Influence of macrophage-derived apolipoprotein E on plasma lipoprotein distribution of apolipoprotein A-I in apolipoprotein E-deficient mice

Two-dimensional multiple-quantum magic angle spinning (MQMAS) NMR and MAS NMR of 11B at various magnetic fields, were applied to elucidate the structure of vitreous (glassy) boron trioxide (v-B2O3), vitreous boron trisulfide (v-B2S3) and crystalline boron trisulfide (c-B2S3). These techniques, when combined with computer simulations of the resulting spectra, provide the isotropic chemical shifts and the quadrupole parameters, as well as a quantitative measure of the intensities of various boron resonances. The MAS NMR of v-B2O3 produced overlapping anisotropic lineshapes corresponding to the -1/2<-->1/2 transition in two distinct types of BO3 units with 3(+/-0.08):] intensity ratio. A combination of MAS and the multiple-quantum method resulted in a better resolved, isotropic 11B spectrum of v-B2O3. A remarkable enhancement of resolution of the MQMAS NMR proved instrumental in finding and identifying various impurities present in v-B2S3 and c-B2S3. In addition to the resonances from boron in two types of BS3 groups, four other structural units, BOS2, BO2S, BO3 and BS4, were elucidated from the spectra of vitreous and crystalline samples. The effects of various experimental parameters, such as the magnitude of the B0 and B1 fields, on the resolution of the MAS and MQMAS techniques are also shown.     

Interaction of apolipoprotein AII with the putative high-density lipoprotein receptor

There is strong evidence to indicate that binding of HDL by cells is due to recognition of apoproteins residing on the surface of the lipoprotein by the putative HDL receptor(s). Although both of the major HDL apoproteins, AI and AII, are recognized by the putative receptor, the nature of the binding interaction and the domains of the apoproteins involved are largely unknown. Previous data from this laboratory led to the proposal of a model to explain how HDL particles containing AII interacted with the HDL receptor in a different manner as compared to HDL particles which contain apoAI but not apoAII [Vadiveloo, P. K., & Fidge, N. H. (1992) Biochem. J. 284, 145-151]. The model predicted that each chain of the apoAII homodimer contained a binding domain capable of interacting with the HDL receptor. This model was tested in the current study by preparing apoAII monomers, complexing them with phospholipid, and determining the ability of these complexes to bind to putative HDL receptors in rat liver plasma membranes (RLPM) and bovine aortic endothelial cell membranes (BAECM) by ligand blotting. The data showed that these complexes were bound by HB1 and HB2 from RLPM, and to the 110-kDa HDL binding protein from BAECM, providing critical evidence to support the model. Further investigation into the binding interaction revealed that apoAII complexed with phospholipid (apoAII-PC) bound more than delipidated apoAII, which bound more than delipidated apoAII monomers. Thus, optimum binding required the presence of lipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of central alpha-helices of human apolipoprotein A-I in the maturation of high-density lipoproteins

We have studied the role of amphipathic alpha-helices in the ability of apoA-I to promote cholesterol efflux from human skin fibroblasts and activate lecithin:cholesterol acyltransferase (LCAT). Three apoA-I mutants were designed, each by deletion of a pair of predicted adjacent central alpha-helices [Delta(100-143), Delta(122-165), Delta(144-186)], and expressed in Escherichia coli. This strategy was used to minimize disruption of the predicted secondary structure of the resulting protein. These three central deletion mutants have been previously shown to be expressed as stable folded proteins but to exhibit altered phospholipid-binding properties. When recombined with phospholipids to form homogeneous LpA-I containing equivalent amounts of POPC and tested for their ability to promote diffusional cholesterol efflux from normal [3H]cholesterol-labeled fibroblasts, each mutant and the wild-type recombinant protein (Rec.-apoA-I) promoted cholesterol efflux with very similar rates at all the concentrations tested. These experiments showed that all LpA-I could acquire cellular cholesterol with similar affinity and binding capacity. However, when the cell-incubated LpA-I were incubated with purified LCAT, two mutants, Delta(122-165) and Delta(144-186), appeared incapable of activating the enzyme. To directly determine their ability to activate LCAT, each mutant and the control were recombined with equivalent amounts of cholesterol and phospholipid and incubated with the purified enzyme. The results show that whereas deletion of residues 100-143 has little effect on LCAT activation, deletion of residues 122-165 or 144-186 results in an inability of the mutants to promote cholesterol esterification. In conclusion, our results show that no specific sequence in the central domain of apoA-I is required for efficient diffusional cholesterol efflux from normal fibroblasts; however, residues 144-186 appear critical for optimum LCAT activation and cholesteryl ester accumulation. Since deletion of residues 144-186 also perturbs phospholipid association and prevents the formation of large LpA-I particles [Frank, P. G., Bergeron, J., Emmanuel, F., Lavigne, J. P., Sparks, D. L., Denèfle, P., Rassart, E., and Marcel, Y. L. (1997) Biochemistry 36, 1798-1806], the data show that this pair of alpha-helices plays an important role in the maturation of HDL. Sequence analysis of these apoA-I helices further identifies specific residues that appear essential to this activity.     

