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R Kisilevsky

Publications and source records attributed to R Kisilevsky.

At least 19 recordsLinked to original sources
PubMed2005

Rapid recycling of cholesterol: the joint biologic role of C-reactive protein and serum amyloid A.

Proteins that are highly conserved throughout evolution are presumed to have critical roles in the survival of the species. The two major acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA) increase up to 1000-fold during inflammation. Both proteins have been highly conserved phylogenetically for at least the last 500 million years. Thus far the physiologic role and the evolutionary significance of each remains uncertain and their potential interactions have been totally ignored despite a vast and accelerating scientific literature on the involvement of each in human disease. CRP is known to bind to phosphocholine in dead eukaryote and some live bacterial cell walls suggesting that CRP facilitates the phagocytosis of fragmented or intact dead cells and/or enhances host bacterial defenses. SAA has recently been shown to increase the rate of export of cholesterol of phagocytosed cell membranes from macrophages fourfold. We postulate that their combined physiological role is to facilitate the rapid endogenous recycling of cell membrane cholesterol and phospholipids during acute inflammation. CRP promotes efficient phagocytosis of dying cells by macrophages; SAA enhances the export of their free cholesterol/phospholipid for reuse in the membranes of the hundreds of billions of new cells required daily during acute inflammation and repair. The evolutionary conservation of these proteins in species from the horseshoe crab and echinoderms to humans suggests that the rapid endogenous recycling of cholesterol and phospholipids during the highly vulnerable period of acute inflammation is critical for their continual survival.

C-Reactive Protein↗
PubMed2001

The in-vitro influence of serum amyloid A isoforms on enzymes that regulate the balance between esterified and un-esterified cholesterol.

The intracellular balance between un-esterified and esterified cholesterol is regulated by two enzyme activities, cholesterol ester hydrolases, which drive the balance in favor of un-esterified cholesterol, and acyl-CoA:cholesterol acyl transferase (ACAT) which acts in the opposite direction. During acute inflammation apo-serum amyloid A (apoSAA) isoforms 1.1 and 2.1 become major constituents of high density lipoprotein and this complex is internalized by macrophages. Mixtures of the two isoforms have been shown to enhance cholesterol esterase activity. Using a purified form of the pancreatic enzyme we have explored the mechanism by which apoSAA may accomplish this stimulation. The pancreatic esterase cleaves cholesteryl-oleate with a Km of 0.255 mM, releasing both cholesterol and oleate. Cholesterol exhibits a product inhibition which is relieved by isoform 2.1 but not 1.1 nor apolipoprotein A-I. The NH2-terminal 16 residues of isoform 2.1 had no effect on the esterase, but the 80 residue peptide constituting its COOH-terminus possessed the stimulatory property. Purified isoforms 1.1, 2.1, 2.2, apolipoprotein A-I, the NH2-terminal 16 residues and COOH-terminal 80 residues of isoform 2.1 were also examined for their effects on macrophage ACAT activity. Isoforms 2.1 and 2.2 produced dose dependent inhibitions of up to 50%, (p<0.001). Isoform 1.1, and apoA-I had no effect on ACAT activity. The NH2-terminal 16 residue peptide of isoform 2.1 reduced the ACAT activity in a dose dependent manner by 74% (p<0.001), whereas the COOH-terminal 80 residues, in contrast to its enhancing effect on the esterase, had no inhibitory effect on ACAT. Such complementary but opposite effects of isoform 2.1 on ACAT and the esterase are consistent with a role for this protein in shifting the balance between unesterified (transportable) and esterified (storage) forms of cholesterol in favor of the latter. They suggest that apoSAA2.1 may mediate cholesterol mobilization at sites of tissue injury.

Animals↗
PubMed2001

Assembly of Alzheimer's amyloid-beta fibrils and approaches for therapeutic intervention.

