©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amyloid -Protein Aggregation Nullifies Its Pathologic Properties in Cultured Cerebrovascular Smooth Muscle Cells (*)

(Received for publication, May 23, 1995; and in revised form, July 12, 1995)

Judianne Davis-Salinas William E. Van Nostrand (§)

From the Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92717-4025

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Alzheimer's disease and related disorders are characterized by deposition of aggregated amyloid beta-protein (Abeta) and accompanying pathologic changes in the neuropil and in the walls of cerebral blood vessels. Abeta induces neurotoxicity in vitro, and this effect is markedly enhanced when the peptide is preaggregated. Recently, we reported that freshly solubilized Abeta can induce cellular degeneration and a striking increase in the levels of cellular amyloid beta-protein precursor and soluble Abeta peptide in cultured cerebrovascular smooth muscle cells (Davis-Salinas, J., Saporito-Irwin, S. M., Cotman, C. W., and Van Nostrand, W. E.(1995) J. Neurochem. 65, 931-934). In the present study, we show that preaggregation of Abeta abolishes the ability of the peptide to induce these cellular pathologic responses in these cells in vitro. These findings suggest that distinct mechanisms for Abeta-induced cytotoxicity exist for cultured neurons and cerebrovascular smooth muscle cells, supporting that different processes may be involved in the parenchymal and cerebrovascular pathology of Alzheimer's disease and related disorders.


INTRODUCTION

Alzheimer's disease (AD) (^1)is characterized by deposition of the 39-42-amino acid amyloid beta-protein (Abeta) in senile plaques within the neuropil and within the walls of cerebral blood vessels(1, 2, 3, 4) . Similar pathologic Abeta depositions are observed in patients with Down's syndrome, hereditary cerebral hemorrhage with amyloidosis Dutch-type, and, to a lesser extent, in normal aging(2, 4, 5) . Abeta is proteolytically derived from its transmembrane parent molecule, the amyloid beta-protein precursor (AbetaPP)(6, 7, 8, 9) . AbetaPP arises from alternative splicing of mRNA encoded by a single gene located on chromosome 21, yielding primarily proteins of 695, 751, and 770 amino acids(10, 11, 12) . The 751 and 770 isoform contain an additional 56-amino acid domain that shows homology to Kunitz-type serine protease inhibitors and were shown to be analogous to the cell-secreted protease inhibitor protease nexin-2 (PN-2)(13, 14) . Normal secretion of AbetaPP involves proteolytic cleavage through the Abeta domain, thus precluding the formation of intact Abeta(15, 16) . Therefore, Abeta must arise through an alternative processing pathway, possibly involving amyloidogenic intermediates generated in an intracellular endosomal/lysosomal compartment(17, 18) . Recent studies have shown that extracellular, soluble Abeta is a product of normal cellular catabolism of AbetaPP and can be found in the medium of cultured cells and in biological fluids(19, 20, 21) . Secreted PN-2/AbetaPP and soluble extracellular Abeta are normally expressed in many cell types of the brain. However, through an undetermined mechanism the soluble Abeta peptide becomes insoluble, aggregated, and deposited in senile plaques and within the walls of the cerebrovasculature.

The deposition of Abeta within the walls of cerebral blood vessels is a pathological trait often seen in patients with AD. Abeta, Abeta, and Abeta have all been reported to be constituents of cerebrovascular amyloid (22, 23, 24, 25) . Several findings have implicated smooth muscle cells as participants in the pathology and production of AbetaPP and Abeta in the cerebrovasculature. For example, immunohistochemical and ultrastructural studies have shown that deposition of Abeta in the walls of the cerebral blood vessels is accompanied by extensive degeneration of the smooth muscle cells, suggesting a toxic effect of the amyloid on these cells in vivo(26, 27, 28, 29) . Immunohistochemical studies have implicated the smooth muscle cells in the production of AbetaPP and Abeta in the cerebrovasculature(27, 28, 29, 30) . In addition, cerebrovascular smooth muscle cells have been shown to synthesize AbetaPP and produce extracellular, soluble Abeta in culture(31, 32) .

