Serum amyloid A forms stable oligomers that disrupt vesicles at lysosomal pH and contribute to the pathogenesis of reactive amyloidosis.

Serum amyloid A (SAA) is an acute-phase plasma protein that functions in innate immunity and lipid homeostasis. SAA is a protein precursor of reactive AA amyloidosis, the major complication of chronic inflammation and one of the most common human systemic amyloid diseases worldwide. Most circulating SAA is protected from proteolysis and misfolding by binding to plasma high-density lipoproteins. However, unbound soluble SAA is intrinsically disordered and is either rapidly degraded or forms amyloid in a lysosome-initiated process. Although acidic pH promotes amyloid fibril formation by this and many other proteins, the molecular underpinnings are unclear. We used an array of spectroscopic, biochemical, and structural methods to uncover that at pH 3.5-4.5, murine SAA1 forms stable soluble oligomers that are maximally folded at pH 4.3 with ∼35% α-helix and are unusually resistant to proteolysis. In solution, these oligomers neither readily convert into mature fibrils nor bind lipid surfaces via their amphipathic α-helices in a manner typical of apolipoproteins. Rather, these oligomers undergo an α-helix to ß-sheet conversion catalyzed by lipid vesicles and disrupt these vesicles, suggesting a membranolytic potential. Our results provide an explanation for the lysosomal origin of AA amyloidosis. They suggest that high structural stability and resistance to proteolysis of SAA oligomers at pH 3.5-4.5 help them escape lysosomal degradation, promote SAA accumulation in lysosomes, and ultimately damage cellular membranes and liberate intracellular amyloid. We posit that these soluble prefibrillar oligomers provide a missing link in our understanding of the development of AA amyloidosis.

Serum amyloid A self-assembles with phospholipids to form stable protein-rich nanoparticles with a distinct structure: A hypothetical function of SAA as a "molecular mop" in immune response.

Serum amyloid A (SAA) is an acute-phase protein whose action in innate immunity and lipid homeostasis is unclear. Most circulating SAA binds plasma high-density lipoproteins (HDL) and reroutes lipid transport. In vivo SAA binds existing lipoproteins or generates them de novo upon lipid uptake from cells. We explored the products of SAA-lipid interactions and lipoprotein remodeling in vitro. SAA complexes with palmitoyl-oleoyl phosphocholine (POPC) were analyzed for structure and stability using circular dichroism and fluorescence spectroscopy, electron microscopy, gel electrophoresis and gel filtration. The results revealed the formation of 8-11nm lipoproteins that were∼50% α-helical and stable at near-physiological conditions but were irreversibly remodeled at Tm∼52°C. Similar HDL-size nanoparticles formed spontaneously at ambient conditions or upon thermal remodeling of parent lipoproteins containing various amounts of proteins and lipids, including POPC and cholesterol. Therefore, such HDL-size particles formed stable kinetically accessible structures in a wide range of conditions. Based on their size and stoichiometry, each particle contained about 12 SAA and 72 POPC molecules, with a protein:lipid weight ratio circa 2.5:1, suggesting a structure distinct from HDL. High stability of these nanoparticles and their HDL-like size suggest that similar lipoproteins may form in vivo during inflammation or injury when SAA concentration is high and membranes from dead cells require rapid removal. We speculate that solubilization of membranes by SAA to generate lipoproteins in a spontaneous energy-independent process constitutes the primordial function of this ancient protein, providing the first line of defense in clearing cell debris from the injured sites.

Flexibility and Solvation of Amyloid-ß Hydrophobic Core.

