Self-repelling bipedal exploration process.

A self-repelling two-leg (biped) spider walk is considered where the local stochastic movements are governed by two independent control parameters ß_{d} and ß_{h}, so that the former controls the distance (d) between the legs positions, and the latter controls the statistics of self-crossing of the traversed paths. The probability measure for local movements is supposed to be the one for the "true self-avoiding walk" multiplied by a factor exponentially decaying with d. After a transient behavior for short times, a variety of behaviors have been observed for large times depending on the value of ß_{d} and ß_{h}. Our statistical analysis reveals that the system undergoes a crossover between two (small and large ß_{d}) regimes identified in large times (t). In the small ß_{d} regime, the random walkers (identified by the position of the legs of the spider) remain on average in a fixed nonzero distance in the large time limit, whereas in the second regime (large ß_{d}), the absorbing force between the walkers dominates the other stochastic forces. In the latter regime, d decays in a power-law fashion with the logarithm of time. When the system is mapped to a growth process (represented by a height field which is identified by the number of visits for each point), the roughness and the average height show different behaviors in two regimes, i.e., they show a power law with respect to t in the first regime and logt in the second regime. The fractal dimension of the random walker traces and the winding angle are shown to consistently undergo a similar crossover.

Plant-based oil-sorbents harbor native microbial communities effective in spilled oil-bioremediation under nitrogen starvation and heavy metal-stresses.

Cultivation on selective media revealed that the oil-sorbents, wheat straw, corncobs and sugarcane bagasse harbor hydrocarbonoclastic, diazotrophic and heavy metal-resistant microorganisms. Nitrogen-free media containing 1.0% crude oil lost between 32.2 and 37.5% of this oil, after 8 months when they have been inoculated with such microorganism-loaded sorbents. The used wheat straw, corncobs and sugarcane bagasse samples, 1.0 g each, absorbed respectively, 1.9, 1.1 and 2.5 g oil samples, and lost 24.3-39.2% of these amounts, after they had been incubated for 8 months. Total genomic DNA's from culture media and sorbents revealed various nitrogenase-coding nifH-genes. Pure hydrocarbonoclastic microbial isolates tolerated certain concentrations of, Hg2+, Cd2+, Pb2+, AsO43- and AsO33-. Some of those isolates even grew excellently with up to 1000 ppm of Pb2+ and 36,000 ppm of AsO43- also in the presence of oil. Tested strains removed the tested heavy metals, Hg2+, Cd2+ and Pb2+ from the media and thus, reduced their toxicity against the hydrocarbon-degraders. It was concluded that plant-based sorbents, not only remove oil physically, but also harbor microbial communities effective in spilled oil-bioremediation under multiple stresses. Although each community consisted of one to three species only, the consortia which reached in numbers millions of CFU ml-1 enrich the oily media with fixed nitrogen, and remove heavy metals which otherwise inhibit the oil-degrading microorganisms.

Bioremediation of Atmospheric Hydrocarbons via Bacteria Naturally Associated with Leaves of Higher Plants.

Bacteria associated with leaves of sixteen cultivated and wild plant species from all over Kuwait were analyzed by a culture-independent approach. This technique depended on partial sequencing of 16S rDNA regions in total genomic DNA from the bacterial consortia and comparing the resulting sequences with those in the GenBank database. To release bacterial cells from leaves, tough methods such as sonication co-released too much leaf chloroplasts whose DNA interfered with the bacterial DNA. A more satisfactory bacterial release with a minimum of chloroplast co-release was done by gently rubbing the leaf surfaces with soft tooth brushes in phosphate buffer. The leaves of all plant species harbored on their surfaces bacterial communities predominated by hydrocarbonoclastic (hydrocarbon-utilizing) bacterial genera. Leaves of 6 representative plants brought about in the laboratory effective removal of volatile hydrocarbons in sealed microcosms. Each individual plant species had a unique bacterial community structure. Collectively, the phyllospheric microflora on the studied plants comprised the genera Flavobacterium, Halomonas, Arthrobacter, Marinobacter, Neisseria, Ralstonia, Ochrobactrum. Exiguobacterium, Planomicrobium, Propionibacterium, Kocuria, Rhodococcus and Stenotrophomonas. This community structure was dramatically different from the structure we determined earlier for the same plants using the culture-dependent approach, although in both cases, hydrocarbonoclastic bacteria were frequent.

