Relationship between Amphipathic β Structures in the β Domain of Apolipoprotein B and the Properties of the Secreted Lipoprotein Particles in McA-RH7777 Cells.

Our previous studies demonstrated that the first 1000 amino acid residues (the βα domain) of human apolipoprotein (apo) B-100, termed apoB:1000, are required for the initiation of lipoprotein assembly and the formation of a monodisperse stable phospholipid (PL)-rich particle. The objectives of this study were (a) to assess the effects on the properties of apoB truncates undergoing sequential inclusion of the amphipathic β strands in the 700 N-terminal residues of the β domain of apoB-100 and (b) to identify the subdomain in the β domain that is required for the formation of a microsomal triglyceride transfer protein (MTP)-dependent triacylglycerol (TAG)-rich apoB-containing particle. Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following.

Phospholipid transfer protein plays a major role in the initiation of apolipoprotein B-containing lipoprotein assembly in mouse primary hepatocytes.

In this study, we tested the hypothesis that phospholipid transfer protein (PLTP) is a plausible mediator of phospholipid (PL) transfer to the N-terminal 1000 residues of apoB (apoB:1000) leading to the initiation of apoB-containing lipoprotein assembly. To this end, primary hepatocytes from wild type (WT) and PLTP knock-out (KO) mice were transduced with adenovirus-apoB:1000 with or without co-transduction with adenovirus-PLTP, and the assembly and secretion of apoB:1000-containing lipoproteins were assessed. PLTP deficiency resulted in a 65 and 72% reduction in the protein and lipid content, respectively, of secreted apoB:1000-containing lipoproteins.

Associations between abnormal rod-mediated dark adaptation and health and functioning in older adults with normal macular health.

Purpose: Delayed rod-mediated dark adaptation (DA) is characteristic of early age-related macular degeneration (AMD) and also can be observed in some older adults in normal macular health. We examine cross-sectional associations between rod-mediated DA and risk factors for AMD in older adults in normal macular health.

Methods: The sample consisted of adults aged ≥60 years old in normal macular health per grading of fundus photos using an established disease classification system.

Aging and transplantation - a topic for biomedicine or bioethics?

The aged patient stands at the nexus of significant biomedical and bioethical issues in transplantation. This in itself can be seen as a microcosm of an imminent global tempest, stemming from expanding numbers and longer lives of the aged population. As a candidate for receiving organ and tissue transplants, the geriatric patient is challenging because they present unique physiology for medical management.

Apolipoprotein B-containing lipoprotein assembly in microsomal triglyceride transfer protein-deficient McA-RH7777 cells.

Microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein (apo) B-containing lipoproteins. Previously, we demonstrated that the N-terminal 1,000 residues of apoB (apoB:1000) are necessary for the initiation of apoB-containing lipoprotein assembly in rat hepatoma McA-RH7777 cells and that these particles are phospholipid (PL) rich. To determine if the PL transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB:1000-containing lipoproteins, we employed microRNA-based short hairpin RNAs (miR-shRNAs) to silence Mttp gene expression in parental and apoB:1000-expressing McA-RH7777 cells.

Anti-inflammatory and recycling properties of an apolipoprotein mimetic peptide, Ac-hE18A-NH(2).

Apolipoprotein E (apoE) exerts prominent anti-inflammatory effects and undergoes recycling by target cells. We previously reported that the peptide Ac-hE18A-NH(2), composed of the receptor binding domain (LRKLRKRLLR) of apoE covalently linked to the Class A amphipathic peptide 18A, dramatically lowers plasma cholesterol and lipid hydroperoxides and enhances paraoxonase activity in dyslipidemic animal models. The objective of this study was to determine whether this peptide, analogous to apoE, exerts anti-inflammatory effects and undergoes recycling under in vitro conditions.

Charged amino acid residues 997-1000 of human apolipoprotein B100 are critical for the initiation of lipoprotein assembly and the formation of a stable lipidated primordial particle in McA-RH7777 cells.

We previously demonstrated that a portion, or perhaps all, of the residues between 931 and 1000 of apolipoprotein (apo) B100 are required for the initiation of apoB-containing particle assembly. Based on our structural model of the first 1000 residues of apoB (designated as apoB:1000), we hypothesized that this domain folds into a three-sided lipovitellin-like "lipid pocket" via a hairpin-bridge mechanism. We proposed that salt bridges are formed between four tandem charged residues 717-720 in the turn of the hairpin bridge and four tandem complementary residues 997-1000 located at the C-terminal end of the model.

Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells.

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P.

Apolipoprotein receptor 2 and X11 alpha/beta mediate apolipoprotein E-induced endocytosis of amyloid-beta precursor protein and beta-secretase, leading to amyloid-beta production.

