Molecular dynamics study of water channels in natural and synthetic amyloid-ß fibrils.

We compared all-atom explicit solvent molecular dynamics simulations of three types of Aß(1-40) fibrils: brain-seeded fibrils (2M4J, with a threefold axial symmetry) and the other two, all-synthetic fibril polymorphs (2LMN and 2LMP, made under different fibrillization conditions). Fibril models were constructed using either a finite or an infinite number of layers made using periodic images. These studies yielded four conclusions. First, finite fibrils tend to unravel in a manner reminiscent of fibril dissolution, while infinite fibrils were more stable during simulations. Second, salt bridges in these fibrils remained stable in those fibrils that contained them initially, and those without salt bridges did not develop them over the time course of the simulations. Third, all fibrils tended to develop a "stagger" or register shift of ß-strands along the fibril axis. Fourth and most importantly, the brain-seeded, 2M4J, infinite fibrils allowed bidirectional transport of water in and out of the central longitudinal core of the fibril by rapidly developing gaps at the fibril vertices. 2LMP fibrils also showed this behavior, although to a lesser extent. The diffusion of water molecules in the fibril core region involved two dynamical states: a localized state and directed diffusion in the presence of obstacles. These observations provided support for the hypothesis that Aß fibrils could act as nanotubes. At least some Aß oligomers resembled fibrils structurally in having parallel, in-register ß-sheets and a sheet-turn-sheet motif. Thus, our findings could have implications for Aß cytotoxicity, which may occur through the ability of oligomers to form abnormal water and ion channels in cell membranes.

Design and characterization of a membrane permeable N-methyl amino acid-containing peptide that inhibits Abeta1-40 fibrillogenesis.

Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of beta-sheet containing fibrils. In a previous publication, we demonstrated that the self-association of the Alzheimer's beta-amyloid (Abeta) peptide is inhibited by peptides homologous to the central core domain of Abeta, but containing N-methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, beta-strand conformation, the alternating position of N-methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen-bonding capabilities due to the replacement of the amide protons by N-methyl groups. Here, we demonstrate, through two-dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N-methyl amino acids, Abeta16-20m or Ac-K(Me)LV(Me)FF-NH2, does indeed have the intended structure of an extended beta-strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine-HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm, in contrast to the nonmethylated congener, Abeta16-20 (Ac-KLVFF-NH2). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Abeta16-20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through beta-strand domains.

A designed Zn2+-binding amphiphilic polypeptide: energetic consequences of pi-helicity.

The pi-helix is a secondary structure with 4.4 amino acids per helical turn. Although it was proposed in 1952, no experimental support for its existence was obtained until the mid-1980s. While short peptides are unlikely to assume a marginally stable secondary structure spontaneously, they might do so in the presence of appropriate structural constraints. In this paper, we describe a peptide that is designed to assume a pi-helical conformation when stabilized by cetyltrimethylammonium bromide (CTAB) micelles and Zn(2+). In the designed peptide, lipophilic amino acids are placed such that it would be amphiphilic in the pi-helical, but not in the alpha-helical, conformation. Also, two His residues are incorporated with i, i + 5 spacing, designed to allow binding of Zn(2+) in a pi-helical but not an alpha-helical conformation. The peptide was found to form moderately stable monolayers at the air-water interface, with a collapse pressure that almost doubled when there was Zn(2+) in the subphase. Also, CTAB micelles induced a marked increase in the helicity of the peptide. In 50% TFE, the peptide had a CD spectrum consistent with an alpha-helical structure. The addition of 1 mM Zn(2+) to this solvent caused a saturable decline in ellipticity to approximately half of its original value. The peptide also bound Zn(2+) when it was bound to CTAB micelles, with Zn(2+) again inducing a decrease in ellipticity. The peptide had slightly greater affinity for Zn(2+) in the presence of the CTAB than in a 50% TFE solution (K(d) = 3.1 x 10(-4) M in CTAB and 2.3 x 10(-4) M in TFE). van't Hoff analysis indicated that thermal denaturation of the peptide in 50% TFE containing 1 mM Zn(2+) was associated with both enthalpic and entropic changes that were greater than those in the absence of Zn(2+). These observations are all consistent with the proposal that the peptide assumed a pi-helical conformation in the presence of Zn(2+) and CTAB micelles, and has allowed the stability of this rare conformation to be assessed.