Recently, we described the effects of synthetic Abeta peptides on primary cultured human leptomeningeal smooth muscle (HLSM) cells. Incubation of Abeta with HLSM cells caused extensive cellular degeneration accompanied by striking increases in the levels of cellular AbetaPP and extracellular, soluble Abeta peptide(32) . However, the effects seen with Abeta were not observed when HLSM cells were incubated with the shorter Abeta and Abeta isoforms, suggesting that the longer Abeta peptide is the pathologic isoform in the cerebrovasculature. These data suggested a novel product-precursor mechanism which could result in the adverse production and accumulation of potentially amyloidogenic Abeta fragments and the spread of the cerebrovascular pathology. Since extracellular, soluble Abeta peptide is a normal product of cellular metabolism(19, 20, 21) , this creates the paradox as to how Abeta could contribute to the cellular pathology of AD and related disorders.

Previous studies have reported that the application of Abeta peptides to primary cultures of rat and human embryo cortical and hippocampal neuronal cells will cause toxicity(33, 34) . Abeta peptides can exist in both soluble and insoluble, aggregated forms. Subsequent studies have shown that the neurotoxicity observed in vitro appears to reside in the insoluble, aggregated forms of Abeta(35, 36) . Since aggregated forms of Abeta are also observed in the cerebrovasculature of patients with AD and related disorders(1, 2, 3, 4, 5) , we investigated the ability of preaggre-gated Abeta to elicit the cellular pathologic changes in cultured HLSM cells.


EXPERIMENTAL PROCEDURES

AbetaPeptides

Abeta was synthesized and characterized as described by Burdick et al.(37) . The lyophilized peptide was resuspended in sterile water to a concentration of 0.25 mM. The Abeta peptide was subjected to several cycles of freezing and incubating at 37 °C to promote aggregation(36) . The freshly solubilized and aggregated forms of the Abeta peptide were characterized by light microscopy and by turbidity spectrophotometric measurements at 400 nm as described(38) .

Cell Culture

Primary cultures of HLSM cells were established and characterized as described previously(31) . The cultures were maintained in 12-well culture dishes with Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Inc.), 20 ng/ml insulin-like growth factor-I, 1 µg/ml hydrocortisone, and antibiotics. For experiments, the near-confluent cultures of HLSM cells were placed in serum-free medium containing 0.1% bovine serum albumin for 24 h prior to treatment. Freshly solubilized or preaggregated Abeta was added to the cultures in fresh serum-free medium and incubated at 37 °C for various lengths of time. Cells were routinely viewed and photographed using an Olympus phase-contrast microscope. Cell viability was quantitated using a fluorescent live/dead cell assay (Molecular Probes) as described by the manufacturer. The cultures were viewed under a Nikon fluorescence microscope, and the number of live and dead cells were scored from at least three separate wells for each sample.

Immunoblot Analysis of AbetaPP

HLSM cells were incubated for 6 days in the presence of 25 µM freshly solubilized or an equivalent amount of preaggregated Abeta; the medium was collected and the cells rinsed with phosphate-buffered saline. The cells were then solubilized in a buffer consisting of 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, containing 1% SDS, 5 mM EDTA, 500 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml chymostatin. The cell lysates were centrifuged at 14,000 g for 10 min to remove insoluble material. The protein concentrations of the resulting supernatants were determined by the method of Bradford(39) . The culture medium and lysate samples were stored at -30 °C until analysis. Aliquots of cell lysate and culture medium samples were electrophoresed on nonreducing SDS-10% polyacrylamide gel, and the proteins were transferred onto Hybond nitrocellulose membranes (Amersham Corp.). The unoccupied sites on the membranes were blocked with 5% non-fat milk. The membranes were then probed with monoclonal antibody P2-1 (5 µg/ml), which specifically recognizes an epitope near the amino terminus of AbetaPP and then incubated with a secondary peroxidase-coupled sheep anti-mouse IgG antibody at a 1:700 dilution. The peroxidase activity on the membrane was detected using an enhanced chemiluminescence system (Amersham), and the blots were exposed to Kodak X-Omat AR film. The absolute levels of secreted and cellular AbetaPP were quantitated from the immunoblots by scanning laser densitometry and comparison with scans of standard curve immunoblots containing known amounts of purified AbetaPP.