The homeostasis of amyloid-beta (Abeta) in the brain is critical to the pathogenesis of Alzheimer's disease (AD). Abeta is a fragment of amyloid-beta precursor protein (APP) generated in neurons by two proteases, beta- and gamma-secretases. APP and beta-secretase, both present on cell surface, are endocytosed into endosomes to produce Abeta.

Lipoprotein-like particles and cholesteryl esters in human Bruch's membrane: initial characterization.

Purpose: To isolate and characterize cholesteryl ester-containing, lipoprotein-like particles (LLPs) from normal aged human Bruch's membrane (BrM)/choroid (Ch).

Methods: From BrM/Ch of 20 eyes of 10 donors aged >60 years, LLPs were released by high-salt buffer, fractionated by density gradient ultracentrifugation, and characterized by determining cholesterol, triglyceride, and phospholipid concentration (by enzymatic colorimetry and fluorometry); cholesteryl ester composition (by electrospray ionization mass spectrometry, ESI/MS); and particle morphology (by negative stain electron microscopy). Apolipoprotein (apo) gene expression was determined with RT-PCR, Western blot analysis, and immunofluorescence of retinal-choroidal cryosections.

Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy.

The principal extracellular lesions of age-related maculopathy (ARM), the leading cause of vision loss in the elderly, involve Bruch's membrane (BrM), a thin vascular intima between the retinal pigment epithelium (RPE) and its blood supply. With age, 80-100 nm solid particles containing esterified cholesterol (EC) accumulate in normal BrM, and apolipoprotein B (apoB) immunoreactivity is detectable in BrM- and ARM-associated lesions. Yet little evidence indicates that increased plasma cholesterol is a risk factor for ARM.

Assembly of lipoprotein particles containing apolipoprotein-B: structural model for the nascent lipoprotein particle.

Apolipoprotein B (apoB) is the major protein component of large lipoprotein particles that transport lipids and cholesterol. We have developed a detailed model of the first 1000 residues of apoB using standard sequence alignment programs (ClustalW and MACAW) and the MODELLER6 package for three-dimensional homology modeling. The validity of the apoB model was supported by conservation of disulfide bonds, location of all proline residues in turns and loops, and conservation of the hydrophobic faces of the two C-terminal amphipathic beta-sheets, betaA (residues 600-763) and betaB (residues 780-1000).

Model class A and class L peptides increase the production of apoA-I-containing lipoproteins in HepG2 cells.

Class A peptides inhibit atherosclerosis and protect cells from class L peptide-mediated lysis. Because the cytolytic process is concentration dependent, we hypothesized that at certain concentrations both classes of peptides exert similar effect(s) on cells. To test this hypothesis, we studied the effects of a class L peptide (18L = GIKKFLGSIWKFIKAFVG) and a class A peptide, 18A-Pro-18A (18A = DWLKAFYDKVAEKLKEAF) (37pA), on apolipoprotein and lipoprotein production in HepG2 cells.

Apolipoprotein B-containing lipoprotein particle assembly: lipid capacity of the nascent lipoprotein particle.

We previously proposed that the N-terminal 1000-residue betaalpha(1) domain of apolipoprotein B (apoB) forms a bulk lipid pocket homologous to that of lamprey lipovitellin. In support of this "lipid pocket" hypothesis, we demonstrated that apoB:1000 (residues 1-1000) is secreted by a stable transformant of McA-RH7777 cells as a monodisperse particle with high density lipoprotein 3 (HDL(3)) density. In contrast, apoB:931 (residues 1-931), missing only 69 residues of the sequence homologous to lipovitellin, was secreted as a particle considerably more dense than HDL(3).

Trans polyunsaturated fatty acids have more adverse effects than saturated fatty acids on the concentration and composition of lipoproteins secreted by human hepatoma HepG2 cells.

The objective of this study was to assess the relative long-term effects of linoleic (cis, cis 18:2), linolelaidic (trans, trans 18:2), and palmitic (16:0) acids on hepatic lipoprotein production in HepG2 cells. All fatty acids increased the mass of triglycerides (TG) in the medium and the incorporation of [(3)H]-glycerol into secreted TG; the increase was more pronounced with linoleic acid than with linolelaidic and palmitic acids. The net accumulation in the medium of apolipoprotein (apo) A-I was not affected by the fatty acids tested and moderate changes in that of apoB resulted in apoB/apoA-I mass ratios of 1.

The N-terminal 1000 residues of apolipoprotein B associate with microsomal triglyceride transfer protein to create a lipid transfer pocket required for lipoprotein assembly.

Apolipoprotein (apo) B, the major protein component of the atherogenic low-density lipoprotein (LDL), has a pentapartite structure, NH2-betaalpha1-beta1-alpha2-beta2-alpha3-COOH, the beta domains containing multiple amphipathic beta strands and the alpha domains containing multiple amphipathic alpha helixes. We recently reported that the first 1000 residues of human apoB-100 have sequence and amphipathic motif homologies to the lipid-pocket of lamprey lipovitellin (LV) [Segrest, J. P.