Indigenous hydrocarbon-utilizing bacterioflora in oil-polluted habitats in Kuwait, two decades after the greatest man-made oil spill.

Kuwaiti habitats with two-decade history of oil pollution were surveyed for their inhabitant oil-utilizing bacterioflora. Seawater samples from six sites along the Kuwaiti coasts of the Arabian Gulf and desert soil samples collected from seven sites all over the country harbored oil-utilizing bacteria whose numbers made up 0.0001-0.01% of the total, direct, microscopic counts. The indigenous bacterioflora in various sites were affiliated to many species. This was true when counting was made on nitrogen-containing and nitrogen-free media. Seawater samples harbored species belonging predominantly to the Gammaproteobacteria and desert soil samples contained predominantly Actinobacteria. Bacterial species that grew on the nitrogen-free medium and that represented a considerable proportion of the total in all individual bacterial consortia were diazotrophic. They gave positive acetylene-reduction test and possessed the nifH genes in their genomes. Individual representative species could utilize a wide range of aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy. Quantitative determination showed that the individual species consumed crude oil, n-octadecane and phenanthrene, in batch cultures. It was concluded that the indigenous microflora could be involved in bioremediation programs without bioaugmentation or nitrogen fertilization. Irrigation would be the most important practice in bioremediation of the polluted soil desert areas.

Serum levels of zinc, copper, vitamin B12, folate and immunoglobulins in individuals with giardiasis.

BACKGROUND: Giardia lamblia is one of the most important intestinal parasites. The aim of this study was to measure serum levels of IgA, IgE, zinc, copper, vitamin B12 and folate in individuals with giardiasis in comparison to normal subjects. METHODS: The study was carried out among 49 Giardia positive and 39 age and sex matched healthy volunteers. Examination of stool samples was done by direct wet smear and formol-ether concentration method. Serum samples were obtained for further laboratory examination. IgA levels were measured by Single Radial Immune Diffusion (SRID). IgE levels were measured by ELISA kit. Zinc and copper levels was measured by Ziestchem Diagnostics Kit and colorimetric endpoint-method respectively. Vitamin B12 and folate levels were measured by DRG Diagnostics Kit and Enzyme Immunoassay method respectively. All data were analyzed using SPSS version 17. RESULTS: There was a statistically significant difference in IgA, IgE, copper and zinc levels between positive and negative groups (P<0.05). There was no significant difference between vitamin B12 and folate levels between the two groups. Mean values of Giardia positive and negative groups for IgA were 309.26 and 216.89 mg/dl, IgE 167.34 and 35.49 IU/ml, copper 309.74 and 253.61 µg/dl and zinc 69.41 and 144.75 µg/dl respectively. CONCLUSION: The results showed levels of IgA may correlate more closely with giardiasis than IgE. Regarding trace elements, giardiasis elevated serum copper levels, while it decreased serum zinc. Finally, there was no significant difference in serum levels of vitamin B12 and folic acid between the two groups.

Phytoremediation of mercury in pristine and crude oil contaminated soils: Contributions of rhizobacteria and their host plants to mercury removal.