RESULTS AND DISCUSSION

Senile plaques found in brains of patients with AD and related disorders are composed of insoluble Abeta aggregates(3, 4) . Previous studies have shown that incubation of preaggregated synthetic Abeta peptides with primary rat or human cortical and hippocampal neuronal cultures causes enhanced neurotoxicity compared with freshly solubilized Abeta peptides(33, 34, 35, 36) . These findings suggest that Abeta aggregation is an important contributing factor to neuronal degeneration and toxicity. The amyloid deposits found in the cerebrovasculature of patients with AD and related disorders also contain aggregated forms of Abeta and that are associated with degenerating smooth muscle cells in the vessel walls(23, 24, 25, 26, 27, 28, 29, 30) . Since aggregation of Abeta is important in causing neurotoxicity, in the present study we investigated the effects of aggregated Abeta on the cellular degeneration of cerebrovascular smooth muscle cells in vitro.

Synthetic Abeta peptide was preaggregated as described by Pike et al.(35, 36) . The extent of aggregation was quantitatively assessed by spectrophotometric turbidity measurements (38) . As shown in Fig. 1A, the preaggregated Abeta exhibited a pronounced optical density compared with the freshly solubilized peptide indicative of aggregates. Similarly, light microscopic analysis of the preparation showed large Abeta aggregates (Fig. 1B) that were absent in preparations of freshly solubilized peptide (data not shown).


Figure 1: Characterization of preaggregated Abeta. Aggregated forms of Abeta were prepared as described by Pike et al.(35, 36) . A, the turbidity of a 250 µM sample of freshly solubilized Abeta (first lane) and an equivalent amount of Abeta that was preaggregated (second lane) were measured at 400 nm in microtiter plate reader as described(38) . B, an aliquot of preaggregated Abeta was photographed using a phase-contrast microscope. Scale bar = 40 µm.



We performed studies to determine if the preaggregated form of Abeta could induce cellular degeneration in the cultured HLSM cells. The cells were incubated for 6 days in the absence or presence of 25 µM freshly solubilized Abeta or an equivalent amount of Abeta that was preaggregated and then the cells were characterized by light microscopy. HLSM cells incubated with the freshly solubilized Abeta (Fig. 2B) showed signs of extensive morphological degeneration compared with the untreated cells (Fig. 2A) as recently described(32) . Surprisingly, HLSM cells incubated with the preaggregated Abeta exhibited no signs of degeneration (Fig. 2C). In parallel studies the viability of the untreated and treated HLSM cells was quantitated using a fluorescent live/dead cell assay as described under ``Experimental Procedures.'' As shown in Fig. 2D, HLSM cells incubated with the preaggregated Abeta peptide showed no loss of viability similar to the untreated cells. However, the HLSM cells incubated with the freshly solubilized Abeta peptide showed a approx60% loss in cell viability. It is noteworthy that incubation of the same preparations of preaggregated and freshly solubilized Abeta peptides with primary rat neuronal cultures produced opposite effects to those seen in the HLSM cells; preaggregated Abeta caused a pronounced increase in neurotoxicity compared with the freshly solubilized peptide (data not shown). Together, these studies suggest that preaggregated Abeta does not induce cytotoxicity in cultured HLSM cells. On the other hand, preaggregation of the Abeta is an important contributing factor to eliciting neurotoxicity(35, 36) . This disparity suggests that different mechanisms are involved in Abeta-induced toxicity in cultured neurons and cerebrovascular smooth muscle cells.


