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R Kisilevsky

Publications and source records attributed to R Kisilevsky.

At least 37 records · Page 2Linked to original sources
PubMed1998

A method based on ICP-MS for the analysis of Alzheimer's amyloid plaques.

Inductively coupled plasma mass spectrometry (ICP-MS) was combined with flow injection (FI) and a selective extraction procedure (which isolates Alzheimer's amyloid plaques) for the multielemental analysis of plaque cores. FI-ICP-MS was also used to analyze the various reagents involved in the sample preparation to determine whether they were the source of the various elements detected in the plaque samples. An external calibration with matrix-matched standards (in terms of salt concentration) was carried out in all cases. The concentrations of Cr, Mn, Ni, Cu, and Pb were in the 0.2-0.8 mg L-1 range whereas that of Al, Fe, and Zn were 2-20 mg L-1 in the plaque sample. These values can be translated into a microgram per gram level in the plaque core by multiplying them by 29-500 (the exact factor depends on the weight of the plaques, which were not dried to prevent the loss of volatile elements). Although spectroscopic interferences arising from matrix elements of the sample cannot be ruled out in the case of Al, Cr, Ni, Cu, and Fe, the large levels detected (especially for Al and Fe), compared to the much lower or undetectable levels in the various reagents, strongly suggest the accumulation of these elements by the AD patient during life rather than contamination during sample processing.

Alzheimer Disease↗
PubMed1998

Comparison of the binding and endocytosis of high-density lipoprotein from healthy (HDL) and inflamed (HDL(SAA)) donors by murine macrophages of four different mouse strains.

Serum amyloid A (SAA) is a plasma acute phase protein and the precursor of the AA-fibril protein deposited in AA-amyloidosis. SAA is bound mainly to high-density lipoproteins (HDL(SAA)). Previous investigations have demonstrated that peritoneal macrophages (mO) from mice are capable of binding and endocytosing HDL(SAA). This observation may indicate a pathway by which SAA enters the mO and where its intracellular metabolism may be followed by degradation and/or amyloidogenesis. Since binding and internalization defects of lipoproteins may be associated with different diseases, it is possible that mouse strain susceptibility to amyloidosis is associated with qualitative differences in the binding and internalization of HDL(SAA). To test this hypothesis a series of binding and internalization experiments was performed in vitro with mO from four different mouse strains, CD-1, A/J, C57BL/6J and C3H/HeJ, which differ in their susceptibility to AA-amyloidosis. Using colloidal gold-labelled lipoproteins, it was evident by light and electron microscopy that mO from all four mouse strains are capable of binding and internalizing HDL (without SAA) and HDL(SAA). HDL and HDL(SAA) were found in such compartments of the receptor-mediated pathway as coated pits, coated vesicles, endosomes and multivesicular bodies and in lipid droplets; no qualitative differences were observed. Therefore, it is unlikely that a defect in binding and uptake of HDL(SAA) is related to the different susceptibilities of these mouse strains to develop AA-amyloidosis. However, the results do not exclude the possibility that differences in the intracellular processing of SAA following endocytosis of HDL(SAA) is involved in this differing susceptibility.

Amyloidosis↗
PubMed1998

A high resolution ultrastructural comparison of isolated and in situ murine AA amyloid fibrils.

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

Animals↗
PubMed1998

Sarcoid, amyloid, and acute myocardial failure.

We report the case of a 58-year-old woman, with an 8-year history of pulmonary sarcoidosis, who died in acute myocardial failure after cardiac catheterization. Systemic amyloid, likely of the AL type, was found to involve her myocardial interstitium and intramyocardial coronary arteries, as well as her spleen, thyroid, kidneys, and bone marrow. Her acute myocardial failure was thought to be the result of focal myocardial infarcts secondary to amyloid vasculopathy and of superimposed chronic restrictive myocardial function caused by amyloid. Five previously reported cases of concurrent sarcoidosis and AA-type amyloid remind us that there is significant experimental data implicating the inflammatory process of sarcoidosis in the causation of AA-type amyloidosis. The concurrence of sarcoidosis and AL-type systemic amyloid seen in this patient was reported in the literature once before, and we think the concurrence was a coincidence.

Amyloidosis↗
PubMed1997

Can deposition of amyloid be prevented in Alzheimer's disease?