The rhizospheric soils of three tested legume crops: broad beans (Vicia faba), beans (Phaseolus vulgaris) and pea (Pisum sativum), and two nonlegume crops: cucumber (Cucumis sativus) and tomato, (Lycopersicon esculentum) contained considerable numbers (the magnitude of 10(5)g(-1) soil) of bacteria with the combined potential for hydrocarbon-utilization and mercury-resistance. Sequencing of the 16S rRNA coding genes of rhizobacteria associated with broad beans revealed that they were affiliated to Citrobacter freundii, Enterobacter aerogenes, Exiquobacterium aurantiacum, Pseudomonas veronii, Micrococcus luteus, Brevibacillus brevis, Arthrobacter sp. and Flavobacterium psychrophilum. These rhizobacteria were also diazotrophic, i.e. capable of N(2) fixation, which makes them self-sufficient regarding their nitrogen nutrition and thus suitable remediation agents in nitrogen-poor soils, such as the oily desert soil. The crude oil attenuation potential of the individual rhizobacteria was inhibited by HgCl(2), but about 50% or more of this potential was still maintained in the presence of up to 40 mgl(-1) HgCl(2). Rhizobacteria-free plants removed amounts of mercury from the surrounding media almost equivalent to those removed by the rhizospheric bacterial consortia in the absence of the plants. It was concluded that both the collector plants and their rhizospheric bacterial consortia contributed equivalently to mercury removal from soil.

The potential of oil-utilizing bacterial consortia associated with legume root nodules for cleaning oily soils.

The surfaces of root nodules of Vicia faba and Lupinus albus (legume crops), were colonized with bacterial consortia which utilized oil and fixed nitrogen. Such combined activities apparently make those periphytic consortia efficient contributors to bioremediation of oily nitrogen-poor desert soils. This was confirmed experimentally in this study. Thus, cultivating V. faba, L. albus and, for comparison, Solanum melongena, a nonlegume crop, separately in oily sand samples resulted in more oil attenuation than in an uncultivated sample. This effect was more pronounced with the legume crops than with the nonlegume crop. Furthermore, in flask cultures, V. faba plants with nodulated roots exhibited a higher potential for oil attenuation in the surrounding water than plants with nodule-free roots. Denaturation gradient gel electrophoresis (DGGE) of polymerase chain reaction amplified 16S rRNA coding genes revealed that periphytic bacteria had DGGE bands not matching those of the oil-utilizing rhizospheric bacteria. Legume nodules also contained endophytic bacteria whose 16S rDNA bands did not match those of Rhizobium nor those of all other individual periphytic and rhizospheric strains. It was concluded that legume crops host on their roots bacterial consortia with a satisfactory potential for oil phytoremediation.

Potential of hexadecane-utilizing soil-microorganisms for growth on hexadecanol, hexadecanal and hexadecanoic acid as sole sources of carbon and energy.

Bacteria and fungi in pristine and oily desert soil samples were counted on inorganic medium aliquots containing 0.5% hexadecane, hexadecanol, hexadecanal or hexadecanoic acid, as sole sources of carbon and energy. It was found that the carbon and energy source most commonly utilized by soil bacteria was the alkane n-hexadecane, and by soil fungi hexadecanoic acid. Representative microorganisms were isolated and identified. The most predominant bacteria in all soil samples belonged to the genera Micrococcus and Pseudomonas; less dominant bacteria belonged to the group of nocardioforms. The most frequent fungal genera were Aspergillus and Penicillium, while Microsporium and Ulocladium were minor fungi. Irrespective of the substrate on which the microbial strains had initially been isolated, the majority of the isolated microorganisms could grow, albeit to a varying degree, on an inorganic medium containing any of the remaining three substrates as sole carbon and energy sources. Bacterial strains preferred the alkane as a carbon and energy source over any of its oxidation products, while fungal strains preferred to grow mainly on the fatty acids. Quantitative analysis by gas liquid chromatography revealed that the predominant bacterial and fungal isolates had a potential for the attenuation of the alkane and its immediate oxidation products in the medium. In view of the continuous release of hydrocarbon oxidation products by oil-utilizing microorganisms in oily environments, it is interesting that the indigenous microflora contribute to the uptake and utilization of all such intermediate compounds, thus, having a potential for efficient self-cleaning and bioremediation of oily soils.