Amyloid fibril deposits found in Alzheimer disease patients are composed of amyloid-ß (Aß) protein forming a number of hydrophobic interfaces that are believed to be mostly rigid. We have investigated the µs-ms time-scale dynamics of the intra-strand hydrophobic core and interfaces of the fibrils composed of Aß1-40 protein. Using solid-state (2)H NMR line shape experiments performed on selectively deuterated methyl groups, we probed the 3-fold symmetric and 2-fold symmetric polymorphs of native Aß as well as the protofibrils of D23N Iowa mutant, associated with an early onset of Alzheimer disease. The dynamics of the hydrophobic regions probed at Leu-17, Leu-34, Val-36, and Met-35 side chains were found to be very pronounced at all sites and in all polymorphs of Aß, with methyl axis motions persisting down to 230-200 K for most of the sites. The dominant mode of motions is the rotameric side chain jumps, with the Met-35 displaying the most complex multi-modal behavior. There are distinct differences in the dynamics among the three protein variants, with the Val-36 site displaying the most variability. Solvation of the fibrils does not affect methyl group motions within the hydrophobic core of individual cross-ß subunits but has a clear effect on the motions at the hydrophobic interface between the cross-ß subunits, which is defined by Met-35 contacts. In particular, hydration activates transitions between additional rotameric states that are not sampled in the dry protein. Thus, these results support the existence of water-accessible cavity recently predicted by molecular dynamics simulations and suggested by cryo-EM studies.

Thermal stability of human plasma electronegative low-density lipoprotein: A paradoxical behavior of low-density lipoprotein aggregation.

Low-density lipoprotein (LDL) aggregation is central in triggering atherogenesis. A minor fraction of electronegative plasma LDL, termed LDL(-), plays a special role in atherogenesis. To better understand this role, we analyzed the kinetics of aggregation, fusion and disintegration of human LDL and its fractions, LDL(+) and LDL(-). Thermal denaturation of LDL was monitored by spectroscopy and electron microscopy. Initially, LDL(-) aggregated and fused faster than LDL(+), but later the order reversed. Most LDL(+) disintegrated and precipitated upon prolonged heating. In contrast, LDL(-) partially retained lipoprotein morphology and formed soluble aggregates. Biochemical analysis of all fractions showed no significant degradation of major lipids, mild phospholipid oxidation, and an increase in non-esterified fatty acid (NEFA) upon thermal denaturation. The main baseline difference between LDL subfractions was higher content of NEFA in LDL(-). Since NEFA promote lipoprotein fusion, increased NEFA content can explain rapid initial aggregation and fusion of LDL(-) but not its resistance to extensive disintegration. Partial hydrolysis of apoB upon heating was similar in LDL subfractions, suggesting that minor proteins importantly modulate LDL disintegration. Unlike LDL(+), LDL(-) contains small amounts of apoA-I and apoJ. Addition of exogenous apoA-I to LDL(+) hampered lipoprotein aggregation, fusion and precipitation, while depletion of endogenous apoJ had an opposite effect. Therefore, the initial rapid aggregation of LDL(-) is apparently counterbalanced by the stabilizing effects of minor proteins such as apoA-I and apoJ. These results help identify key determinants for LDL aggregation, fusion and coalescence into lipid droplets in vivo.

Myeloperoxidase-mediated Methionine Oxidation Promotes an Amyloidogenic Outcome for Apolipoprotein A-I.

High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo.

Molecular basis for increased risk for late-onset Alzheimer disease due to the naturally occurring L28P mutation in apolipoprotein E4.

The apolipoprotein (apo) E4 isoform has consistently emerged as a susceptibility factor for late-onset Alzheimer disease (AD), although the exact mechanism is not clear. A rare apoE4 mutant, apoE4[L28P] Pittsburgh, burdens carriers with an added risk for late-onset AD and may be a useful tool for gaining insights into the role of apoE4 in disease pathogenesis. Toward this end, we evaluated the effect of the L28P mutation on the structural and functional properties of apoE4. ApoE4[L28P] was found to have significantly perturbed thermodynamic properties, to have reduced helical content, and to expose a larger portion of the hydrophobic surface to the solvent. Furthermore, this mutant is thermodynamically destabilized and more prone to proteolysis. When interacting with lipids, apoE4[L28P] formed populations of lipoprotein particles with structural defects. The structural perturbations brought about by the mutation were accompanied by aberrant functions associated with the pathogenesis of AD. Specifically, apoE4[L28P] promoted the cellular uptake of extracellular amyloid ß peptide 42 (Aß42) by human neuroblastoma SK-N-SH cells as well as by primary mouse neuronal cells and led to increased formation of intracellular reactive oxygen species that persisted for at least 24 h. Furthermore, lipoprotein particles containing apoE4[L28P] induced intracellular reactive oxygen species formation and reduced SK-N-SH cell viability. Overall, our findings suggest that the L28P mutation leads to significant structural and conformational perturbations in apoE4 and can induce functional defects associated with neuronal Aß42 accumulation and oxidative stress. We propose that these structural and functional changes underlie the observed added risk for AD development in carriers of apoE4[L28P].