Amyloid is a generic term referring to extracellular fibrillar protein deposits defined by their unique tinctorial, ultrastructural, and protein conformational properties. At least 17 different forms have been identified. In each form the deposit consists of a disease-specific (or pathologic process-specific) protein and a set of common components. The disease-specific protein serves as the basis for the classification of the amyloids. In inflammation-associated (AA) amyloid it can be demonstrated that interactive processes between serum amyloid A (SAA), the AA precursor, and the common components, are likely responsible for AA amyloid deposition. Understanding the details of these interactions provide targets for therapeutic interference that are successful in vivo. Analogous interactions take place between the common components and the beta-protein and beta-protein precursor responsible for the congophilic angiopathy and neuritic plaque amyloid in Alzheimer's disease. Interference with beta-protein/common component interaction in vitro both prevents and reverses beta-protein fibril assembly, indicating that successful delivery of effective agents across the blood-brain barrier should prevent and possibly reverse amyloid deposition in Alzheimer's disease.

Alzheimer Disease↗
PubMed1997

Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse.

Acute inflammation results in a profound change in the apolipoprotein composition of high density lipoprotein (HDL). Several isoforms of the serum amyloid A (SAA) family, SAA1 and SAA2, become major components of HDL. This structural relationship has suggested that acute phase SAA plays some as yet unidentified role in HDL function, possibly related to cholesterol transport, during the course of acute inflammation. Using subcutaneous AgNO3 to induce a sterile abscess changes in plasma cholesterol and SAA were monitored over the subsequent 144 h. Total plasma cholesterol began to increase within 12 h of the induction of inflammation and reached a peak in 24 h. Thereafter its plasma levels fell returning to normal values by 96-120 h. The bulk of the increase in plasma cholesterol was found in the free cholesterol fraction of HDL. This pattern of cholesterol increase corresponds to the established temporal changes for acute phase SAA (AP-SAA). AP-SAA levels increased within 8 h of the induction of inflammation and reached a peak at 24 h. They began to decrease by 48 h with small quantites still present 120 h later. In concert, but inversely, with the changes in AP-SAA the apoA-I, apoA-II, and apo-E, content of HDL decreased during the AP-SAA increases and increased as AP-SAA levels fell. The plasma appearance of cholesterol from the periphery, and central parts of the inflammatory site was assessed by the use of radiolabelled cholesterol. The peripherally placed cholesterol rapidly reached a peak plasma concentration within 24 h of injection. Cholesterol placed in the central part of the sterile abscess, a site relatively inaccessible to the vasculature required 48 h to reach its peak and was 5-times lower than that placed peripherally. The influence of AP-SAA on neutral cholesterol ester hydrolase (nCEH) activity in mouse liver homogenates, mouse peritoneal macrophage homogenates, and a purified porcine pancreatic enzyme with nCEH activity was also assessed. Following optimization with regard to pH, bile salt concentration, protein concentration and incubation time, mouse peritoneal macrophages had a significantly higher nCEH specific activity than that found in liver (7-8 fold). Purified AP-SAA, assessed over a concentration range of 0-10 microg/ml, enhanced nCEH activity at concentrations above 2 microg/ml. The nCEH activity, regardless of its source, increased by 3-7 fold in the presence of AP-SAA. Equivalent concentrations of apolipoprotein A-I (apo A-I) and bovine serum albumin (BSA) failed to alter the activity of nCEH. The effect of AP-SAA on a purified form of nCEH suggests that AP-SAA may have a direct effect on the activity of this enzyme. The temporal correlation of circulating AP-SAA and plasma cholesterol and the significant stimulation of nCEH by AP-SAA (but not apoA-I or BSA) provides further evidence that AP-SAA plays a role in cholesterol metabolism during the course of acute inflammation.

Animals↗
PubMed1997

Characterization of high affinity binding between laminin and the acute-phase protein, serum amyloid A.