Niacin decreases removal of high-density lipoprotein apolipoprotein A-I but not cholesterol ester by Hep G2 cells. Implication for reverse cholesterol transport

Increasing the expression of c-FMS (colony-stimulating factor 1 receptor) by introduction of a transgene reduced the concentration of retinoic acid or 1,25-dihydroxy vitamin D3 needed to cause myeloid or monocytic cell differentiation and hypophosphorylation of the retinoblastoma tumor suppressor protein (RB) typically associated with cell cycle G0 arrest and differentiation of HL-60 human myelo-monoblastic precursor cells. The data are consistent with a model in which signals originating with retinoic acid and c-FMS integrate to cause differentiation, RB hypophosphorylation, and G0 arrest. Furthermore, these two signals can compensate for each other. Three HL-60 sublines described previously (A. Yen et al., Exp. Cell Res., 229: 111-125, 1996) expressing low (wild-type HL-60), intermediate, and high cell surface c-FMS were treated with various concentrations of retinoic acid. The lowest concentration tested, 10(-8) M, induced significant differentiation of only the high c-FMS-expressing cells, with no accompanying hypophosphorylated RB or G0 arrest. The low and intermediate c-FMS expressing cells showed no induced differentiation, hypophosphorylation of RB, or G0 arrest. A 10-fold higher retinoic acid concentration, 10(-1) M, induced significant differentiation of both intermediate and high c-FMS-expressing cells. It induced RB hypophosphorylation only in high c-FMS-expressing cells but with no accompanying G0 arrest in any of the cells. The highest retinoic acid concentration, 10(-6) M, elicited differentiation, hypophosphorylation of RB, and G0 arrest in low, intermediate, and high c-FMS-expressing cells. As the concentration of retinoic acid increased, cell differentiation, hypophosphorylation of RB, and G0 arrest were progressively elicited within this ensemble of cells with different c-FMS expression levels. Thus, for example, at the lowest concentration of retinoic acid, expression of high enough c-FMS still allowed differentiation. At higher concentrations, progressively less c-FMS was needed for differentiation. The apparent threshold for the sum of the retinoic acid plus c-FMS originated signals to elicit differentiation, hypophosphorylation of RB, and G0 arrest increased, in that order. Thus retinoic acid-induced cell differentiation, RB hypophosphorylation, and G0 arrest have different signal threshold requirements. 1,25-Dihydroxy vitamin D3, also a ligand for a member of the steroid thyroid hormone receptor superfamily, caused monocytic differentiation with a similar c-FMS dependency, indicating that these effects characterize both myeloid and monocytic differentiation.     

Apolipoprotein A-I structural modification and the functionality of reconstituted high density lipoprotein particles in cellular cholesterol efflux

The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; however, the mechanisms by which this occurs are not completely understood. To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes. These particles were characterized structurally and for their efficiency as acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) domains of apoA-I. The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC:protein) mole ratio. Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes. The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species. Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL. The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts. All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 microg of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux. These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.     