Amyloid plaques are the principal features of Alzheimers disease (AD) pathology and are considered to be a major factor in the disease process. These fibrillar deposits are composed primarily of the 40-42 residue amyloid-beta (Abeta) peptide which is a proteolytic product of a larger membrane precursor protein. Electron microscopy and X-ray diffraction have revealed that the mature amyloid fibrils are assembled as a highly beta-sheet polymer that has a well-defined protofilament quaternary structure. This organization is observed for amyloid fibrils from a wide variety of disorders and appears to represent a structural superfamily. Amyloid plaques also contain a number of other components such as proteoglycans that contain highly sulfated glycosaminoglycan (GAG) chains. These amyloid-associated elements may contribute to the aggregation and/or stabilization of Abeta as insoluble fibrils. We have recently developed an aggressive model for Abeta plaque formation in transgenic mice that exhibits an "early-onset" phenotype. Immunocytochemistry has demonstrated that even with this rapid progression, Abeta deposits within the neuropil and cerebrovascular system all co-localize with heparan sulfate proteoglycans (HSPG). These findings indicate a number of structural features that can be targeted as potential sites for the development of amyloid inhibitors. In addition, the use of small compounds that interfere with the proteoglycan-amyloid pathway may be effective therapeutic agents that can be assessed through the use of these transgenic models.

Alzheimer Disease↗
PubMed2001

Beta-amyloid fibrils of Alzheimer's disease: pathologically altered, basement membrane-associated microfibrils?

Beta amyloid fibrils were examined in situ in the cerebral cortex of brains from patients with Alzheimer's disease using high resolution ultrastructural and immunohistochemical techniques. The main body of the fibril was identical with that of microfibrils and was made up of a core containing amyloid P component (AP), and a surface layer. Beta amyloid protein (Abeta) in the form of 1 nm wide flexible filaments was associated with the external surface of the microfibril. In cerebrovascular amyloid angiopathy the fibrils were formed at the outer surface of the vascular basement membrane. Overproduction of microfibrils has been reported at the basement membrane of "leaky" capillaries including the glomerular capillary in disease or leaky alveolar-capillary walls of normal lungs. Similarly, in Alzheimer's disease overproduction of microfibril-like beta amyloid fibrils in amyloid angiopathy coincided with breakdown of the blood-brain barrier of the cerebromicrovasculature. Thus, in the above three locations, the presence of abundant microfibrils, or microfibril-like structures, may be related to plasma which leaks out of the circulation into the adjoining vascular basement membrane. AP is an essential constituent of microfibrils and since the only site where AP is available in the cerebral cortex is in leaky microvasculature, a chronic, steady supply of AP into perivascular areas may be the cause of overproduction of microfibrils. Brain "microfibrils" may further be altered pathologically into beta amyloid fibrils by the addition of Abeta. The origin of the fibrils in senile plaques may also be the microvasculature since in the area of the plaques no source of AP is apparent.

Alzheimer Disease↗
PubMed2000

Synthesis of 4-deoxy-4-fluoro analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose and their effects on cellular glycosaminoglycan biosynthesis.

4-Deoxy-4-fluoro analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose were synthesized and evaluated as inhibitors of hepatic glycosaminoglycan biosynthesis. 2-Acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-fluoro-D-glucopyranose (16) exhibited a reduction of [3H]GlcN and [35S]SO4 incorporation into hepatocyte cellular glycosaminoglycans to 12 and 18%, respectively, of the control cells, at 1.0 mM. Similarly, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-fluoro-D-galactopyranose (31) exhibited a reduction of [3H]GlcN and [35S]SO4 incorporation to 1 and 9%, respectively, of the control cells, at 1.0 mM. Unlike 16, 31 exhibited a reduction of [14C]Leu incorporation into cellular protein to 57% of control cells, at 1.0 mM.

Acetylgalactosamine↗
PubMed2000

Synthesis of 4-deoxy analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-xylose and their effects on glycoconjugate biosynthesis.

4-Deoxy analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-xylose were synthesized and evaluated as inhibitors of glycoconjugate biosynthesis. Methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside (11) showed a reduction in [3H]GlcN and [14C]Leu incorporation into hepatocyte cellular glycoconjugates by 89 and 88%, of the control cells, respectively, at 20 mM, whereas the free sugars, 2-acetamido-2,4-dideoxy-alpha,beta-D-xylo-hexopyranoses (15), showed a reduction of [3H]GlcN and [14C]Leu incorporation by 75 and 64%, respectively, at 20 mM. The acetylated analogues of 11 and 15, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-beta-D-xylo-hexopyranoside and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha,beta-D-xylo-hexopyra noses, showed a greater inhibition of [3H]GlcN and [14C]Leu incorporation at 1 mM compared with their non-acetylated counterparts, but were toxic to hepatocytes at concentrations of 10 and 20 mM. Corresponding derivatives of 2-acetamido-2,4-dideoxy-L-threo-pentopyranose showed no biological effect up to 20 mM, suggesting that the C-6 substituent is important for the biological activity.