Serum amyloid A isoforms, apoSAA1 and apoSAA2, are acute-phase proteins of unknown function and can be precursors of amyloid AA peptides (AA) found in animal and human amyloid deposits. These deposits are often a complication of chronic inflammatory disorders and are associated with a local disturbance in basement membrane (BM). In the course of trying to understand the pathogenesis of this disease laminin, a major BM glycoprotein, has been discovered to bind saturably, and with high affinity to murine acute-phase apoSAA. This interaction involves a single class of binding sites, which are ionic in nature, conformation-dependent, and possibly involve sulfhydryls. Binding activity was significantly enhanced by Zn2+, an effect possibly mediated through Cys-rich zinc finger-like sequences on laminin. Collagen type IV also bound apoSAA but with lower affinity. Unexpectedly, no binding was detected for perlecan, a BM proteoglycan previously implicated in AA fibrillogenesis, although a low affinity interaction cannot be excluded. Entactin, another BM protein that functions to cross-link the BM matrix and is normally complexed with laminin, could inhibit laminin-apoSAA binding suggesting apoSAA does not bind to normal BM. Since laminin binds apoSAA with high affinity and has previously been shown to codeposit with AA amyloid fibrils, we postulate that laminin interacts with apoSAA and facilitates nucleation events leading to fibrillogenesis. This work also provides further support for the hypothesis that a disturbance in BM metabolism contributes to the genesis of amyloid. The specificity and avidity of the laminin-apoSAA interaction also implies that it may be a normal event occurring during the inflammatory process, which mediates one or more of the functions recently proposed for apoSAA.

Animals↗
PubMed1997

Ultrastructural organization of hemodialysis-associated beta 2-microglobulin amyloid fibrils.

Fibrils of hemodialysis-associated beta 2-microglobulin amyloid were examined by high resolution electron microscopy and immunohistochemical labeling. The amyloid containing tissues obtained through autopsy were prepared for thin section observations. In contrast to other forms of amyloid, the most conspicuous feature of these fibrils were their curved conformations. The fibril core showed ultrastructural and immunohistochemical features in common with the core of connective tissue microfibrils and of previously observed fibrils of experimental murine AA amyloidosis and familial amyloid polyneuropathy (FAP). The core was wrapped in a layer of 3 nm wide ribbon-like "double tracked" structures identified as chondroitin sulfate proteoglycan (CSPG) with immunogold labeling as well as from the results of previous in vitro experiments. Finally, the outer surface of the fibril was associated with a loose assembly of 1 nm wide filaments immunohistochemically identified as beta 2-microglobulin. This is similar to the manner in which AA protein and transthyretin filaments are associated with their respective fibrils. The results of this study provide an additional example for the concept that amyloid fibrils in general are microfibril-like structures externally associated with amyloid protein filaments. An unusual feature of the fibrils of hemodialysis-associated amyloid, however, is the presence of a peripheral layer composed of CSPG rather than of heparan sulfate proteoglycan (HSPG) as in the case of the other two amyloids above. These chondroitin sulfate chains in the outer CSPG layer may be less effective in providing rigidity to the fibril core, thus allowing for the curved conformations of beta 2-microglobulin amyloid fibrils.

Aged↗
PubMed1997

A beta amyloidogenesis: unique, or variation on a systemic theme?

For more than a century amyloid was considered to be an interesting, unique, but inconsequential pathologic entity that rarely caused significant clinical problems. We now recognize that amyloid is not one entity. In vivo it is a uniform organization of a disease, or process, specific protein co-deposited with a set of common structural components. Amyloid has been implicated in the pathogenesis of diseases affecting millions of patients. These range from Alzheimer's disease, adult-onset diabetes, consequences of prolonged renal dialysis, to the historically recognized systemic forms associated with inflammation and plasma cell disturbances. Strong evidence is emerging that even when deposited in local organ sites significant physiologic effects may ensue. With emphasis on A beta amyloid, we review the present definition, classification, and general in vivo pathogenetic events believed to be involved in the deposition of amyloids. This encompasses the need for an adequate amyloid precursor protein pool, whether precursor proteolysis is required prior to deposition, amyloidogenic amino acid sequences, fibrillogenic nucleating particles, and an in vivo microenvironment conducive to fibrillogenesis. The latter includes several components that seem to be part of all amyloids. The role these common components may play in amyloid accumulation, why amyloids tend to be associated with basement membranes, and how one may use these findings for anti-amyloid therapeutic strategies is also examined.

Alzheimer Disease↗
PubMed1996

Laminin interactions important for basement membrane assembly are promoted by zinc and implicate laminin zinc finger-like sequences.