Hapivirins and diprovirins: novel θ-defensin analogs with potent activity against influenza A virus.

θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.

Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a dominant inherited kidney disorder characterized by lipoprotein thrombi in glomerular capillaries. Single-amino-acid mutations in apoE have been associated with the development of the disease, although the mechanism is unknown. In an effort to gain mechanistic insight linking the presence of such mutations and the development of LPG, we evaluated the effects of three of the most common apoE3 variants associated with this disease, namely R145P(Sendai), R147P(Chicago), and R158P(Osaka or Kurashiki), on the structural and conformational integrity of the protein. All three variants were found to have significantly reduced helical content, to expose a larger portion of hydrophobic surface to the solvent, and to be significantly thermodynamically destabilized, often lacking functionally relevant unfolding intermediates. Furthermore, all variants were aggregation prone and had enhanced sensitivity to protease digestion. Finally, although the variants were able to form discoidal lipoprotein particles, discrete subpopulations of poorly formed or aberrant particles were evident. Furthermore, these lipoprotein particles were thermodynamically destabilized and aggregation prone. Overall, our data suggest that these mutations induce a generalized unfolding of the N-terminal domain of apoE3 toward a molten-globule-like structure. ApoE3 N-terminal domain unfolding due to mutation may constitute a common mechanism underlying the protein's association with the pathogenesis of LPG.

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis.

Fusion of modified LDL in the arterial wall promotes atherogenesis. Earlier we showed that thermal denaturation mimics LDL remodeling and fusion, and revealed kinetic origin of LDL stability. Here we report the first quantitative analysis of LDL thermal stability. Turbidity data show sigmoidal kinetics of LDL heat denaturation, which is unique among lipoproteins, suggesting that fusion is preceded by other structural changes. High activation energy of denaturation, E(a) = 100 ± 8 kcal/mol, indicates disruption of extensive packing interactions in LDL. Size-exclusion chromatography, nondenaturing gel electrophoresis, and negative-stain electron microscopy suggest that LDL dimerization is an early step in thermally induced fusion. Monoclonal antibody binding suggests possible involvement of apoB N-terminal domain in early stages of LDL fusion. LDL fusion accelerates at pH < 7, which may contribute to LDL retention in acidic atherosclerotic lesions. Fusion also accelerates upon increasing LDL concentration in near-physiologic range, which likely contributes to atherogenesis. Thermal stability of LDL decreases with increasing particle size, indicating that the pro-atherogenic properties of small dense LDL do not result from their enhanced fusion. Our work provides the first kinetic approach to measuring LDL stability and suggests that lipid-lowering therapies that reduce LDL concentration but increase the particle size may have opposite effects on LDL fusion.

Janus USPION modular platform (JUMP) for theranostic ultrasound-mediated targeted intratumoral microvascular imaging and DNA/miRNA delivery.