Figure 2: Toxicity of soluble and preaggregated Abeta peptides for cultured primary HLSM cells. Primary cultures of HLSM cells were incubated with 25 µM of freshly solubilized Abeta or an equivalent amount of Abeta that was preaggregated for 6 days and then photographed using phase-contrast microscopy. A, no peptide; B, freshly solubilized Abeta; and C, preaggregated Abeta. Magnification = 50. D, cultures of HLSM cells were incubated in the absence of peptide (first lane), with 25 µM freshly solubilized Abeta (second lane), or an equivalent amount of Abeta that was preaggregated (third lane) for 18 days and viability of the cells was assessed using a fluorescent live/dead cell assay as described under ``Experimental Procedures.''



We recently reported that incubation of freshly solubilized Abeta with cultured HLSM cells caused a striking increase in the levels of cellular AbetaPP which coincided with the cellular degeneration(32) . Therefore, we determined if similar increases in cellular AbetaPP levels are observed when the HLSM cells are incubated with preaggregated Abeta. Representative immunoblots of the secreted and cellular AbetaPP are shown in Fig. 3, A and B, respectively, and quantitation of the AbetaPP levels are presented in Fig. 3C. HLSM cells incubated with 25 µM of the freshly solubilized Abeta exhibited a approx10-fold increase in the levels of cellular AbetaPP compared with untreated cells (Fig. 3, B and C). Greater than 90% of the AbetaPP in the cell lysates resided in a washed membrane fraction (data not shown). In contrast, HLSM cells incubated with an equivalent amount of Abeta that was preaggregated showed no increase in the levels of cellular AbetaPP. Neither preaggregated nor freshly solubilized Abeta caused an appreciable change in the levels of secreted AbetaPP in the HLSM cells (Fig. 3, A and C). Together, these studies indicate that preaggregated Abeta does not cause increased levels of cellular AbetaPP in cultured HLSM cells.


Figure 3: Quantitation of the effects of soluble and preaggregated Abeta on secreted and cellular AbetaPP in cultured HLSM cells. HLSM cells were incubated in the absence or presence of 25 µM freshly solubilized Abeta or an equivalent amount of Abeta that was preaggregated for 6 days and then equal aliquots of each culture medium sample (A) or each cell lysate sample (B) were subjected to electrophoresis on nonreducing SDS-10% polyacrylamide gels and subsequently analyzed by immunoblotting using mAbP2-1 as described under ``Experimental Procedures.'' C, the absolute levels of secreted and cellular AbetaPP in the HLSM cells incubated alone or with the freshly solubilized or preaggregated Abeta were determined by quantitative immunoblotting as described under ``Experimental Procedures.'' Data for each condition represents the mean ± S.D. from geq8 separate experiments.



Our previous studies showed that freshly solubilized Abeta could induce several cellular pathologic responses in cultured HLSM cells, including cellular degeneration with a concomitant marked increase in cellular AbetaPP and soluble Abeta peptide(32) . The present studies, however, demonstrate that preaggregated Abeta is incapable of inducing these pathologic responses in the cultured HLSM cells. These findings suggest the possibility that soluble, unaggregated Abeta may interact with a ``receptor'' or some other molecule on the surface of HLSM cells to initiate the molecular cascades involved with the observed pathologic responses. In this scenario, preaggregated Abeta may be incapable of interacting with this cell surface component to initiate the pathologic responses. Alternatively, soluble Abeta may be required to assemble into an aggregated structure on the surface of the HLSM cells in a manner that is different than the structure of aggregates that assemble in solution in the absence of cells. Regardless, the present findings indicate that preaggregated forms of Abeta are not cytotoxic to HLSM cells, whereas they have pronounced toxicity in neuronal cultures. This suggests the intriguing notion that different mechanisms of Abeta cytotoxicity exist for distinct cell types that associated with the neuronal or cerebrovascular pathologies of AD and related disorders.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AG00538 and HL49566, Research Career Development Award HL03229, and Grant-in-aid Award 94006240 from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, University of California, Irvine, CA 97217-4025. Tel.: 714-824-2556; Fax: 714-824-8598.