Laminin is an abundant basement membrane (BM) glycoprotein which regulates specific cellular functions and participates in the assembly and maintenance of the BM superstructure. The assembly of BM is believed to involve the independent polymerization of collagen type IV and laminin, as well as high affinity interactions between laminin, entactin/nidogen, perlecan, and collagen type IV. We report here that Zn2+ can influence laminin binding activity, in vitro. Laminin contains 42 cysteine-rich repeats of which 12 contained nested zinc finger consensus sequences. Recently, the entactin binding site was mapped to one of these zinc finger-containing repeats on the laminin gamma chain (Mayer, U., Nischt, R., Poschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., and Timpl, R. (1993) EMBO J. 12, 1879-1885). Based on these observations, the effect of a series of essential ions (Ca2+, Cd2+, Cu2+, Mg2+, Mn2+, and Zn2+) on laminin binding activity was evaluated. Zn2+ was found to be the most effective at enhancing laminin-entactin and laminin-collagen type IV binding. Laminin-bound Zn2+ was detected by flame atomic absorption spectroscopy at a maximum of 8 mol/mol of laminin. Furthermore, Ca2+-dependent laminin polymerization was unaffected by Zn2+, an observation consistent with the lack of zinc finger-containing repeats in the terminal globular domains required for polymerization. We conclude that Zn2+-laminin complexes may generate high affinity binding sites which contribute to BM cross-linking important for its assembly and homeostasis. Zinc is likely a cofactor for 2 kinds of cross-linking interactions; one involving direct binding between laminin and collagen type IV and the other a ternary complex of laminin-entactin-collagen type IV.

Amino Acid Sequence↗
PubMed1996

Proteoglycans and amyloid fibrillogenesis.

A brief discussion of the general structure of proteoglycans is followed by a description of the diverse nature of amyloids. Using the murine form of inflammation-associated (AA) amyloid, we have examined the temporal and anatomical relationship between the heparan sulfate proteoglycan, its mRNA and AA amyloid deposition in vivo. The in vitro effect of heparan sulfate on the secondary structure of amyloid precursors, and on amyloid peptides, suggests that this interaction is important in amyloidogenesis. The relationship of these two components likely reflects a more general process taking place between basement membrane proteins (which may be synthesized by a variety of cell types within and outside the CNS) and amyloid precursors. A general definition of in vivo amyloid deposits emerges from these considerations as do concepts for interfering with amyloidogenesis. Preliminary results showing the effect of small molecule aliphatic sulfonates and sulfates on in vitro amyloid beta-protein fibrillogenesis and AA amyloidogenesis in vivo supports the general process presented and suggests therapeutic strategies for treating amyloid-based diseases.

Amino Acid Sequence↗
PubMed1996

Anti-amyloid drugs: potential in the treatment of diseases associated with aging.

The requirements for amyloidogenesis, as it is currently understood, include an adequate amyloid precursor pool, a nidus for fibrillogenesis, interactions with a set of common components (most of which are involved in basement membrane structure) and amyloid turnover. These factors serve as the basis for therapeutic attack. General strategies focusing on each of these factors are presented with examples from the experimental and clinical literature. These include reducing the amyloid precursor protein pool in familial amyloid polyneuropathy by liver transplantation, inhibiting nidus formation in familial Mediterranean fever by the use of colchicine, inhibiting amyloid precursor protein/heparan sulphate interaction in experimental inflammation-associated amyloidosis by the use of novel small molecule anionic sulphates and sulphonates, and the use of new analogues of doxorubicin in light chain amyloidosis to accelerate amyloid removal. The potential significance of local and systemic amyloid deposits is discussed in the light of new information on the genetics of Alzheimer's disease, observations made in patients receiving long term dialysis for renal failure, and the potential involvement of amyloid deposits in the pathogenesis of non-insulin-dependent diabetes mellitus.

Aged↗
PubMed1996

A high resolution ultrastructural study of experimental murine AA amyloid.