High density lipoprotein particle size restriction in apolipoprotein A-I(Milano) transgenic mice

Human carriers of apolipoprotein A-I(Milano) (Arg173 --> Cys substitution in apolipoprotein A-I) are characterized by an HDL deficiency in which small, dense HDL accumulate in plasma. Because affected individuals are heterozygous for this mutation, the full impact of apolipoprotein A-I(Milano) (apoA-I(Milano)) on HDL-cholesterol metabolism is unknown. In this study, apoA-I(Milano) transgenic mice were used to evaluate the extent of apoA-I(Milano) dimerization and HDL particle size restriction in the absence of wild-type apoA-I. Murine apoA-I knockout mice were utilized to express apoA-I(Milano) and human apoA-II in the presence of wild-type, human apoA-I (apoA-IMilano/A-Iwt/A-II) and in its absence (apoA-IMilano/A-II). Plasma HDL-cholesterol concentrations were similar (30 mg/dl) in both lines of apoA-I(Milano) transgenic mice. In the apoA-IMilano/A-Iwt/A-II phenotype, 14% of the apoA-I(Milano) formed homodimers and 33% formed heterodimers with apoA-II. ApoA-I(Milano) homodimers increased by 71% in the apoA-IMilano/A-II transgenics and was associated with an abundance of small, 7.6-nm HDL3-sized particles compared to the 9.5, 8.3, and 7.6-nm-sized particles in apoA-IMilano/A-Iwt/A-II mice. The unesterified cholesterol/cholesteryl ester mole ratio of HDL was elevated by 45% in apoA-IMilano/A-Iwt/A-II mice and by 90% in apoA-IMilano/A-II transgenics compared to wild-type (human apoA-I/A-II). Both apoA-I(Milano) transgenics possessed normal levels of plasma LCAT activity, but endogenous cholesterol esterification rates were reduced by 50% compared to controls. Thus, HDL particle size restriction was not the result of impaired LCAT activation; rather, dimerization of apoA-I(Milano) limited the esterification of cholesterol on endogenous HDL. In the absence of wild-type apoA-I, the more extensive dimerization of apoA-I(Milano) severely limited cholesteryl ester accumulation on plasma HDL accounting for the abundance of small, 7.6-nm HDL3 particles in apoA-IMilano/A-II mice.     

Apolipoprotein A-I complexed with phospholipid promotes hepatic lipoprotein and apolipoprotein secretion in the perfused hamster liver

BACKGROUND: Apolipoprotein A-I within high density lipoprotein (HDL) plays a significant role in the process of reverse cholesterol transport from peripheral tissues to the liver. However, additional roles are not well defined for it in hepatic cholesterol metabolism. We have previously shown in the hamster that dietary cholesterol supplementation resulted in enhancement of apolipoprotein A-I (Apo A-I) in secreted nascent hepatic very low density lipoprotein (VLDL), suggesting that apolipoprotein A-I itself may play a role in hepatic lipoprotein secretion. METHODS: Using the isolated hamster liver with Apolipoprotein A-I perfusion, we then examined the hypothesis that Apo A-I alone or in association with phosphotidylcholine (PC) i.e., Apo A-I/PC as a HDL-like particle, has effects upon hepatic lipoprotein and bile secretion. Ultracentrifugation was performed on perfusate samples at 3 hours on control vs treated livers (Apo A-I/PC, Apo A-I, or PC) to access lipid and protein concentration in VLDL, low density lipoprotein (LDL) and HDL. Four to thirty percent gradient SDS polyacrylamide electrophoresis (PAGE) and Western blot analysis were used on delipidated lipoprotein fractions and microsomes to assess apolipoproteins Apo B, A-I, II, and E. RESULTS: We found that perfusion of reconstituted HDL vesicles containing human apolipoprotein A-I and PC (Apo A-I/PC) 10 mg and 10 mg, respectively, in 22 mL for 3 hours into isolated hamster liver increased cholesterol (CH) and triglyceride (TG) components in secreted HDL; 45- and 6-fold, and in LDL; 15- and 2-fold, respectively. No significant changes occurred in VLDL or in biliary lipids. Concomitantly, Apo A-I/PC perfusion increased Apo E and Apo A-II and HDL and Apo B in LDL, while Apo E decreased in VLDL. Apo A-I/PC perfusion did not change the apolipoprotein content of hepatic microsomes of the perfused liver. Perfusion of apolipoprotein A-I (without PC) or PC (without apolipoprotein A-I) had none of these effects. CONCLUSION: These results indicate that the perfused discoidal apolipoprotein A-I/PC particle affects hepatic lipoprotein assembly and secretion, whereby both lipid and apolipoprotein components are enhanced in secreted HDL and LDL of hepatic origin.     

