A 1000-fold increase in the concentration of serum amyloid A (SAA) protein occurs within 24 hours of LPS injection in the mouse. We have isolated a cDNA clone and its corresponding genomic phage for a third, previously unreported SAA protein. The sequence of the cDNA, the gene's exons and neighboring DNA are presented along with the mapping evidence supporting the gene structure.

• Shirahama T, Skinner M, Cohen AS
• Heterogeneous participation of the hepatocyte population in amyloid protein AA synthesis.
• Cell Biol Int Rep. 1984; 8: 849-56
• Display abstract

• Amyloid protein AA is believed to be synthesized in the liver in a form of its precursor, the acute phase reactant, SAA. To identify precisely the cell involved in SAA biosynthesis, immunohistochemical reaction to anti-mouse AA was analysed on the liver sections from CBA/J mice which had received a single casein injection and were sacrificed at predetermined times up to 48 hours. Two different primary localizations of the reaction were revealed. One was in the cytoplasm of certain hepatocytes. This reaction peaked at 6-8 hours after the casein injection in the intensity of the reaction and in the number of the cells involved. The hepatocytes with positive cytoplasmic reaction were scattered over the lobule and even at the peak did not exceed 20% of the total hepatocyte population. The other localization of the reaction covered linearly the surface of the hepatocyte cords, and peaked at 12-16 hours after the casein injection.

• Skogen B, Sletten K, Lea T, Natvig JB
• Heterogeneity of human amyloid protein AA and its related serum protein, SAA.
• Scand J Immunol. 1983; 17: 83-8
• Display abstract

• The heterogeneity of the human amyloid proteins SAA and AA was studied. Both proteins could be separated into several fractions by ion-exchange chromatography. Amino acid analysis of the ion-exchange-chromatographed fractions of protein AA showed that the main difference was in the length of the polypeptide. Thus, it seems that the original AA preparation consists of a mixture of AA proteins with length ranging from 66 to 78 amino acid residues. By enzymatic degradation of three different forms of SAA with kallikrein, fragments were formed with a molecular weight very similar to that of protein AA.

• Matsumura A et al.
• A novel amyloid fibril protein isolated from senescence-accelerated mice.
• Lab Invest. 1982; 47: 270-5
• Display abstract

• A protein was isolated from amyloid fibrils extracted from the liver of an inbred strain of mice (senescence-accelerated mouse) characterized by a high frequency of age-associated systemic amyloidosis. This 5200-dalton protein has a different electrophoretic mobility from the murine protein AA. Its amino acid composition also differs from that of murine protein AA and from the amyloid protein in SJL/J, the only strain for which the spontaneous occurring amyloid has been characterized biochemically and immunochemically. Sequence analysis of the protein also revealed a blocked N-terminus. Immunochemically, the protein did not react with antisera against mouse immunoglobulin components. The antiserum against the protein did not react with murine protein AA, mouse immunoglobulin components, or with mouse normal liver protein. Thus, we have characterized an amyloid protein that is distinguishable from previously reported murine amyloid proteins and is not related to immunoglobulins. The possible relationship between this protein, designated ASSAM, and ASc in human senile cardiac amyloidosis is considered.

• Bausserman LL, Saritelli AL, Herbert PN, McAdam KP, Shulman RS
• NH2-terminal analysis of four of the polymorphic forms of human serum amyloid A proteins.
• Biochim Biophys Acta. 1982; 704: 556-9
• Display abstract

• We recently demonstrated that the serum amyloid A proteins (SAA) occur in six related polymorphic forms of indistinguishable molecular weights and COOH-terminal sequence. We have now obtained very homogeneous preparations of four of these proteins and shown that their amino acid compositions are similar but not identical. Two of these, SAA1 and SAA4, have the same 20 NH2-terminal residues despite striking differences in electrophoretic mobility and solution properties. SAA5 and SAA2, respectively, lack one and three of the NH2-terminal residues common to SAA1 and SAA4. The data are consistent with the postulate that some of the SAA polymorphs are products of different genes.

• Wegelius O
• The resolution of amyloid substance.
• Acta Med Scand. 1982; 212: 273-5

• Sanchez Ramos JA et al.
• [Amyloidosis: the biochemical nature of amyloid substance. Amyloidogenesis]
• Rev Clin Esp. 1982; 167: 5-10

• Marhaug G, Sletten K, Husby G
• Characterization of amyloid related protein SAA complexed with serum lipoproteins (apoSAA).
• Clin Exp Immunol. 1982; 50: 382-9
• Display abstract

• The amyloid related protein SAA was isolated from the serum lipoproteins of three patients with connective tissue disease, one of them having amyloidosis, and from a pool of normal control sera. The bulk of SAA (apoSAA) was complexed to high density lipoprotein (HDL), but significant amounts of apoSAA were also detected in the other lipoprotein fractions. Electrofocusing revealed five-six subspecies of SAA which were distributed in similar proportions in HDL and LDL. Three of these SAA subspecies made up almost all of the apoSAA present in HDL and LDL in the sera from the patients as well as in the control. A small portion of SAA not complexed to lipoproteins was isolated corresponding to a molecular weight higher than 200,000. No particular 'amyloid prone' SAA was found in serum from the patient with amyloidosis.