An essential and distinguishing feature of all amyloids is the presence of fibrillar structures of approximately 10-nm width. The precise nature of the fibril is not yet clearly understood, particularly in situ, and the ultrastructure of isolated fibrils differs significantly from that of fibrils observed in situ. The fibrils are generally believed to be composed of a protein specific to each type of amyloid, but increasing evidence suggests additional associations with other components such as heparan sulfate proteoglycan (HSPG) and amyloid P component (AP). Experimental AA amyloidosis was induced in mice by amyloid enhancing factor and an inflammatory stimulus (subcutaneous AgNO3); fibrils were thereafter examined in detail. Particular attention was paid to ultrastructural characteristics known to represent particular molecular components of basement membranes such as HSPG and AP. Additionally, rabbit anti-mouse AA antisera was used with 5-nm and 1-nm gold particles to establish the location of the AA protein in-situ. Amyloid fibrils could be identified in their mature form as well as at apparent intermediate stages of formation. The fibril contained an apparent core which is composed of an assembly of 3.5-nm wide pentosomal particles having the characteristics of AP. Wound around the AP assembly in a helical fashion is a "double tracked" ribbon-like entity, 3 nm wide, having the morphologic characteristics of chondroitin sulfate proteoglycan (CSPG). Covering the surface of this structure is a second ribbon-like double track structure, but this one is wider (4.6 nm vs 3.0 nm) than the CSPG. These have the ultrastructural characteristics of HSPG. Routine fixation and tissue preparation techniques that usually remove HSPG from microfibrils did not do so with amyloid fibrils, suggesting an alteration in affinity between these components. The AA protein could be identified as a 1 - to 2-nm filament network on the most exterior surface of the fibril. The ultrastructure of AA amyloid fibrils in situ resembles that of connective tissue microfibrils, and, in addition to AA protein, is likely composed of HSPG, CSPG, and AP. Amyloid fibrils can be distinguished from microfibrils by the apparently stronger binding of HSPG to the surface of the amyloid fibril and the presence of the AA filaments. A model of the in situ organization of AA amyloid fibrils is proposed.

Amyloidosis↗
PubMed1996

Effect of poly(vinylsulfonate) on murine AA amyloid: a high-resolution ultrastructural study.

In experimental murine inflammation-associated amyloidosis (AA amyloidosis), an interaction between heparan sulfate and serum amyloid A (SAA), the AA precursor, has been demonstrated and is believed to play an important role in AA amyloidogenesis. Poly(vinylsulfonate) sodium salt (PVS) can arrest AA amyloid induction and cause established amyloid deposits to regress. PVS is thought to have this property by virtue of limited anionic structural similarities it has to heparan sulfate. In the present study, a comparison has been made of the in situ light microscopic and high-resolution ultrastructure of amyloid deposits before and after PVS treatment. As shown recently in situ, AA fibrils from untreated mice are composed of an outer layer of heparan sulfate proteoglycan and a 1- to 2-nm filament network of AA protein. This layer encloses a microfibril-like structure composed of chondroitin sulfate proteoglycan wound around a core of amyloid P component. After treatment with PVS, both the heparan sulfate proteoglycan and the AA filament network are lost from the fibrils, and the more central portion disintegrates into the chondroitin sulfate proteoglycan with associated amyloid P subunits. These findings add further support to the concept that heparan sulfate proteoglycan is important in amyloid fibril structure, and interference with its binding interactions with the amyloid filament protein provides a point of therapeutic attack.

Amyloidosis↗
PubMed1995

Influence of monosaccharide derivatives on liver cell glycosaminoglycan synthesis: 3-deoxy-D-xylo-hexose (3-deoxy-D-galactose) and methyl (methyl 4-chloro-4-deoxy-beta-D-galactopyranosid) uronate.

An improved, convenient synthesis of 3-deoxy-D-xylo-hexose (3-deoxy-D-galactose) has been developed, and the chemical synthesis of a novel monosaccharide derivative, methyl (methyl 4-chloro-4-deoxy-beta-D-galactopyranosid)uronate (compound 10), is described. Using primary hepatocytes in culture, each was used to explore its effect on glycosaminoglycan (GAG) synthesis. In the absence of analogues hepatocytes synthesize primarily (92-95%) heparan sulphate. At 1 mM, 3-deoxy-D-galactose had little observable effect on either liver cell GAG or protein synthesis. At 10 mM and 20 mM, 3-deoxy-D-galactose reduced [3H]glucosamine and 35SO4 incorporation into hepatocyte cellular GAGs to, respectively, 75% and 60% of the control cells. This inhibition of GAG synthesis occurred without any effect on hepatocyte protein synthesis, indicating that 3-deoxy-D-galactose's effect on GAG synthesis is not mediated through an inhibition of proteoglycan core protein synthesis. Furthermore, GAGs in the presence of 20 mM of the analogue were significantly reduced in size, 17 kDa vs. 66 kDa in untreated cells. These results reflect either impaired cellular GAG chain elongation, and/or altered GAG chain degradation. Compound 10 exhibited a concentration-dependent inhibition of both hepatocyte cellular GAG and protein synthesis. At concentrations of 5, 10 and 20 mM, compound 10 inhibited GAG and protein synthesis by 20, 65 and 90%, respectively. Exogenous uridine was able to restore partially the inhibition of protein synthesis, but was unable to reverse the effect of compound 10 on GAG synthesis. These results show that part of the effect of compound 10 on GAG synthesis is not mediated by an inhibition of proteoglycan core protein synthesis. GAGs in the presence of compound 10 are half as large as those in the absence of this compound (33 and 66 kDa, respectively). These results again may reflect either impaired cellular GAG chain elongation and/or altered GAG chain degradation. Potential metabolic routes for each analogue's effect are presented.