Human apolipoprotein A-I gene promoter mutation influences plasma low density lipoprotein cholesterol response to dietary fat saturation

Numerous studies have demonstrated an association between polycyclic aromatic hydrocarbons (PAHs) and lymphocyte toxicity. The present study shows that, consistent with its effects on Ca2+ homeostasis, benzo[a]pyrene (BaP) induces apoptosis in Daudi cells. Terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis at 18 h revealed a significant increase in the number of cells undergoing apoptosis in response to BaP (75%), BaP-7, 8-dihydrodiol (110%), and BaP-7,8-9,10-diol epoxide (BPDE) (215%) over DMSO vehicle control cultures. By 36 h, the trend toward increasing numbers of apoptotic cells continued with the parent compound producing a 125% increase over control values and the 7, 8-dihydrodiol and BPDE metabolites producing 195% and 370% increases over controls, respectively. DNA fragmentation assays demonstrated the presence of internucleosomal cleavage products consistent with the increasing numbers of TUNEL-positive cells responding to PAHs at 18 and 36 h. Analysis of poly(ADP-ribose) polymerase (PARP) protein in BaP- and BaP-7,8-dihydrodiol-treated cells strongly suggested the involvement of cysteine proteases by the appearance of an 85-kD fragment derived from hydrolytic cleavage of PARP, a phenomenon that has been associated with apoptosis in many systems. Immunoblot analysis demonstrated that both BaP and its 7,8-dihydrodiol metabolite affected a pathway involving Bcl-2 and Bax cytosolic proteins. Daudi cells undergoing apoptosis at 36 h in response to 10 microM BaP, the parent compound, expressed moderately reduced amounts of Bcl-2 (78% of vehicle controls). At the same time point, the 7,8-dihydrodiol and BDPE metabolites at 3 microM resulted in Bcl-2 protein expression that was 52% of that seen in vehicle controls. Parallel samples analyzed for expression of Bax protein displayed a 130% increase over vehicle control in Bax expression in response to the parent compound, while the 7,8-dihydrodiol metabolite produced a 257% increase in Bax. Furthermore, the effects on increased Bax expression were observed as early as 3 h after PAH exposure. The apoptotic response to PAHs in Daudi cells was sensitive to 4-h pretreatment with 0.3 microM alpha-naphthoflavone (ANF), a known inhibitor of cytochrome P450. In TUNEL assays of cells exposed to PAHs following pretreatment with ANF, at 18 h there was a significant reduction in the number of cells undergoing apoptosis in response to ANF compared to cells that were not pretreated with the compound. The effect of the parent compound at 18 h was completely blocked with ANF pretreatment, while ANF exerted a relatively weaker, but significant, effect on BaP-7, 8-dihydrodiol-induced apoptosis. With regard to modulation of expression of apoptosis-related proteins, Bax expression was restored to that observed in vehicle-control cultures at all time points tested (3, 18, and 36 h). Bcl-2 expression was most responsive to ANF at later time points following PAH exposure (18 and 36 h); however, Bcl-2 appeared to be more sensitive to the effects of ANF alone. Taken together, these data suggest that modulation of Bcl-2 family proteins, perhaps secondary to altered Ca2+ homeostasis, plays an important role in human B cell apoptosis induced by BaP.     