• Westermark P, Sletten K
• A serum AA-like protein as a common constituent of secondary amyloid fibrils.
• Clin Exp Immunol. 1982; 49: 725-31

• Bausserman LL, Herbert PN, McAdam KP
• Serum amyloid A polymorphism in a subject with a naturally occurring inflammatory response.
• Clin Chim Acta. 1982; 118: 201-7

• Missmahl HP, Bohmer R
• [Amyloid, amyloid precipitates, amyloid diseases]
• Fortschr Med. 1981; 99: 1341-2

• Takahashi Y, Muro H, Goto M, Shirasawa H
• Purification of amyloid fibrils and protein AA from mouse amyloid deposits induced by caseinate and M. butyricum.
• Acta Pathol Jpn. 1981; 31: 27-33
• Display abstract

• Amyloidosis was induced in mice by the simultaneous injection of sodium caseinate and heat-killed M. butyricum. Amyloid fibrils were isolated by collagenase digestion, 1 M NaCl extraction and repeated washing with 0.15 M NaCl. The amyloid fibril fraction was practically free of contaminations such as collagen, chromatin and membranes as judged by electron microscopic morphometry. The protein AA was purified from the isolated fibrils to an apparent homogeneity as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis using one step gel filtration from Sephacryl S-200 in the presence of 6 M guanidine-HCl and 50 mM dithiothreitol. The molecular weight of the peptides of the protein AA were 8,500 and 10,000.

• Strachan AF, Johnson PM
• Amyloid proteins.
• Lancet. 1980; 1: 1420-1420

• Bausserman LL, Herbert PN, McAdam KP
• Heterogeneity of human serum amyloid A proteins.
• J Exp Med. 1980; 152: 641-56
• Display abstract

• Serum amyloid A proteins (SAA), presumed precursors of the tissue amyloid A proteins (AA) characteristic of secondary amyloidosis, have been isolated from the plasma high-density lipoproteins (HDL) of normals after etiocholanolone-induced inflammation and from patients with Wegener's granulomatosis, systemic lupus erythematosis, juvenile rheumatoid arthritis, Waldenstrom's macroglobulinemia, and Goodpasture's syndrome. At least six polymorphic forms of SAA wer identified among the low molecular weight proteins of HDL, and these comprosed up to 27% of the total HDL protein. Gel and ion-exchange chromatography permitted isolation of the SAA polymorphs in homogeneous form. Their amino acid compositions were very similar, they were indistinguishable in cationic and sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems, and each had the terminal sequency COOH-Tyr-Lys-Phe-. Charge heterogeneity in anionic-urea polyacrylamide gel electropherograms was unaffected by neuaminidase treatment, and none of the SAA protein bands stained with the periodate-Schiff reagent. The two major SAA polymorphs, designated SAA4 and SAA5 according to their order of elution from DEAE-cellulose, had different NH2-terminal sequences. Manual Edman degradation demonstrated NH2-arg-ser-phe-phe- for SAA4 and NH2-ser-phe-phe- for SAA5. This NH2-terminal heterogeneity corresponds to that most frequently reported for AA and suggests that microheterogeneity in SAA may underlie that already documented in AA. Sufficient quantitites of the other SAA polymorphs were not available for similar analyses, but the amino acid compositions do not indicate that NH2-terminal heterogeneity accounts for all of the observed polymorphism. Artifactual polymorphism also appears unlikely, and the heterogeneiy of SAA may reflect origin from more than one cell type with or without posttranslational modificaton. We calculate from quantitative COOH-terminal analyses that SAA is of 11,000-11,900 mol wt. Primary structure studies have shown AA t be a single chain protein of 76 residues, and SAA, therefore, appears to contain a peptide of 33 amino acids that is missing from AA.

• Franklin EC, Lavie G, Zucker-Franklin D
• Current concepts of the structure and pathogenesis of the amyloid diseases.
• Ric Clin Lab. 1980; 10: 3-7

• Lovisetto P, Gilardi E, Frascisco M, Marchi L, Mairano D
• [Amyloidosis. I. Recent findings concerning amyloid substance]
• Minerva Med. 1980; 71: 1793-800
• Display abstract