Point mutation in essential genes with loss or mutation of the second allele: relevance to the retention of tumor-specific antigens.

Antigens that are tumor specific yet retained by tumor cells despite tumor progression offer stable and specific targets for immunologic and possibly other therapeutic interventions. Therefore, we have studied two CD4(+) T cell-recognized tumor-specific antigens that were retained during evolution of two ultraviolet-light-induced murine cancers to more aggressive growth. The antigens are ribosomal proteins altered by somatic tumor-specific point mutations, and the progressor (PRO) variants lack the corresponding normal alleles. In the first tumor, 6132A-PRO, the antigen is encoded by a point-mutated L9 ribosomal protein gene. The tumor lacks the normal L9 allele because of an interstitial deletion from chromosome 5. In the second tumor, 6139B-PRO, both alleles of the L26 gene have point mutations, and each encodes a different tumor-specific CD4(+) T cell-recognized antigen. Thus, for both L9 and L26 genes, we observe "two hit" kinetics commonly observed in genes suppressing tumor growth. Indeed, reintroduction of the lost wild-type L9 allele into the 6132A-PRO variant suppressed the growth of the tumor cells in vivo. Since both L9 and L26 encode proteins essential for ribosomal biogenesis, complete loss of the tumor-specific target antigens in the absence of a normal allele would abrogate tumor growth.

Inhibition of beta-amyloid(40) fibrillogenesis and disassembly of beta-amyloid(40) fibrils by short beta-amyloid congeners containing N-methyl amino acids at alternate residues.

A potential goal in the prevention or therapy of Alzheimer's disease is to decrease or eliminate neuritic plaques composed of fibrillar beta-amyloid (Abeta). In this paper we describe N-methyl amino acid containing congeners of the hydrophobic "core domain" of Abeta that inhibit the fibrillogenesis of full-length Abeta. These peptides also disassemble preformed fibrils of full-length Abeta. A key feature of the inhibitor peptides is that they contain N-methyl amino acids in alternating positions of the sequence. The most potent of these inhibitors, termed Abeta16-22m, has the sequence NH(2)-K(Me-L)V(Me-F)F(Me-A)E-CONH(2). In contrast, a peptide, NH(2)-KL(Me-V)(Me-F)(Me-F)(Me-A)-E-CONH(2), with N-methyl amino acids in consecutive order, is not a fibrillogenesis inhibitor. Another peptide containing alternating N-methyl amino acids but based on the sequence of a different fibril-forming protein, the human prion protein, is also not an inhibitor of Abeta40 fibrillogenesis. The nonmethylated version of the inhibitor peptide, NH(2)-KLVFFAE-CONH(2) (Abeta16-22), is a weak fibrillogenesis inhibitor. Perhaps contrary to expectations, the Abeta16-22m peptide is highly soluble in aqueous media, and concentrations in excess of 40 mg/mL can be obtained in buffers of physiological pH and ionic strength, compared to only 2 mg/mL for Abeta16-22. Analytical ultracentrifugation demonstrates that Abeta16-22m is monomeric in buffer solution. Whereas Abeta16-22 is susceptible to cleavage by chymotrypsin, the methylated inhibitor peptide Abeta16-22m is completely resistant to this protease. Circular dichroic spectroscopy of Abeta16-22m indicates that this peptide is a beta-strand, albeit with an unusual minimum at 226 nm. In summary, the inhibitor motif is that of alternating N-methyl and nonmethylated amino acids in a sequence critical for Abeta40 fibrillogenesis. These inhibitors appear to act by binding to growth sites of Abeta nuclei and/or fibrils and preventing the propagation of the network of hydrogen bonds that is essential for the formation of an extended beta-sheet fibril.