Acetylglucosamine↗
PubMed2000

Review: amyloidogenesis-unquestioned answers and unanswered questions.

Current assumptions and conclusions in several active areas of amyloid research are examined to see how consistent the data from chosen in vitro and in vivo model systems are with clinical and anatomic observations. These areas include the assembly of amyloid-like fibrils in vitro, the nucleation phenomenon, amyloid fibril structure in vivo and in vitro, common structural components of the amyloids, and the regression of tissue amyloid and proteolysis of amyloid proteins. Divergences and congruencies are highlighted, which in turn suggests caution in the interpretation of present data, greater collaboration and communication among investigators, and, additional areas and techniques for investigation.

Amyloid↗
PubMed2000

Immunoglobulin light chains, glycosaminoglycans, and amyloid.

Immunoglobulin light chains are the precursor proteins for fibrils that are formed during primary amyloidosis and in amyloidosis associated with multiple myeloma. As found for the approximately 20 currently described forms of focal, localized, or systemic amyloidoses, light chain-related fibrils extracted from physiological deposits are invariably associated with glycosaminoglycans, predominantly heparan sulfate. Other amyloid-related proteins are either structurally normal, such as beta2-microglobulin and islet amyloid polypeptide, fragments of normal proteins such as serum amyloid A protein or the precursor protein of the beta peptide involved in Alzheimer's disease, or are inherited forms of single amino acid variants of a normal protein such as found in the familial forms of amyloid associated with transthyretin. In contrast, the primary structures of light chains involved in fibril formation exhibit extensive mutational diversity rendering some proteins highly amyloidogenic and others non-pathological. The interactions between light chains and glycosaminoglycans are also affected by amino acid variation and may influence the clinical course of disease by enhancing fibril stability and contributing to resistance to protease degradation. Relatively little is currently known about the mechanisms by which glycosaminoglycans interact with light chains and light-chain fibrils. It is probable that future studies of this uniquely diverse family of proteins will continue to shed light on the processes of amyloidosis, and contribute as well to a greater understanding of the normal physiological roles of glycosaminoglycans.

Amyloid↗
PubMed1999

The heparin/heparan sulfate-binding site on apo-serum amyloid A. Implications for the therapeutic intervention of amyloidosis.

Serum amyloid A isoforms, apoSAA1 and apoSAA2, are apolipoproteins of unknown function that become major components of high density lipoprotein (HDL) during the acute phase of an inflammatory response. ApoSAA is also the precursor of inflammation-associated amyloid, and there is strong evidence that the formation of inflammation-associated and other types of amyloid is promoted by heparan sulfate (HS). Data presented herein demonstrate that both mouse and human apoSAA contain binding sites that are specific for heparin and HS, with no binding for the other major glycosaminoglycans detected. Cyanogen bromide-generated peptides of mouse apoSAA1 and apoSAA2 were screened for heparin binding activity. Two peptides, an apoSAA1-derived 80-mer (residues 24-103) and a smaller carboxyl-terminal 27-mer peptide of apoSAA2 (residues 77-103), were retained by a heparin column. A synthetic peptide corresponding to the CNBr-generated 27-mer also bound heparin, and by substituting or deleting one or more of its six basic residues (Arg-83, His-84, Arg-86, Lys-89, Arg-95, and Lys-102), their relative importance for heparin and HS binding was determined. The Lys-102 residue appeared to be required only for HS binding. The residues Arg-86, Lys-89, Arg-95, and Lys-102 are phylogenetically conserved suggesting that the heparin/HS binding activity may be an important aspect of the function of apoSAA. HS linked by its carboxyl groups to an Affi-Gel column or treated with carbodiimide to block its carboxyl groups lost the ability to bind apoSAA. HDL-apoSAA did not bind to heparin; however, it did bind to HS, an interaction to which apoA-I contributed. Results from binding experiments with Congo Red-Sepharose 4B columns support the conclusions of a recent structural study which found that heparin binding domains have a common spatial distance of about 20 A between their two outer basic residues. Our present work provides direct evidence that apoSAA can associate with HS (and heparin) and that the occupation of its binding site by HS, and HS analogs, likely caused the previously reported increase in amyloidogenic conformation (beta-sheet) of apoSAA2 (McCubbin, W. D., Kay, C. M., Narindrasorasak, S., and Kisilevsky, R. (1988) Biochem. J. 256, 775-783) and their amyloid-suppressing effects in vivo (Kisilevsky, R., Lemieux, L. J., Fraser, P. E., Kong, X., Hultin, P. G., and Szarek, W. A. (1995) Nat. Med. 1, 143-147), respectively.