Rationale: High mortality in pancreatic cancer (PDAC) and triple negative breast cancer (TNBC) highlight the need to capitalize on nanoscale-design advantages for multifunctional diagnostics and therapies. DNA/RNA-therapies can provide potential breakthroughs, however, to date, there is no FDA-approved systemic delivery system to solid tumors. Methods: Here, we report a Janus-nanoparticle (jNP)-system with modular targeting, payload-delivery, and targeted-imaging capabilities. Our jNP-system consists of 10 nm ultrasmall superparamagnetic iron oxide nanoparticles (USPION) with opposing antibody-targeting and DNA/RNA payload-protecting faces, directionally self-assembled with commercially available zwitterionic microbubbles (MBs) and DNA/RNA payloads. Results: Sonoporation of targeted jNP-payload-MBs delivers functional reporter-DNA imparting tumor-fluorescence, and micro-RNA126 reducing non-druggable KRAS in PDAC-Panc1 and TNBC-MB231 xenografted tumors. The targeting jNP-system enhances ultrasound-imaging of intra-tumoral microvasculature using less MBs/body weight (BW). The jNP-design enhances USPION's T2*-magnetic resonance (MR) and MR-imaging of PDAC-peritoneal metastases using less Fe/BW. Conclusion: Altogether, data advance the asymmetric jNP-design as a potential theranostic Janus-USPION Modular Platform - a JUMP forward.

Effects of phospholipase A(2) and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets.

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A(2) (PLA(2)), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial ß-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA.

Interactions of alpha-, beta-, and theta-defensins with influenza A virus and surfactant protein D.

We have reported that the alpha-defensins human neutrophil peptides (HNP)-1 and HNP-2 neutralize and aggregate influenza A virus (IAV) and promote uptake of IAV by neutrophils. These alpha-defensins were also shown to bind to surfactant protein (SP)-D and reduce its antiviral activity. In this study, we examined retrocyclin (RC)1 and RC2, humanized versions of the antiviral theta-defensins found in the leukocytes of certain nonhuman primates. RC1 was just as effective as HNP-1-3 in neutralizing IAV, and RC2 and RC101 (an analog of RC1) were more effective. In contrast, human beta-defensins (HBDs) showed less neutralizing activity. Human defensins 5 and 6 (mainly produced by intestinal Paneth cells) had viral neutralizing activity similar to HNP-1-3. Like HNP-1-3, RCs induced viral aggregation and promoted the uptake of IAV by neutrophils. We used surface plasmon resonance to evaluate binding of defensins to SP-D. HBDs, HD6, and HNP-4 bound minimally to SP-D. HNP-1-3 and RCs bound SP-D with high affinity; however, unlike HNP-1 and HNP-2, RCs did not inhibit SP-D antiviral activity. HBDs also did not inhibit antiviral activity of SP-D. Given their strong neutralizing activity and compatibility with SP-D, RCs may provide attractive prototypes for designing therapeutics that can prevent or treat respiratory infections caused by IAV.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins.

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Critical role for cross-linking of trimeric lectin domains of surfactant protein D in antiviral activity against influenza A virus.

Collectins are multimeric host defence lectins with trimeric CRDs (carbohydrate-recognition domains) and collagen and N-terminal domains that form higher-order structures composed of four or more trimers. Recombinant trimers composed of only the CRD and adjacent neck domain (termed NCRD) retain binding activity for some ligands and mediate some functional activities. The lung collectin SP-D (surfactant protein D) has strong neutralizing activity for IAVs (influenza A viruses) in vitro and in vivo, however, the NCRD derived from SP-D has weak viral-binding ability and lacks neutralizing activity. Using a panel of mAbs (monoclonal antibodies) directed against the NCRD in the present study we show that mAbs binding near the lectin site inhibit antiviral activity of full-length SP-D, but mAbs which bind other sites on the CRD do not. Two of the non-blocking mAbs significantly increased binding and antiviral activity of NCRDs as assessed by haemagglutination and neuraminidase inhibition and by viral neutralization. mAb-mediated cross-linking also enabled NCRDs to induce viral aggregation and to increase viral uptake by neutrophils and virus-induced respiratory burst responses by these cells. These results show that antiviral activities of SP-D can be reproduced without the N-terminal and collagen domains and that cross-linking of NCRDs is essential for antiviral activity of SP-D with respect to IAV.