Pharmacokinetic differences between a T cell-tolerizing and a T cell-activating peptide.

Vaccination with a peptide representing a CTL epitope from the human papillomavirus (HPV)16 E7 protein induces a specific CTL response that prevents the outgrowth of HPV16 E7-expressing tumors. In contrast, vaccination with a peptide encoding an adenovirus type 5 (Ad5) E1A CTL epitope results in CTL tolerance and enhanced growth of an Ad5 E1A-expressing tumor. It is unclear why these peptides induce such opposite effects. To determine whether a difference in pharmacokinetics can explain the functional contrasts, tritiated Ad5 E1A and HPV16 E7 peptides were injected into mice. Results show that the tolerizing peptide spread through the body 16 times faster than the activating peptide and was cleared at least 2 times faster. The HPV16 E7 peptide kinetics correlated with the kinetics of HPV16 E7-specific CTL induction. In contrast, Ad5 E1A peptide injection resulted in physical deletion of preexisting Ad5 E1A-specific CTLs within 24 h after injection. This tolerization occurred at the time when the peptide reached its maximum peptide concentration in the organs. These data suggest that ubiquitous expression of the tolerizing Ad5 E1A peptide within a short period of time causes activation-induced cell death of Ad5 E1A-specific CTLs. Therefore, information on the pharmacokinetics of peptides is vital for the safety and efficacy of peptide-based vaccines.

Review: model peptides and the physicochemical approach to beta-amyloids.

beta-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of beta-amyloid fibrils poses a challenge because of the limited solubility of beta-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of beta-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as beta-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10-35 of human beta-amyloid and indicates that in fibrils, this peptide assumes a parallel beta-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Abeta peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.

Enhanced growth of primary tumors in cancer-prone mice after immunization against the mutant region of an inherited oncoprotein.

One major objective of tumor immunologists is to prevent cancer development in individuals at high risk. (TG.AC x C57BL/6)F1 mice serve as a model for testing the feasibility of this objective. The mice carry in the germline a mutant ras oncogene that has an arginine at codon 12 instead of glycine present in the wild-type, and after physical (wounding) or chemical promotion, these mice have a high probability for developing papillomas that progress to cancer. Furthermore, F1 mice immunized with Arg(12) mutant ras peptide in complete Freund's adjuvant (CFA) develop T cells within 10 d that proliferate in vitro on stimulation with the Arg(12) mutant ras peptide. Within 14 d, these mice have delayed-type hypersensitivity to the peptide. Immunization with CFA alone or with a different Arg(12) mutant ras peptide in CFA induced neither response. To determine the effect of immunization on development of tumors, mice immunized 3 wk earlier were painted on the back with phorbol 12-myristate 13-acetate every 3 d for 8 wk. The time of appearance and the number of papillomas were about the same in immunized and control mice, but the tumors grew faster and became much larger in the mice immunized with the Arg(12) mutant ras peptide. Thus, the immunization failed to protect against growth of papillomas. The peptide-induced CD4(+) T cells preferentially recognized the peptide but not the native mutant ras protein. On the other hand, mice immunized with Arg(12) mutant ras peptide and bearing papillomas had serum antibodies that did bind native mutant ras protein. Together, these studies indicate that active immunization of cancer-prone individuals may result in immune responses that fail to eradicate mutant oncogene-expressing tumor cells, but rather induce a remarkable enhancement of tumor growth.

Two-dimensional structure of beta-amyloid(10-35) fibrils.