Plasma apolipoproteins and risk for age related maculopathy.

AIM: To determine if elevated plasma levels of atherogenic and/or anti-atherogenic lipoproteins are risk factors for developing age related maculopathy (ARM). METHODS: In a cross sectional study in a university clinic setting, 129 patients (72 women and 57 men) underwent colour fundus photography, acuity and contrast sensitivity assessment, and electroimmunoassays of plasma apolipoproteins B (apoB) and A-I (apoA-I), the principal proteins of low density and high density lipoproteins, respectively. Maculopathy stage was assigned using the AREDS grading system. RESULTS: Levels of apoB in no ARM, mild, intermediate, and advanced ARM groups were 93.3, 91.8, 95.2, and 98.2 mg/dl, respectively. Levels of apoA-I were 147.4, 148.6, 141.0, and 144.9 mg/dl in the same groups. There was no significant association between these measures, typical for age, and maculopathy stage. CONCLUSION: Although drusen associated with ARM and ageing contain cholesterol and apoB, like the lipid rich core of an atherosclerotic plaque, the results of this study and our previous work in toto make the prospects of a plasma origin for these lesion constituents increasingly untenable. This conclusion is consistent with an emerging hypothesis that a large lipoprotein of intraocular origin is an important pathway for constituent retinal lipid processing and the biogenesis of drusen.

Structure of apolipoprotein B-100 in low density lipoproteins.

There is general consensus that amphipathic alpha-helices and beta sheets represent the major lipid-associating motifs of apolipoprotein (apo)B-100. In this review, we examine the existing experimental and computational evidence for the pentapartite domain structure of apoB. In the pentapartite nomenclature presented in this review (NH(2)-betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3)-COOH), the original alpha(1) globular domain (Segrest, J. P. et al. 1994. Arterioscler. Thromb. 14: 1674;-1685) is expanded to include residues 1;-1,000 and renamed the betaalpha(1) domain. This change reflects the likelihood that the betaalpha(1) domain, like lamprey lipovitellin, is a globular composite of alpha-helical and beta-sheet secondary structures that participates in lipid accumulation in the co-translationally assembled prenascent triglyceride-rich lipoprotein particles. Evidence is presented that the hydrophobic faces of the amphipathic beta sheets of the beta(1) and beta(2) domains of apoB-100 are in direct contact with the neutral lipid core of apoB-containing lipoproteins and play a role in core lipid organization. Evidence is also presented that these beta sheets largely determine LDL particle diameter. Analysis of published data shows that with a reduction in particle size, there is an increase in the number of amphipathic helices of the alpha(2) and alpha(3) domains associated with the surface lipids of the LDL particle; these increases modulate the surface pressure decreases caused by a reduction in radius of curvature. The properties of the LDL receptor-binding region within the overall domain structure of apoB-100 are also discussed. Finally, recent three-dimensional models of LDL obtained by cryoelectron microscopy and X-ray crystallography are discussed. These models show three common features: a semidiscoidal shape, a surface knob with the dimensions of the betaC globular domain of lipovitellin, and planar multilayers in the lipid core that are approximately 35 A apart; the multilayers are thought to represent cholesteryl ester in the smectic phase. These models present a conundrum: are LDL particles circulating at 37 degrees C spheroidal in shape, as generally assumed, or are they semidiscoidal in shape, as suggested by the models? The limited evidence available supports a spheroidal shape.

Body fat, fat distribution and serum lipids, lipoproteins and apolipoproteins in African-American and Caucasian-American prepubertal children.