(^1)
The abbreviations used are: AD, Alzheimer's disease; Abeta, amyloid beta-protein; AbetaPP, amyloid beta-protein precursor; PN-2, protease nexin-2; HLSM cells, human leptomeningeal smooth muscle cells.


REFERENCES

  1. Glenner, G. G., and Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 120,885-890 [Medline] [Order article via Infotrieve]
  2. Glenner, G. G., and Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 122,1131-1135 [Medline] [Order article via Infotrieve]
  3. Masters, C. L., Multhaup, G., Simms, G., Pottgiesser, J., Martins, R. N., and Beyreuther, K. (1985) EMBO J. 4,2757-2763 [Abstract]
  4. Masters, C. L., Simms, G., Weinman, N. A., Multhaup, G., McDonald, B. L., and Beyreuther, K. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,4245-4269 [Abstract]
  5. van Duinen, S. G., Castano, E. M., Prelli, F., Bots, G. T. A. M., Luyendijk, W., and Frangione, B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,5991-5994 [Abstract]
  6. Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, K., and Muller-Hill, B. (1987) Nature 325,733-736 [CrossRef][Medline] [Order article via Infotrieve]
  7. Goldgaber, D., Lerman, M. I., McBride, O. W., Saffiotti, U., and Gajdusek, D. C. (1987) Science 235,877-880 [Medline] [Order article via Infotrieve]
  8. Tanzi, R. E., Gusella, J. F., Watkins, P. C., Bruns, G. A. P., St. George-Hyslop, P., Van Keuren, M. L., Patterson, D., Pagan, S., Kurnit, D. M., and Neve, R. L. (1987) Science 235,880-884 [Medline] [Order article via Infotrieve]
  9. Robakis, N. K., Ramakrishna, N., Wolfe, G., and Wisniewski, H. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,4190-4194 [Abstract]
  10. Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller, F., and Cordell, B. (1988) Nature 331,525-527 [CrossRef][Medline] [Order article via Infotrieve]
  11. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., Villa-Komaroff, L., Gusella, J. F., and Neve, R. L. (1988) Nature 331,528-530 [CrossRef][Medline] [Order article via Infotrieve]
  12. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S., and Ito, H. (1988) Nature 331,530-532 [CrossRef][Medline] [Order article via Infotrieve]
  13. Van Nostrand, W. E., Wagner, S. L., Suzuki, M., Choi, B. H., Farrow, J. S., Geddes, J. W., Cotman, C. W., and Cunningham, D. D. (1989) Nature 341,546-549 [CrossRef][Medline] [Order article via Infotrieve]
  14. Oltersdorf, T., Fritz, L. C., Schenk, D. B., Lieberburg, I., Johnson-Wood, K. L., Beattie, E. C., Ward, P. J., Blacher, R. W., Dovey, H. F., and Sinha, S. (1989) Nature 341,144-1471 [CrossRef][Medline] [Order article via Infotrieve]
  15. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, T., McClure, D., and Ward, P. J. (1990) Science 248,1122-1124 [Medline] [Order article via Infotrieve]
  16. Wang, R., Meschia, J. F., Cotter, R. J., and Sisodia, S. S. (1991) J. Biol. Chem. 266,16960-16964 [Abstract/Free Full Text]
  17. Estus, S., Golde, T. E., Kunishita, T., Blades, D., Lowery, D., Eisen, M., Usiak, M., Qu, X., Tabira, T., Greenberg, B. D., and Younkin, S. G. (1992) Science 255,726-728 [Medline] [Order article via Infotrieve]