Beta-amyloid (Abeta) peptides are the main protein component of the pathognomonic plaques found in the brains of patients with Alzheimer's disease. These heterogeneous peptides adopt a highly organized fibril structure both in vivo and in vitro. Here we use solid-state NMR on stable, homogeneous fibrils of Abeta(10-35). Specific interpeptide distance constraints are determined with dipolar recoupling NMR on fibrils prepared from a series of singly labeled peptides containing (13)C-carbonyl-enriched amino acids, and skipping no more that three residues in the sequence. From these studies, we demonstrate that the peptide adopts the structure of an extended parallel beta-sheet in-register at pH 7.4. Analysis of DRAWS data indicates interstrand distances of 5.3 +/- 0.3 A (mean +/- standard deviation) throughout the entire length of the peptide, which is compatible only with a parallel beta-strand in-register. Intrastrand NMR constraints, obtained from peptides containing labels at two adjacent amino acids, confirm the secondary structural findings obtained using DRAWS. Using peptides with (13)C incorporated at the carbonyl position of adjacent amino acids, structural transitions from alpha-helix to beta-sheet were observed at residues 19 and 20, but using similar techniques, no evidence for a turn could be found in the putative turn region comprising residues 25-29. Implications of this extended parallel organization for Abeta(10-35) for overall fibril formation, stability, and morphology based upon specific amino acid contacts are discussed.

Familial British dementia: expression and metabolism of BRI.

Vidal et al. (1999. Nature 399: 776-778) discovered that the underlying genetic lesion in familial British dementia (FBD) is a T-A transversion at the termination codon of a membrane protein, termed BRI. The mutation creates an arginine codon; translational read-through generates a novel protein, termed BRI-L, that is extended by 11 amino acids at the carboxyl-terminus. BRI-L is the precursor of the ABri peptide, a component of amyloid deposits in FBD brain. We demonstrate that both BRI and its mutant counterpart are constitutively processed by furin, resulting in the secretion of carboxyl-terminal peptide derivatives that correspond to all, or part of, ABri. Notably, elevated levels of peptides are generated from the mutant BRI precursor, suggesting that subtle conformational alterations at the carboxyl-terminus may influence furin-mediated processing. We have examined BRI/BRI-L processing by other members of the prohormone convertase (PC) family (PACE4, LPC, PC 5/6) and found that these enzymes also process BRI, albeit inefficiently. Moreover, BRI-L processing by the other PC members is severely compromised. Finally, our electron microscopic studies reveal that synthetic ABri peptides assemble into insoluble beta-pleated fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates amyloidogenic peptides that initiate the pathogenesis of FBD.

Furin mediates enhanced production of fibrillogenic ABri peptides in familial British dementia.

The genetic lesion underlying familial British dementia (FBD), an autosomal dominant neurodegenerative disorder, is a T-A transversion at the termination codon of the BRI gene. The mutant gene encodes BRI-L, the precursor of ABri peptides that accumulate in amyloid deposits in FBD brain. We now report that both BRI-L and its wild-type counterpart, BRI, were constitutively processed by the proprotein convertase, furin, resulting in the secretion of carboxyl-terminal peptides that encompass all or part of ABri. Elevated levels of peptides were generated from the mutant BRI precursor. Electron microscopic studies revealed that synthetic ABri peptides assembled into irregular, short fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates fibrillogenic peptides that initiate the pathogenesis of FBD.

Peptide model of a highly conserved, N-terminal domain of apolipoprotein E is able to modulate lipoprotein binding to a member of the class A scavenger receptor family.

Apolipoprotein E plays a critical role in plasma lipoprotein clearance. Peptide models of a highly conserved, N-terminal domain of this protein have been shown to increase the binding of low density lipoprotein (LDL) to fibroblast cell surfaces independently of the low density lipoprotein receptor. Here we provide data to show that these peptides not only increase the binding of LDL, but also of high density lipoprotein, though not acetylated LDL. We also have data suggesting that this novel activity is mediated, at least in part, by a member of the scavenger receptor family, SR-AI. Furthermore, we show that this activity is also prominent in macrophages, a cell relevant to atherogenesis. In addition, this current paper provides evidence suggesting that this complex binding activity is initiated by a peptide-receptor interaction, and that our peptides are able to induce activity at physiologically relevant concentrations. This study provides evidence for a possible novel receptor interaction and further anti-atherogenic properties of apolipoprotein E and raises the possibility of a therapeutic potential of our peptide models.