OBJECTIVE: The purpose of the present study was to determine the impact of body fat mass and fat distribution on serum lipids, lipoproteins and apolipoproteins in African-American and Caucasian-American prepubertal children. SUBJECTS: Study participants included 62 African-American children (age 8.3+/-1.4 y; body mass 37.3+/-13.6 kg; height 133+/-11 cm) and 39 Caucasian children (age 8.6+/-1.2 y; body mass 34.1+/-11.0 kg; height 131+/-9 cm). METHODS: Venous blood samples were obtained after a 12 h overnight fast and serum was analyzed for total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), triacylglycerol (TAG), apolipoprotein A-I (ApoA-I), apolipoprotein B (ApoB) and lipoprotein (a) (Lp(a)) concentrations. Body composition and body fat distribution were measured by dual-energy X-ray absorptiometry and computed tomography, respectively. RESULTS: African-American children had lower TAG (46+/-20 vs 61+/-32 mg/dl, P=0.015) and higher Lp(a) (34+/-25 vs 17+/-28 mg/dl, P=0.001) and HDL-C (44+/-11 vs 39+/-8 mg/dl, P=0.041). There were no ethnic differences in TC, ApoA-I and ApoB (P=0.535, P=0.218, P=0.418, respectively). The ethnic difference in TAG and Lp(a) was not explained by total fat or abdominal fat. The ethnic difference in HDL-C was explained by visceral fat and TAG. CONCLUSION: In prepubertal children, neither body fat nor fat distribution explain the ethnic difference in TAG or Lp(a), but visceral fat and TAG may contribute to differences in HDL-C.

Cationic domain 141-150 of apoE covalently linked to a class A amphipathic helix enhances atherogenic lipoprotein metabolism in vitro and in vivo.

We previously showed 1 that a peptide, Ac-hE18A-NH(2), in which the arginine-rich heparin-binding domain of apolipoprotein E (apoE) [residues 141;-150] (LRKLRKRLLR), covalently linked to 18A (DWLKAFYDKVAEKLKEAF; a class A amphipathic helix with high lipid affinity), enhanced LDL uptake and clearance. Because VLDL and remnants contain more cholesterol per particle than LDL, enhanced hepatic clearance of VLDL could lead to an effective lowering of plasma cholesterol. Therefore, in the present article we compared the ability of this peptide to mediate/facilitate the uptake and degradation of LDL and VLDL in HepG2 cells. The peptide Ac-hE18A-NH(2), but not Ac-18A-NH(2), enhanced the uptake of LDL by HepG2 cells 5-fold and its degradation 2-fold. The association of the peptides with VLDL resulted in the displacement of native apoE; however, only Ac-hE18A-NH(2) but not Ac-18A-NH(2) caused markedly enhanced uptake (6-fold) and degradation (3-fold) of VLDL. Ac-hE18A-NH(2) also enhanced the uptake (15-fold) and degradation (2-fold) of trypsinized VLDL Sf 100;-400 (containing no immuno-detectable apoE), indicating that the peptide restored the cellular interaction of VLDL in the absence of its essential native ligand (apoE). Pretreatment of HepG2s with heparinase and heparitinase abrogated all peptide-mediated enhanced cellular activity, implicating a role for cell-surface heparan sulfate proteoglycans (HSPG). Intravenous administration of Ac-hE18A-NH(2) into apoE gene knockout mice reduced plasma cholesterol by 88% at 6 h and 30% at 24 h after injection. We conclude that this dual-domain peptide associates with LDL and VLDL and results in rapid hepatic uptake via a HSPG-facilitated pathway.

Insulin resistance, dietary cholesterol, and cholesterol concentration in postmenopausal women.

Questions remain concerning the effect of variations in cholesterol intake on plasma cholesterol concentration, as well as on the role of factors modulating the metabolic impact of this dietary intervention. To define the impact of wide variations in dietary cholesterol intake on plasma total and low-density lipoprotein (LDL) cholesterol concentrations, as well as testing the hypothesis that resistance to insulin-mediated glucose disposal would accentuate the increase in plasma total and LDL cholesterol concentrations in response to a given increment in dietary cholesterol intake, we performed a prospective, randomized study comparing diets varying in cholesterol content in 65 healthy, postmenopausal women, 31 defined as insulin-resistant and 34 as insulin-sensitive. The changes in total and LDL cholesterol in response to increments in dietary cholesterol of up to approximately 800 mg/day were modest in magnitude, without evidence of a statistically significant diet-induced increase in cholesterol concentration, or of any difference in the responses of insulin-resistant as compared with insulin-sensitive women. These results indicate that relatively large increments in dietary cholesterol intake had little effect on total or LDL cholesterol concentrations in healthy, postmenopausal women, irrespective of whether they were insulin-resistant or insulin-sensitive.