• The considerable progress made recently in the study of amyloid substance have led to the identification of numerous organised protein components in typical microfibrillar structures. In spite of the biochemical heterogeneity of fibril proteins, it is still possible to find similar chemicophysical and tintorial features in the various types of amyloid, probably due, at least in part, to the common Beta type molecular configuration, a structure proper to fibril proteins. In so-called primary amyloidosis and in that associated with myelomatous diseases, the principal protein component consists of AL protein, correlated with the light immunoglobulin chains, with which analogies have been observed both in the amino acid sequence and in antigenic characteristics. In secondary amyloidosis, AA protein, which is unrelated to immunoglobulins or other known human proteins, is prevalent. AA protein probably derives from a serum globulin, SAA, whose blood levels increase during numerous pathological processes, particularly in those of neoplastic or inflammatory type. The origin of serum protein, which might be either a normal tissue component released under stimulus or a reagent of the acute phase synthesised ex novo, and its function, which is probably of immunomodulator or more specifically immunosuppressive type, are still to be defined. In all forms of amyloidosis studied, a common observation is the presence of AP protein, organised in pentagonal structures. This protein would appear to derive from a serum component defined as SAP, with a marked affinity for amyloid fibrils. Also identifiable are other forms of amyloid such as APUD-amyloid, which probably derives from polypeptide hormones, and AS amyloid, which is present in some organs of elderly patients and is biochemically identifiable at cardiac level with A(SCA) protein. Still awaiting definition in amyloid tumours or amyloidomas is the precise chemical composition of deposited proteins.

• Holck M, Husby G, Sletten K, Natvig JB
• The amyloid P-component (protein AP): an integral part of the amyloid substance?
• Scand J Immunol. 1979; 10: 55-60
• Display abstract

• The P-component of amyloid (protein AP) appears to be present in all types of amyloid substance regardless of the clinical category of amyloidosis or the chemical class of the amyloid fibril. The role of protein AP in the formation of amyloid substance has not been established. In a patient with primary amyloidosis, significant amounts of protein AP were found closely associated with the amyloid fibril proteins and was released from the latter only after dissociation and reduction of the amyloid fibril preparation. EDTA seemed to be very effective in releasing protein AP, and it is thought that the close association between the amyloid fibrils and protein AP is calcium-dependent. The very close association between the amyloid fibrils and protein AP suggests that the latter is an integral part of the amyloid substance.

• Skinner M, Shirahama T, Benson MD, Cohen AS
• Murine amyloid protein AA in casein-induced experimental amyloidosis.
• Lab Invest. 1977; 36: 420-7
• Display abstract

• Amyloidosis was induced in mice by 25 subcutaneous injections of casein. The splenic amyloid fibrils were identified by electron microscopy to be closely associated with reticular cells. After isolation of the fibrils by simple physical techniques, their ultrastructure revealed single filaments of 80 to 100 A width, which were rigid, nonbranching, and of indeterminate length. This is comparable to previous studies on human preparations. The amyloid fibrils were dissociated by solution in guanidine and chromatography. The resultant amyloid fibril protein was characterized as to its molecular weight, amino acid analysis, and amino-terminal sequence. It was thus definitely identified as protein AA, the major component of secondary amyloidosis. An antibody to this protein, murine AA, identified a cross-reacting mouse serum protein SAA and indicated a species specificity when tested against human preparations. A comparison is made with the AA protein in another murine model as well as AA proteins from human, guinea pig, monkey, and mink amyloidosis.

• Husby G, Natvig JB, Sletten K, Nordstoga K, Anders RF
• An experimental model in mink for studying the relation between amyloid fibril protein AA and the related serum protein SAA.
• Scand J Immunol. 1975; 4: 811-6
• Display abstract

• Experimental amyloidosis was induced in mink by repeated injections with endotoxin. Amyloid fibrils extracted from liver and spleen were fractionated by gel filtration after treatment with guanidine-hydrochloride and a reducing agent, dithiothreitol. An elution profile very similar to that of human amyloid fibrils, having protein AA as a major component, was obtained. The mink amyloid protein eluted at a position similar to that of human protein AA was by amino acid composition and partial sequence studies shown to be very similar to the latter protein and was called mink protein AA. In addition, a protein AA-related component (protein SAA) was found in increased amounts in serum of amyloidotic mink, providing further evidence of the homology with human amyloids. Experimental amyloidosis in mink represents a suitable model for studying amyloid proteins and related serum components.

• Lian JB, Benson MD, Skinner M, Cohen AS
• A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA.
• Arch Biochem Biophys. 1975; 171: 197-205

• Skinner M, Cathcart ES, Cohen AS, Benson MD
• Isolation and identification by sequence analysis of experimentally induced guinea pig amyloid fibrils.
• J Exp Med. 1974; 140: 871-6

• Baranov VN
• [Fine structure of amyloid]
• Biull Eksp Biol Med. 1973; 76: 119-21

• Kazimierczak J
• Ultrastructural observations on the first amyloid to be found in the spleen of casein treated mice.
• Acta Pathol Microbiol Scand [A]. 1972; 233: 141-50

• Benditt EP, Eriksen N
• Chemical classes of amyloid substance.
• Am J Pathol. 1971; 65: 231-52

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