Amino Acid Sequence↗
PubMed1999

Basement membranes, microfibrils and beta amyloid fibrillogenesis in Alzheimer's disease: high resolution ultrastructural findings.

It is known that beta amyloid fibrils are deposited at the basement membrane of the cerebromicrovasculature in the brains of patients with Alzheimer's disease, and the assembly of the fibrils may be in continuation with the core of senile plaques. The fibrils accumulate in a manner similar to that in which microfibrils accumulate in the glomerular basement membrane of the rat kidney during long-term experimental diabetes, and in the alveolar-capillary basement membrane of the normal lung. beta amyloid fibrils in-situ are known to be about 10 nm wide tubular structures and they closely resemble connective tissue microfibrils. Our recent high resolution ultrastructural studies combined with immunogold labeling demonstrated that beta amyloid fibrils in-situ are indeed microfibril-like structures, and the beta protein is associated with their surface in the form of loose assemblies of 1 nm wide flexible filaments. Thus, the result of this study indicates that in-situ a major component of the beta amyloid deposit is the microfibril-like structure. The elucidation of the mechanism of cerebral beta amyloid fibrillogenesis in Alzheimer's disease may therefore require understanding the mechanism of 'normal' microfibrils biogenesis.

Alzheimer Disease↗
PubMed1999

Experimental AA amyloidogenesis is associated with differential expression of extracellular matrix genes.

An abnormality in basement membrane metabolism has been postulated to play an important role in the pathogenesis of experimental murine AA amyloidosis. The potential contribution of the structural basement membrane proteins laminin, type IV collagen and entactin to amyloidogenesis in this model was investigated with a kinetic analysis of the expression of the corresponding genes during amyloid formation. Splenic AA amyloid deposition was stimulated by the concomitant administration of subcutaneous silver nitrate, as an inflammatory stimulus, and intravenous amyloid enhancing factor. Using a reverse transcription-polymerase chain reaction assay, a differential pattern of expression of these genes was observed at the mRNA level. Whereas laminin B1 mRNA levels did not change at any time during amyloidogenesis, a 2.2 to 3 fold induction of laminin B2, entactin and alpha 1-type IV collagen mRNAs coincided with the initial detection of splenic amyloid deposits at 48 hours post-stimulation, as detected by immunohistochemistry. Temporal and spatial codeposition of laminin and type IV collagen with amyloid was demonstrated by immunohistochemistry. A 1.4, 2.3 and 2.2-fold increase in laminin B2, entactin and alpha 1-type IV collagen mRNA levels, respectively, was detected at 24 hours post-stimulation, a point at which amyloid deposits could not be detected. Neither inflammation nor amyloid enhancing factor alone influenced laminin, entactin or type IV collagen expression at the protein or mRNA level. These observations suggest that the laminin B2 chain and alpha 1-type IV collagen chain account, at least in part, for the observed laminin and collagen IV immunoreactivity in AA amyloid deposits and that entactin may also be a component of the amyloid deposit. The onset of the induction of laminin B2, entactin and alpha 1-type IV collagen gene expression prior to the appearance of amyloid deposits, and our previous data with the heparan sulfate proteoglycan, perlecan, suggests these basement membrane proteins may play a role in the initial stages of AA fibrillogenesis.

Amyloid↗
PubMed1999

Laminin interactions with the apoproteins of acute-phase HDL: preliminary mapping of the laminin binding site on serum amyloid A.