Biophysical properties of apolipoprotein E4 variants: implications in molecular mechanisms of correction of hypertriglyceridemia.

In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated with hypertriglyceridemia. It has been shown that overexpression of human wild-type (WT) apoE4 in apoE-deficient mice induces hypertriglyceridemia. In contrast, overexpression of an apoE4 variant, apoE4-mut1 (apoE4(L261A, W264A, F265A, L268A, V269A)), does not induce hypertriglyceridemia and corrects hypercholesterolemia. Furthermore, overexpression of another variant, apoE4-mut2 (apoE4(W276A, L279A, V280A, V283A)), induces mild hypertriglyceridemia and does not correct hypercholesterolemia. To better understand how these mutations improve the function of apoE4, we investigated the conformation and stability of apoE4-mut1 and apoE4-mut2 and their binding to dimyristoyl phosphatidylcholine (DMPC) vesicles and to triglyceride (TG)-rich emulsion particles. We found that the mutations introduced in apoE4-mut1 lead to a more stable and compactly folded conformation of apoE4. These structural changes are associated with a slower rate of solubilization of DMPC vesicles by apoE4-mut1 and reduced binding of the protein to emulsion particles compared with WT apoE4. Under conditions of apoE4 overexpression, the reduced binding of apoE4-mut1 to TG-rich lipoprotein particles may facilitate the lipolysis of these particles and may alter the conformation of the lipoprotein-bound apoE in a way that favors the efficient clearance of the lipoprotein remnants. Mutations introduced in apoE4-mut2 result in smaller structural alterations compared with those observed in apoE4-mut1. The slightly altered structural properties of apoE4-mut2 are associated with slightly reduced binding of this protein to TG-rich lipoprotein particles and milder hypertriglyceridemia as compared with WT apoE4.

Serum amyloid A sequesters diverse phospholipids and their hydrolytic products, hampering fibril formation and proteolysis in a lipid-dependent manner.

Serum amyloid A action in immune response and deposition in inflammation-linked amyloidosis involve SAA-lipid interactions. We show that SAA sequesters neutral and anionic phospholipids and their hydrolytic products to form nanoparticles, suggesting a synergy with phospholipase A2. The lipid charge and shape affect SAA protection from proteolysis, aggregation and fibrillogenesis.

A Set of Handwriting Features for Use in Automated Writer Identification.

A writer's biometric identity can be characterized through the distribution of physical feature measurements ("writer's profile"); a graph-based system that facilitates the quantification of these features is described. To accomplish this quantification, handwriting is segmented into basic graphical forms ("graphemes"), which are "skeletonized" to yield the graphical topology of the handwritten segment. The graph-based matching algorithm compares the graphemes first by their graphical topology and then by their geometric features. Graphs derived from known writers can be compared against graphs extracted from unknown writings. The process is computationally intensive and relies heavily upon statistical pattern recognition algorithms. This article focuses on the quantification of these physical features and the construction of the associated pattern recognition methods for using the features to discriminate among writers. The graph-based system described in this article has been implemented in a highly accurate and approximately language-independent biometric recognition system of writers of cursive documents.

Human plasma high-density lipoproteins are stabilized by kinetic factors.