  18. Golde, T. E., Estus, S., Younkin, L. H., Selkoe, D. J., and Younkin, S. G. (1992) Science 255,728-730 [Medline] [Order article via Infotrieve]
  19. Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D. B., Teplow, D. B., and Selkoe, D. J. (1992) Nature 359,322-325 [CrossRef][Medline] [Order article via Infotrieve]
  20. Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., Swindlehurst, C., McCormack, R., Wolfert, R., Selkoe, D., Lieberburg, I., and Schenk, D. (1992) Nature 359,325-327 [CrossRef][Medline] [Order article via Infotrieve]
  21. Shoji, M., Golde, T. E., Ghiso, J., Cheung, T. T., Estus, S., Shaffer, L. M., Cai, X.-D., McKay, D. M., Tintner, R., Frangione, B., and Younkin, S. G. (1992) Science 258,126-129 [Medline] [Order article via Infotrieve]
  22. Gravina, S. A., Ho, L., Eckman, C. B., Long, K. E., Otvos, L., Younkin, L. H., Suzuki, N., and Younkin, S. G. (1995) J. Biol. Chem. 270,7013-7016 [Abstract/Free Full Text]
  23. Roher, A. E., Lowenson, J. D., Clarke, S., Woods, A. S., Cotter, R. J., Gowing, E., and Ball, M. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,10836-10840 [Abstract]
  24. Joachim, C. L., Duffy, L. K., Morris, J. H., and Selkoe, D. J. (1988) Brain Res. 474,100-111 [Medline] [Order article via Infotrieve]
  25. Prelli, F., Castano, E., Glenner, G. G., and Frangione, B. (1988) J. Neurochem. 51,648-651 [Medline] [Order article via Infotrieve]
  26. Coria, F., Larrondo-Lillo, M., and Frangione, B. (1989) J. Neuropathol. Exp. Neurol. 48,368-375
  27. Kawai, M., Kalaria, R. N., Cras, P., Siedlak, S. L., Velasco, M. E., Shelton, E. R., Chan, H. W., Greenberg, B. D., and Perry, G. (1993) Brain Res. 623,142-146 [CrossRef][Medline] [Order article via Infotrieve]
  28. Wisniewski, H. M., Frackowiak, J., Zoltowska, A., and Kim, K. S. (1994) Amyloid 1,8-16
  29. Wisniewski, H. M., and Weigel, J. (1994) Acta Neuropathol. 87,233-241 [CrossRef][Medline] [Order article via Infotrieve]
  30. Rozemuller, A. J. M., Roos, R. A. C., Bots, G. T. A. M., Kamphorst, W., Eikelenboom, P., and Van Nostrand, W. E. (1993) Am. J. Pathol. 142,1449-1457 [Abstract]
  31. Van Nostrand, W. E, Rozemuller, A. J. M., Chung, R., Cotman, C. W., and Saporito-Irwin, S. M. (1994) Amyloid 1,1-7
  32. Davis-Salinas, J., Saporito-Irwin, S. M., Cotman, C. W., and Van Nostrand, W. E. (1995) J. Neurochem. 65,931-934 [Medline] [Order article via Infotrieve]
  33. Yankner, B. A., Duffy, L. K., and Kirschner, D. A. (1990) Science 250,279-282 [Medline] [Order article via Infotrieve]
  34. Mattson, M. P., Cheng, B., Davis, D., Bryant, K., Lieberburg, I., and Rydel, R. E. (1992) J. Neurosci. 12,376-389 [Abstract]
  35. Pike, C. J., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1991) Brain Res. 563,311-314 [CrossRef][Medline] [Order article via Infotrieve]
  36. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1993) J. Neurosci. 13,1676-1687 [Abstract]
  37. Burdick, D., Soreghan, B., Kwon, M., Kosmoski, J., Knauer, M., Henschen, A., Yates, J., Cotman, C., and Glabe, C. (1992) J. Biol. Chem. 267,546-554 [Abstract/Free Full Text]
  38. Jarrett, J. T., and Lansbury, P. T. (1992) Biochemistry 31,12345-12352 [Medline] [Order article via Infotrieve]
  39. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.