During AA amyloidosis, the major basement membrane components, collagen type-IV (C-IV), entactin, laminin and perlecan codeposit both spatially and temporally with AA fibrils. Our previous work demonstrated that laminin, and collagen type-IV, can associate with high affinity to a mixture of mouse acute-phase serum amyloid A isoforms (apoSAA1, apoSAA2 and apoSAA3). However, laminin also bound to residual HDL from which apoSAAs were extracted. To characterize further laminin binding specificity for acute-phase HDL apolipoproteins, we have systematically isolated the acute-phase apoSAAs, apoA-I, apoA-II and the apoCs (I, II and III) by reverse phase high pressure liquid chromatography (RP-HPLC), and tested their laminin binding activities individually by ELISA. All the apoSAAs tested bound laminin saturably and with high affinity (Kd approximately 2 nM). In addition, apoA-I also showed laminin binding activity (Kd approximately 4.6 nM). Specific binding for apoA-II and the apo-Cs was not detected. To localize the laminin binding site on apoSAA, we generated defined CNBr fragments of apoSAA1 and apoSAA2, purified them by RP-HPLC, and tested their laminin binding activity by ELISA. A 53 residue peptide corresponding to residues 24-76 of apoSAA2 had the highest laminin binding activity, followed by an 80 residue peptide corresponding to residues 24-103 of apoSAA1, both of which contain a 30 residue sequence that has changed little during evolution. In addition, a 7 residue peptide (residues 17-23), which is common to both apoSAA1 and apoSAA2, also had laminin binding activity. We postulate that laminin facilitates AA amyloidogenesis by sequestering apoSAA and providing a "surface" on which heparan sulfate-dependent fibrillogenic nucleation events can take place.

Acute-Phase Proteins↗
PubMed1999

New clothes for amyloid enhancing factor (AEF): silk as AEF.

Amyloid enhancing factor (AEF) is an activity that appears naturally during the course of persistent inflammation and precedes, by 24-48 h, AA amyloid deposition in appropriate murine models. AEF is defined by its biological properties, namely, when administered intravenously or intraperitoneally to a mouse, it primes the recipient for the rapid induction of AA amyloid when they are given an inflammatory stimulus. Available evidence indicates that AEF is protein in nature, but a specific molecular species (if a singular species exits) has not been identified. Past work (Ganowiak et al., Biochem. Biophys. Res. Commun. 199:306-312, 1994) has shown that AEF activity may be imparted to two different proteins (IAPP and beta-protein) provided each is organized in the form of an amyloid fibril. Since a characteristic property of proteins in amyloid fibrils is their beta-sheet organization, one possibility is that AEF activity, in part, depends on such organization, and other proteins with such properties may also have AEF activity. To investigate this possibility, silk, a protein which contains substantial beta-sheet content, was denatured in LiSCN and allowed to renature slowly under reducing conditions to form a gel. The denatured silk preparation was then sonicated thoroughly to permit intravenous injection and assessed for AEF activity. The modified silk, presented as small fibrils in a beta-sheet conformation as assessed by electron microscopy and circular dichroism, respectively. This silk at 0-50 micrograms/animal was administered intravenously as "AEF" followed immediately by subcutaneous AgNO3 as the inflammatory stimulus. Six days later the spleens were examined for the presence of AA amyloid and following Congo red staining, the amount of amyloid quantified by image analysis. Modified silk without an inflammatory stimulus, and non-sonicated modified silk, failed to induce AA amyloid. Sonicated modified silk followed by AgNO3 induced large quantities of splenic AA amyloid in a dose dependent fashion. Modified silk in quantities as small as 1-5 micrograms/animal can function as AEF. The AEF properties of the modified silk were stable at 4 degrees C for at least 4 weeks (the longest period tested). This procedure may provide a means of standardizing AEF preparations.

Amyloid↗
PubMed1998

Ultrastructure of familial amyloid polyneuropathy amyloid fibrils: examination with high-resolution electron microscopy.

The ultrastructure of familial amyloid polyneuropathy (FAP) amyloid fibrils was examined with high-resolution electron microscopy and immunolabeling. Sural nerve biopsies from FAP (Met 30) patients as well as control tissues were prepared for thin-section observations. Extracellular spaces in the vicinity of myelinated and unmyelinated peripheral nerves were found to be filled with amyloid fibrils as well as with deposits of an "amorphous" material. The fibril was composed of a surface layer and a core. The surface layer was made up of heparan sulfate proteoglycan and was externally associated with a loose assembly of 0.5- to 1-nm-wide filaments. The core was a microfibril-like structure in which amyloid P component was enclosed in a tight helical structure by chondroitin sulfate proteoglycan. Immunogold labeling showed that the peripheral fine filaments were composed of transthyretin. The dimensions of the transthyretin filament suggest that its basic unit is a modified monomer. The deposited amorphous material was a mixture of individual components of the fibril. These results suggest that the main body of FAP amyloid fibrils is similar to that of recently observed fibrils of experimental murine AA and hemodialysis-associated amyloid as well as of connective tissue microfibrils. The differences in the fibrils of these various types of amyloid are in the peripheral filaments which are composed of a protein specific to each type of amyloid.

Amyloid↗