Long-term effects of cis and trans monounsaturated (18:1) and saturated (16:0) fatty acids on the synthesis and secretion of apolipoprotein A-I- and apolipoprotein B-containing lipoproteins in HepG2 cells.

The objective of this study was to compare the long-term effects of oleic (cis 18:1), elaidic (trans 18:1), and palmitic (16:0) acids on hepatic lipoprotein production, using HepG2 cells as an experimental model. The net accumulation in the medium of apolipoprotein A-I (apoA-I) was not significantly altered by fatty acids, whereas that of apoB was increased with oleic and elaidic acids. Oleic acid, and to a lesser extent elaidic and palmitic acids, increased the mass of triglycerides in the medium and the incorporation of [(3)H]glycerol into secreted triglycerides. The incorporation of [(14)C]acetate into cellular and secreted total cholesterol was stimulated by 96% and 83%, respectively, with elaidic acid but was not significantly modified by oleic or palmitic acid. Relative to oleic acid, the secretion of (14)C-labeled phospholipids and triglycerides was decreased 28% to 31% with elaidic and palmitic acids whereas that of free cholesterol and cholesteryl esters was enhanced 93% and 73%, respectively, with elaidic acid but remained unchanged with palmitic acid. Compared with oleic acid, elaidic acid stimulated the secretion of very low density lipoprotein cholesterol (VLDL-Chol), low density lipoprotein cholesterol (LDL-Chol), and high density lipoprotein cholesterol (HDL-Chol) by 43%, 70%, and 34%, respectively, whereas palmitic acid decreased VLDL-Chol but had no significant effect on LDL-Chol and HDL-Chol. The ratios of total cholesterol to HDL-Chol were 3.17, 3.60, and 3.25 with oleic, elaidic, and palmitic acids, respectively; the corresponding ratios of LDL-Chol to HDL-Chol were 0.87, 1.10, and 0.93, respectively. Compared with oleic and palmitic acids, the LDL and HDL particles secreted in the presence of elaidic acid contained higher levels of free cholesterol and cholesteryl esters and a lower content of phospholipids. The phospholipid-to-total cholesterol ratios of HDL were 1.05, 0.40, and 0.76 with oleic, elaidic, and palmitic acids, respectively. Our results indicate that in comparison with cis monounsaturated and saturated fatty acids, trans fatty acids have more adverse effects on the concentration and composition of lipoproteins secreted by HepG2 cells.

Lipolytic remnants of human VLDL produced in vitro. Effect of HDL levels in the lipolysis mixtures on the apoCs to apoE ratio and metabolic properties of VLDL core remnants.