High-density lipoproteins (HDL) are heterogeneous complexes of proteins and lipids that mediate cholesterol removal from the body. Our thermal and chemical denaturation studies of mature spherical HDL isolated from human plasma show that, contrary to the widely held assumption, the particle stability has a kinetic rather than thermodynamic origin. Guanidinum hydrochloride (GdmHCl) concentration jumps at 25 degrees C monitored by circular dichroism (CD) at 222 nm reveal two dominant irreversible kinetic phases in HDL denaturation. The slower phase (relaxation time tau(1) approximately 2 x 10(4) seconds) is observed in 1-6 M GdmHCl, and the faster phase (tau(2) approximately 2 x 10(3) seconds) is detected in 3-6 M GdmHCl. Comparison of the free energy barriers associated with these phases, deltaG* = 16-17 kcal mol(-1), with the near-zero apparent thermodynamic stability inferred from the spectroscopic measurements after prolonged incubation in 0-6 M GdmHCl at 22 degrees C indicates the kinetic origin for HDL stabilization. Electron microscopic analysis of HDL incubated in 0-6 M GdmHCl suggests that the slower kinetic phase involves HDL fusion, while the faster phase involves particle rupture and release of the apolar lipid core. Thermal denaturation experiments indicate high enthalpic barriers for the particle rupture that may arise from the transient disruption of lipid and/or protein packing interactions. These results corroborate our earlier analysis of model discoidal HDL and indicate that a kinetic mechanism provides a universal natural strategy for lipoprotein stabilization. Such a mechanism may facilitate structural integrity of the heterogeneous lipoprotein particles, slow their spontaneous interconversions, and thereby modulate lipoprotein lifetime and functions.

Analysis of gender-specific atherosclerosis susceptibility in transgenic[hCETP]25DS rat model.

Epidemiological and clinical data demonstrate differences in atherosclerotic coronary heart disease prevalence between age-matched men and premenopausal women. Mechanisms underlying relative athero-susceptibility in men and athero-resistance in premenopausal women remain to be elucidated. Lack of informative animal models hinders research. We report here a moderate-expresser line transgenic for human cholesteryl ester transfer protein (CETP) in the Dahl salt-sensitive hypertensive rat strain, Tg25, that recapitulates premenopausal female athero-resistance. Having ascertained identical genetic background, environmental factors, and equivalent CETP hepatic RNA levels, we detect worse hypercholesterolemia, hypertriglyceridemia, coronary plaques and survival outcome in Tg25 male rats compared with Tg25 females. Hepatic transcription profiles of Tg25 males and females normalized to respective gender- and age-matched non-transgenic controls exhibit significant differences. Genes implicated on hierarchical cluster analysis and quantitative real-time RT-PCR pinpoint pathways associated with coronary plaque progression such as inflammation and arachidonic acid epoxygenation, and not just cholesterol metabolism pathways. The data demonstrate gender-specific factors as key modulators of atherosclerosis phenotype and suggest a possible role for the liver in atheroma progression as a large organ source of proatherogenic systemic factors.

A novel approach for latent print identification using accurate overlays to prioritize reference prints.

A novel approach to automated fingerprint matching and scoring that produces accurate locally and nonlinearly adjusted overlays of a latent print onto each reference print in a corpus is described. The technology, which addresses challenges inherent to latent prints, provides the latent print examiner with a prioritized ranking of candidate reference prints based on the overlays of the latent onto each candidate print. In addition to supporting current latent print comparison practices, this approach can make it possible to return a greater number of AFIS candidate prints because the ranked overlays provide a substantial starting point for latent-to-reference print comparison. To provide the image information required to create an accurate overlay of a latent print onto a reference print, "Ridge-Specific Markers" (RSMs), which correspond to short continuous segments of a ridge or furrow, are introduced. RSMs are reliably associated with any specific local section of a ridge or a furrow using the geometric information available from the image. Latent prints are commonly fragmentary, with reduced clarity and limited minutiae (i.e., ridge endings and bifurcations). Even in the absence of traditional minutiae, latent prints contain very important information in their ridges that permit automated matching using RSMs. No print orientation or information beyond the RSMs is required to generate the overlays. This automated process is applied to the 88 good quality latent prints in the NIST Special Database (SD) 27. Nonlinear overlays of each latent were produced onto all of the 88 reference prints in the NIST SD27. With fully automated processing, the true mate reference prints were ranked in the first candidate position for 80.7% of the latents tested, and 89.8% of the true mate reference prints ranked in the top ten positions. After manual post-processing of those latents for which the true mate reference print was not ranked first, these frequencies increased to 90.9% (1st rank) and 96.6% (top ten), respectively. Because the computational process is highly parallelizable, it is feasible for this method to work with a reference corpus of several thousand prints.