Structural analysis of apolipoprotein A-I: effects of amino- and carboxy-terminal deletions on the lipid-free structure

An amino-terminal deletion mutant (residues 1-43) and a carboxy-terminal deletion mutant (residues 187-243) of human apoliprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the importance of the missing residues for the conformation of apo hA-I. Our focus has been to study the lipid-free structure of apo hA-I to understand how discrete domains influence the conformational plasticity of the protein and, by inference, the mechanism of lipid binding. All spectral and physical measurements indicate that both apo delta(1-43)A-I and apo delta(187-243)A-I have folded, tertiary structures. These structures differ in the specific arrangement of helical domains based, in part, on their relative thermodynamic stability, near- and far-UV CD, limited proteolysis, and the accessibility of tryptophans to fluorescence quenchers. In addition, all data indicate that the folded domains of apo hA-I and apo delta(187-243)A-I are very similar. Results from analytical ultracentrifugation suggest that lipid-free apo hA-I and the deletion mutants each exist in a dynamic equilibrium between a loosely folded, helical bundle and an elongated monomeric helical hairpin. The conformational heterogeneity is consistent with significant ANS binding exhibited by all three proteins and could help to explain the facile lipid binding properties of apo hA-I.     

Repetitive elements in the third intron of murine apolipoprotein A-I gene

PURPOSE: To review the long-term experience of helium ion therapy as a therapeutic alternative to enucleation for uveal melanoma, particularly with respect to survival, local control, and morbidity. METHODS AND MATERIALS: 347 patients with uveal melanoma were treated with helium ion RT from 1978-1992. A nonrandomized dose-searching study was undertaken, with doses progressively reduced from 80 GyE in five fractions to 48 GyE in four fractions, given in 3-15 days, mean of 7 days. RESULTS: Local control was achieved in 96% of patients, with no difference in the rate of local control being seen at 80, 70, 60, or 50 GyE in five fractions. At the lowest dose level of 48 GyE in four fractions, the local control rate fell to 87%. Fifteen of 347 patients (4%) had local regrowth in the eye requiring enucleation (12 patients), laser (1 patient) or reirradiation (2 patients). The time of appearance of local regrowth ranged from 4 months to 5 years posttreatment, with 85% occurring within 3 years. Of the 347 patients, 208 are alive as of May 1, 1997. The median follow up of all patients is 8.5 years, range 1-17 years. Kaplan-Maier (K-M) survival is 80% at 5 years, 76% at 10 years, and 72% at 15 years posttreatment. Patients with tumors not involving the ciliary body have a 15-year K-M survival of 80%. The results for patients whose tumors involved the ciliary body are poor, with a 15-year K-M survival of 43%. Seventy-five percent of patients with tumors at least 3.0 mm from the fovea and optic nerve, and initial ultrasound height less than 6.0 mm, retained vision of 20/200 or better posttreatment. Patients with tumors larger than 6 mm in thickness, or with tumors lying close to the optic nerve or fovea, have a reduced chance of retaining useful vision. The enucleation rate is 19%, 3% for local failure and 16% because of complications of the helium RT, particularly neovascular glaucoma, which occurred in 35% of patients. CONCLUSIONS: Local control and retention of the eye are excellent. Complications of therapy reduce vision and eye preservation. Twenty-four percent of patients manifested distant metastases 6 to 146 months posttreatment, mean of 43 months, median of 36 months. Late-appearing distant metastases do not appear to be caused by persistent tumor in the eye. The risk of metastases is high for patients with tumors greater than 7 mm in initial ultrasound height (37%), anterior tumors involving the ciliary body (47%), and in those with local failure (53%). Patients with tumors not involving the ciliary body and initial dimensions less than 10 mm had only an 8% chance of death from melanoma. A search for effective adjuvant therapy is needed for patients at high risk of metastases (large tumors, ciliary body involved, local regrowth in eye).     