Propagating structure of Alzheimer's beta-amyloid(10-35) is parallel beta-sheet with residues in exact register.

The pathognomonic plaques of Alzheimer's disease are composed primarily of the 39- to 43-aa beta-amyloid (Abeta) peptide. Crosslinking of Abeta peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Abeta. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Abeta peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Abeta. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 A. Analysis of these data establish that this central core of Abeta consists of a parallel beta-sheet structure in which identical residues on adjacent chains are aligned directly, i. e., in register. Our data, in conjunction with existing structural data, establish that the Abeta fibril is a hydrogen-bonded, parallel beta-sheet defining the long axis of the Abeta fibril propagation.

Structure-function relationships in side chain lactam cross-linked peptide models of a conserved N-terminal domain of apolipoprotein E.

Bioactive peptides have multiple conformations in solution but adopt well-defined conformations at lipid surfaces and in interactions with receptors. We have used side chain lactam cross-links to stabilize secondary structures in the following peptide models of a conserved N-terminal domain of apolipoprotein E (cross-link periodicity in parentheses): I, H2N-GQTLSEQVQEELLSSQVTQELRAG-COOH (none); III, [sequence; see text] (i to i + 3); IV,[sequence; see text] (i to i + 4); IVa, [sequence, see text] (i to i + 4) (lactams above the sequence, potential salt bridges below the sequence). We previously demonstrated [Luo et al. (1994) Biochemistry 33, 12367-12377; Braddock et al. (1996) Biochemistry 35, 13975-13984] that peptide III, containing lactam cross-links between the i and i + 3 side chains, enhances specific binding of LDL via a receptor other than the LDL-receptor. Peptide III in solution consists of two short alpha helices connected by a non alpha helical segment. Here we examine the hypothesis that the domain modeled by peptide III is one antipode of a conformational switch. To model another antipode of the switch, we introduced two strategic modifications into peptide III to examine structure-function relationships in this domain: (1) the spacing of the lactam cross-links was changed (i to i + 4 in peptides IV and IVa) and (2) peptides IV and IVa contain the two alternative sequences at a site of a possible end-capping interaction in peptide III. The structure of peptide IV, determined by 2D-NMR, is alpha helical across its entire length. Despite the remarkable degree of structural order, peptide IV is biologically inactive. In contrast, peptides III and possibly IVa contain a central interruption of the alpha helix, which appears necessary for biological activity. These and other studies support the hypothesis that this domain is a conformational switch which, to the extent that it models apolipoprotein E itself, may modulate interactions between apo E and its various receptors.

Tumor cells induce cytolytic T cells to a single immunodominant mutant peptide.

Two different approaches have shown that cancers express mutant proteins that may be recognized as tumor-specific antigens. On the one hand, DNA sequences known to be mutant in tumor cells have been used to select for mutant peptides that induce tumor-specific T cells (the so-called "reverse immunologic" approach). On the other, T cells induced by vaccination with whole tumor cells have been used to identify tumor-specific mutations in proteins ("direct immunologic approach"). While both approaches generate tumor-specific T cells that can lyse cancer cells expressing the relevant mutant protein, the present study suggests that there may be crucial differences. Mutant epitopes originally defined from DNA sequences have so far been immunorecessive, and tumor cells themselves generally appear unable to induce specific CD8+ T cells that recognize the encoded mutant gene product. In contrast, we find that mutant epitopes identified by CD8+ T cells stimulated by immunization with whole tumor cells induce cytolytic T cells to such mutant peptides. In fact, much or all of the response appears to be to a single mutant octapeptide that seems to be immunodominant. One possible reason for the failure of immunorecessive antigens to induce a response may be the presence of lower amounts of the antigen in the cancer cell, but other mechanisms are possible as well. For example, in the host bearing a growing tumor, neither purified proteins nor peptides might be known; thus, only immunodominant unique antigens may be able to restimulate and activate tumor-specific memory T cells that localize in the tumor following active immunization.