Animals↗
PubMed1995

Arresting amyloidosis in vivo using small-molecule anionic sulphonates or sulphates: implications for Alzheimer's disease.

Amyloid is a term for extracellular protein fibril deposits that have characteristic tinctorial and structural properties. Heparan sulphate, or the heparan sulphate proteoglycan perlecan, has been identified in all amyloids and implicated in the earliest stages of inflammation-associated (AA) amyloid induction. Heparan sulphate interacts with the AA amyloid precursor and the beta-peptide of Alzheimer's amyloid, imparting characteristic secondary and tertiary amyloid structural features. These observations suggest that molecules that interfere with this interaction may prevent or arrest amyloidogenesis. We synthesized low-molecular-weight (135-1,000) anionic sulphonate or sulphate compounds. When administered orally, these compounds substantially reduced murine splenic AA amyloid progression. They also interfered with heparan sulphate-stimulated beta-peptide fibril aggregation in vitro.

Acute Disease↗
PubMed1995

An interaction between basement membrane and Alzheimer amyloid precursor proteins suggests a role in the pathogenesis of Alzheimer's disease.

BACKGROUND: Extracellular matrix proteins (ECMPs) of the basement membrane type, such as the heparan sulfate proteoglycan perlecan, laminin, entactin, collagen IV, and fibronectin are present in and have been implicated in the genesis of amyloids. As in many forms of amyloid, perlecan, laminin, collagen IV, and fibronectin are present in Alzheimer deposits. We have previously demonstrated high-affinity interactions between Alzheimer amyloid precursor proteins (beta PP-695, -751, and -770), and perlecan or laminin. With a view to examining our hypothesis that beta PP:ECMP interactions are involved in Alzheimer's amyloidogenesis, additional studies have now been performed examining the interactions of the beta PPs with entactin, fibronectin, and collagen IV, the influence each of the ECMPs has on the binding of the others to beta PPs, and the effect of beta PPs on interactions among the various ECMPs. EXPERIMENTAL DESIGN: A modified solid-phase enzyme-linked immunosorbent assay was used to assess the binding of the various ECMPs to the beta PPs. One element was immobilized on plastic, and another element, operationally defined as a ligand, was incubated in solution at various concentrations over the immobilized protein. To evaluate the effect of one ECMP on the binding of other ECMPs to beta PP, the beta PP was immobilized and the binding of the "ligand" ECMP was assessed in the presence of a single concentration of a second "competitor" ECMP. Similarly, in evaluating the effect of beta PPs on the binding of ECMPs to each other, one ECMP was immobilized and the binding of a second ECMP "ligand" was assessed in the presence of a fixed concentration of beta PP "competitor." RESULTS: As in the case of perlecan and laminin, each of the ECMPs bound to the beta PPs with high affinity (Kd values in the nanomolar range). The binding of entactin to beta PPs was stimulated by collagen IV but was markedly inhibited by laminin, perlecan, and fibronectin. Conversely, the presence of entactin inhibited the binding of perlecan, laminin, and fibronectin to beta PPs. Moreover, the presence of beta PPs usually interfered with the binding of ECMPs to each other. Generally, in all binding assays, beta PP-751 and -770, behaved in similar ways, but beta PP-695, the brain-specific form, exhibited unique characteristics. CONCLUSIONS: These binding data may reflect the normal interactions of beta PPs with ECMPs. However, the fact that beta PPs interfere with the normal interactions between ECMPs themselves, a process that spontaneously generates a basement membrane, suggests that aspects of ECMP:beta PP binding may be a pathologic part of the amyloidogenic process in Alzheimer's disease.

Alzheimer Disease↗