To determine the role of high-density lipoprotein (HDL) as an acceptor of lipolytic surface remnants of very low density lipoprotein (VLDL) in the metabolism of VLDL core remnants, we examined the effect of HDL levels in the VLDL lipolysis mixture on 1) the morphology and the apoCs to E ratio in VLDL core remnants and 2) the metabolic properties of VLDL core remnants in human hepatoma cell line HepG2 and human hepatocytes in the primary culture. Normolipidemic VLDL was lipolyzed in vitro by purified bovine milk lipoprotein lipase (LpL) in a lipolysis mixture containing a physiologic level of VLDL and albumin (30 mg VLDL-cholesterol (CH)/dl and 6% albumin) in the absence and presence of either a low HDL level (VLDL-CH:HDL-CH = 3:1) or a high HDL level (VLDL-CH:HDL-CH = 1:4). Lipolysis of VLDL in either the absence or presence of HDL resulted in the hydrolysis of >85% of VLDL-triglycerides (TG) and the conversion of VLDL into smaller and denser particles. In the absence of HDL, heterogeneous spherical particles with numerous surface vesicular materials were produced. In the presence of low or high HDL, spherical particles containing some or no detectable vesicular surface components were produced. The apoCs to apoE ratios, as determined by densitometric scanning of the SDS polyacrylamide gradient gel, were 2.89 in control VLDL and 2.27, 0.91, and 0.22 in VLDL core remnants produced in the absence and in the presence of low and high HDL levels, respectively. In vitro lipolysis of VLDL markedly increased binding to HepG2 cells at 4 degrees C and internalization and degradation by human hepatocytes in primary culture at 37 degrees C. However, the HDL-mediated decrease in the apoCs to apoE ratio had a minimal effect on binding, internalization, and degradation of VLDL core remnants by HepG2 cells and human hepatocytes in primary culture. In order to determine whether HepG2 bound VLDL and VLDL core remnants are deficient in apoCs, (125)I-labeled VLDL and VLDL core remnants were added to HepG2 culture medium at 4 degrees C. The bound particles were released by heparin, and the levels of (125)I-labeled apoCs and apoE, relative to apoB, in the released particles were examined. When compared with those initially added to culture medium, the VLDL and VLDL core remnants released from HepG2 cells had a markedly increased (113%) level of apoE and a reduced (30-39%), but not absent, level of apoCs. We conclude that apoCs, as a minimum structural and/or functional component of VLDL and VLDL core remnants, may not have an inhibitory effect on the binding of VLDL or VLDL core remnants to hepatic apoE receptors.

The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts.

Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) which contains the LDL receptor (LDLR) binding site (residues 129-169). To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH(2), Ac-hE18A-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.

N-terminal domain of apolipoprotein B has structural homology to lipovitellin and microsomal triglyceride transfer protein: a "lipid pocket" model for self-assembly of apob-containing lipoprotein particles.

The process of assembly of apolipoprotein (apo) B-containing lipoprotein particles occurs co-translationally after disulfide-dependent folding of the N-terminal domain of apoB but the mechanism is not understood. During a recent database search for protein sequences that contained similar amphipathic beta strands to apoB-100, four vitellogenins, the precursor form of lipovitellin, an egg yolk lipoprotein, from chicken, frog, lamprey, and C. elegans appeared on the list of candidate proteins. The X-ray crystal structure of lamprey lipovitellin is known to contain a "lipid pocket" lined by antiparallel amphipathic beta sheets. Here we report that the first 1000 residues of human apoB-100 (the alpha(1) domain plus the first 200 residues of the beta(1) domain) have sequence and amphipathic motif homologies to the lipid-binding pocket of lamprey lipovitellin. We also show that most of the alpha(1) domain of human apoB-100 has sequence and amphipathic motif homologies to human microsomal triglyceride transfer protein (MTP), a protein required for assembly of apoB-containing lipoproteins. Based upon these results, we suggest that an LV-like "proteolipid" intermediate containing a "lipid pocket" is formed by the N-terminal portion of apoB alone or, more likely, as a complex with MTP. This intermediate produces a lipid nidus required for assembly of apoB-containing lipoprotein particles; pocket expansion through the addition of amphipathic beta strands from the beta(1) domain of apoB results in the formation of a progressively larger high density lipoprotein (HDL)-like, then very low density lipoprotein (VLDL)-like, spheroidal lipoprotein particle.

Evaluation and management of dyslipoproteinemia in children.