Dipolar recoupling NMR of biomolecular self-assemblies: determining inter- and intrastrand distances in fibrilized Alzheimer's beta-amyloid peptide.

We demonstrate a new method for investigating the structure of self-associating biopolymers using dipolar recoupling NMR techniques. This approach was applied to the study of fibrillar beta-amyloid (Abeta) peptides (the primary component of the plaques of Alzheimer's disease) containing only a single isotopic spin label (13C), by employing the DRAWS (dipolar recoupling with a windowless sequence) technique to measure 13C-13C distances. The 'single-label' approach simplified analysis of DRAWS data, since only interstrand contacts are present, without the possibility of any intrastrand contacts. As previously reported [T.L.S. Benzinger, D.M. Gregory, T.S. Burkoth, H. Miller-Auer, D.G. Lynn, R.E. Botto, S.C. Meredith, Proc. Natl. Acad. Sci. 95 (1998) 13407.], contacts of approximately 5 A were observed at all residues studied, consistent with an extended parallel beta-sheet structure with each amino acid in exact register. Here, we propose that our strategy is completely generalizable, and provides a new approach for characterizing any iterative, self-associating biopolymer. Towards the end of generalizing and refining our approach, in this paper we evaluate several issues raised by our previous analyses. First, we consider the effects of double-quantum (DQ) transverse relaxation processes. Next, we discuss the effects of various multiple-spin geometries on modeling of DRAWS data. Several practical issues are also discussed: these include (1) the use of DQ filtering experiments, either to corroborate DRAWS data, or as a rapid screening assessment of the proper placement of isotopic spin labels; and (2) the comparison of solid samples prepared by either lyophilization or freezing. Finally, data obtained from the use of single labels is compared with that obtained in doubly 13C-labeled model compounds of known crystal structure. It is shown that such data are obtainable in far more complex peptide molecules. These data,taken together, refine the DRAWS method, and demonstrate its precision and utility in obtaining high resolution structural data in complex biomolecular aggregates such as Abeta.

Specificity of ligand-induced conformational change of lipoprotein(a).

The conformation of Lp(a) was probed with a set of omega-aminocarboxylic acids and other analogs of 6-aminohexanoic acid (6-AHA). Using the viscosity-corrected sedimentation coefficient, six additional ligands were shown to induce a major conformational change in Lp(a), from a compact form to an extended form. These were trans-4-(aminomethyl)cyclohexanecarboxylic acid (t-AMCHA), proline, 4-aminobutyric acid, 8-aminooctanoic acid, Nalpha-acetyllysine, and glycine. Lysine, Nepsilon-acetyllysine, glutamic acid, and adipic acid were determined not to cause a conformational change. Urea and guanidine hydrochloride were ineffective at inducing this conformational change at concentrations at which the above ligands did unfold Lp(a). The conformational change was inhibited by 100 mM NaCl and to a lesser extent by 20 mM sodium glutamate. Despite the fact that these two salts have nearly the same ionic strengths, the greater inhibition of the unfolding by NaCl is consistent with a proposed stabilization of interkringle interactions by chloride ions. In 100 mM NaCl, which most closely resembles physiological conditions, only proline, 4-aminobutyric acid, 6-AHA, and t-AMCHA were effective ligands. By analyzing the dimensions of the conformation altering ligands, we propose that a critical variable in determining the effectiveness of a ligand in disrupting Lp(a) is the distance between the carboxyl and amine functions of the ligand. The optimal distance is approximately 6 A, which agrees with the observed 6.6-6.8 A separation of the cationic and anionic centers of known plasminogen and apo(a) lysine binding sites. These studies have implications for the mechanism of Lp(a) particle assembly.

The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

A simulation of hand impairments: effects on upper extremity function and implications toward medical impairment rating and disability determination.

OBJECTIVES: To determine whether simulation of significant impairment of the hand will have a predictable impact on degree of functional loss at the wrist and hand. DESIGN: Single subject repeat measures using before-after trial comparisons and healthy volunteer subjects. SETTING: Occupational therapy section of a large academic medical center. OTHER PARTICIPANTS: Twenty adult volunteer student subjects from an occupational therapy education (OTE) department were included. All were between ages 18 and 43 years, right hand dominant, and in excellent general health. There were 19 women and 1 man, reflecting gender distribution of the OTE student body. INTERVENTION: A simulated fusion of the carpometacarpal (CMC) joint of the thumb was achieved by immobilization in an individually fabricated splint designed to maximally restrict motion at the first CMC joint. Impairment ratings (baseline vs splinted) according to the AMA Guides were obtained by Greenleaf testing, and upper extremity function was quantitatively assessed before and after splinting. MAIN OUTCOME MEASURES: Measures of upper extremity function included grip and pinch strength, wrist torque, and speed of performance on the Valpar Small Tools test, Jebsen Hand Function test, and an exploratory measure, the Functional Life Activity Test (FLAT). RESULTS: Significant impairments were achieved for all subjects after splinting and according to Greenleaf testing. Splinting resulted in significant reductions in grip and pinch strength, wrist torque, and significant slowing of performance on the Valpar, Jebsen, and FLAT tests. Regressions of degree of impairment on degree of functional loss after splinting, and according to each of the above measures, were not significant. CONCLUSIONS: Impairment of the hand was simulated to a mild-to-moderate degree as measured according to the AMA Guides. This imposed significant reductions in motion at key joints of the wrist and hand as well as significant reductions in grip and pinch strength and wrist torque. A corresponding and significant slowing of performance on a variety of measures of upper extremity function of an industrial and nonindustrial nature was also seen. However, and for the first time, correlation and regression reveals that it is not possible to predict degree of functional loss attributable to degree of impairment for the hand. It thus appears that, for mild-to-moderate clinical impairments, the associated impairment rating is a poor estimator of functional loss at the hand and should be used cautiously, if at all, as a criterion for disability determination.

Conformationally specific enhancement of receptor-mediated LDL binding and internalization by peptide models of a conserved anionic N-terminal domain of human apolipoprotein E.

In this paper, we test the hypothesis that peptide models of a highly conserved domain of apolipoprotein E (amino acids 41-60 in human apo E) modulate the binding and internalization of LDL to cell surface receptors in a conformationally specific manner. Three peptides were compared: peptide I containing the natural sequence of amino acids 41-60 of human apo E; peptide III containing side-chain lactam cross-links designed to enhance alpha-helical structure; and peptide II containing cross-links designed to prevent formation of alpha-helices. Peptide III was shown previously to consist of two short alpha-helical domains linked by a turn and to have more alpha-helical content than peptide I, while peptide II was shown to have less helical content than either peptide III or I(Luo et al., 1994). Peptide III induced a 30-fold increase in the specific binding of 125I-LDL to normal human skin fibroblasts and a 60-fold increase in the binding to fibroblasts lacking the LDL-R. This same peptide also restored the binding to normal fibroblasts of 125I-LDL from a patient with familial defective apolipoprotein B, the R3500-->Q mutation. Analysis of binding indicated an increase in the apparent number of binding sites, with little effect on the affinity of 125I-LDL for the cell surface. Heparinase treatment of the cells did not abrogate this effect, suggesting that the increased binding is not mediated by cell surface glycans. LDL internalization but not degradation was also increased by peptide III. Similar but smaller effects were also induced by peptide I. Peptide II was much less active than peptide I or III. Thus, the order of biological activity was the same as the order of alpha-helical content, i.e., peptide III > peptide I > peptide II. These results suggest a hitherto unknown biological function for a highly conserved domain of apolipoprotein E, and this bioactivity was shown by peptide models to be specific to the alpha-helical conformation.