The major goal of the evaluation and management of DLP in children is to provide safe and effective therapy with lifestyle modification. There is a strong rationale for the initiation of DLP treatment in childhood to limit the earliest stages of atherosclerosis, to establish lifelong lifestyle changes in diet and activity, and to limit the acquisition of additional CVD risk factors such as smoking and obesity. The NCEP has recommended screening for children with a parent with total cholesterol of 240 mg/dL or greater or a parent or grandparent with onset of CVD before age 55 years. Clinical evaluation and management are based on an LDL-C level of 130 mg/dL or greater. This approach to screening has a low sensitivity to identify children with DLP. Initial therapy is with a step 1 diet followed by the step 2 diet if necessary. Medications are reserved for older children with LDL-C of 190 mg/dL or greater after diet therapy or 160 mg/dL or greater with other CVD risk factors.

Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates.

Development of a computer program called LOCATE allowed us to show that human apolipoprotein B-100 is composed of five domains, NH2-alpha1-beta1-alpha2-beta2-alpha3-COOH, enriched, alternately, in amphipathic alpha helixes and amphipathic beta strands. Using updated versions of this program, here we compare the complete sequence of human apolipoprotein B-100 with partial sequences from eight additional species of vertebrates (chicken, frog, hamster, monkey, mouse, pig, rat, and rabbit). The lipid-associating amphipathic alpha helixes cluster in domains alpha2 (between residues 2075 +/- 25 and 2575 +/- 25) and alpha3 (between residues 4100 +/- 100 and 4550 +/- 50) in all species for which those regions have been sequenced but with little conservation of individual helixes. Lipid-associating amphipathic beta strands cluster in domains beta1 (approximately residues 827-2000) and beta2 (approximately residue 2571 to residue 4000 +/- 50) in all species for which these regions have been sequenced, with conservation of several individual amphipathic beta strands. Hydrophobic segments are present in apolipoprotein B-100 sequences of all nine species but the frequency of occurrence is no greater than generally found in beta sheet-containing proteins. We conclude that four alternating lipid-associating domains, -beta1-alpha2-beta2-alpha3-COOH, are common supramolecular features of apolipoprotein B-100 in nine vertebrate species.

Effect of ethanol on the synthesis and secretion of apoA-I- and apoB-containing lipoproteins in HepG2 cells.

The short- and long-term effects of ethanol on the production of lipids and apolipoproteins (apo) in HepG2 cells were studied. Short-term incubation with 1% ethanol caused a significant 32% increase in the cellular content of both triglycerides and cholesteryl esters. Under these conditions, the net accumulation in the medium of triglycerides, unesterified cholesterol, cholesteryl esters, apoA-I, and apoE was stimulated by 75%, 41%, 43%, 19%, and 39%, respectively. ApoA-I and apoE mRNA levels increased by 15%. The major short-term effect of ethanol was on the net accumulation of apoB in the medium which was stimulated by 56-100% in the presence of 0.1-1.0% ethanol. Under these conditions, apoB mRNA abundance was elevated by 17-26% and LDL receptor activity was unchanged. The increase in apoB accumulation in the medium was predominantly due to augmented secretion of newly synthesized apoB-100 which was evident at 0.05% ethanol. The secretion of newly synthesized apoA-I was not altered by short-term incubation with < or = 0.5% ethanol. The rate of apoB production was positively correlated with the cellular and secreted cholesteryl esters and secreted triglycerides. Addition of Pfizer CP-113,818, an inhibitor of acyl-CoA:cholesterol acyltransferase, caused a 69% reduction in the secretion of cholesteryl esters and a 24% decrease in that of apoB-100. In contrast to the short-term effect of ethanol, long-term incubation with ethanol resulted in a dose-dependent increase in the secretion of newly synthesized apoA-I without significantly affecting that of apoB-100. The increase in apoA-I secretion was evident at 0.05% ethanol and reached a maximum of 77% at 0.5% ethanol. These observations indicate that in HepG2 cells the effect of ethanol on the production of apoA-I- and apoB- containing lipoproteins is both time- and dose-dependent and is different in these two apolipoproteins.