Evolution of mammalian apolipoprotein A-I and conservation of antigenicity: correlation with primary and secondary structure

We have evaluated the immunoreactivity of 20 monoclonal antibodies (mAbs) directed against human apolipoprotein (apo)A-I with a panel of high density lipoproteins (HDL) from 13 mammalian species. The pattern of cross-reactivity showed that 20 mAbs had different specificity. While not all mAbs recognized apoA-I from all of the different species, the antigenicity of some sequences was well conserved. Thus, mAb A05 cross-reacted with all species except guinea pig and rat. In contrast, the mAb 4H1, which recognized residues 2-8, required a specific proline in position 3, as no immunoreactivity was found in the species missing this amino acid. Furthermore, the presence of a threonine residue in place of serine (in position 6) in the cynomolgus monkey was associated with a 20-fold loss of immunoreactivity in radioimmunometric assay with 4H1. As most of the epitopes were found in CNBr fragments 2 and 3, we sequenced these regions in four species (horse, goat, sheep, and cat) and analyzed the alignment of most known sequences to evaluate their consensus. Except for the rat and the chicken, considerable identity was observed. This permitted us to deduce the involvement of the residues in some antigenic epitopes. In the middle of apoA-I, a conservative mutation Asp103-->Glu was found sufficient to eliminate all reactivity of this epitope for A11 (residues 99-105 ... 12l6-132) in five species (rabbit, cow, goat, sheep, and rat). The residues essential to the expression of two other epitopes overlapping with A11 were also characterized. Edmundson-wheel representation of 18-residue repeated sequences of the different apoA-I species (for the eight amphipatic helices of residues 46-63, 68-85, 101-118, 123-140, 143-160, 167-184, 189-206, and 222-239) showed that secondary structure of apoA-I was more conserved than the antigenic epitopes. The N-terminal region, residues 1 to about 98, is rich in both strictly preserved sequences and epitope expression in most of the species surveyed. This evolutionary conservation of the N-terminal domain suggests an important yet unknown function.     

Effect of long chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine on the interaction with recombinant high density lipoprotein apolipoprotein A-I

The effects of polyunsaturated fatty acids (PUFA) on the structure of recombinant high density lipoprotein (rHDL) was investigated using homogeneous particles containing phosphatidylcholine (PC), [3H]cholesterol, and apolipoprotein A-I (apoA-I). The PC component of the rHDL contained sn -1 16:0 and sn -2 18:1 (POPC), 18:2 (PLPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC). The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.57-1.70 m) compared to those containing POPC (2.83 m). Intrinsic apoA-I tryptophan fluorescence emission intensity and lifetimes were decreased for rHDL containing long chain PUFA compared to POPC and PLPC rHDL. Monoclonal antibody binding studies demonstrated that apoA-I had decreased immunoreactivity with monoclonal antibodies spanning amino acid residues 115-147 in rHDL containing long chain PUFA. PC lipid fluidity, measured as diphenylhexatriene (DPH) fluorescence polarization, was increased in PUFA rHDL compared to POPC rHDL. There also was a strong correlation between the number of sn -2 double bonds in rHDL and DPH fluorescence lifetime (r 2 = 0. 89). LCAT reactivity of the homogeneous size rHDL was ordered POPC = PLPC>PAPC> PEPC>PDPC. We conclude that rHDL with long chain PUFA in the sn -2 position of PC contain apoA-I that is less stable and in a different conformation than that in POPC rHDL and have a fatty acyl region that is more fluid and hydrated. The weaker interaction of apoA-I with PC containing PUFA may lead to hypercatabolism of apoA-I in plasma explaining, in part, the decreased plasma HDL and apoA-I concentrations seen with PUFA diets.     

Modifications in high-density lipoprotein lipid composition and structure alter the plasma distribution of free and liposomal annamycin

Recent studies have shown that changes in lipoprotein cholesterol and triglyceride concentration alters the plasma distribution of free (Ann.) and liposomal annamycin (LAnn) and that the majority of Ann. is associated with high-density lipoproteins (HDL) following the incubation in plasma of LAnn. To demonstrate that alterations in HDL lipid composition and HDL structure may influence the plasma distribution of Ann. and LAnn, Ann. and LAnn (20 micrograms/mL) were incubated in plasma pretreated with dithionitrobenzoate (DTNB, a compound which inhibits the conversion of free cholesterol to esterified cholesterol) 18 h prior to the experiment or in untreated plasma for 60 min at 37 degrees C. In addition, Ann. and LAnn were co-incubated with DTNB in plasma for 60 min at 37 degrees C. Following incubation the plasma was separated into its HDL, low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and lipoprotein-deficient plasma (LPDP) fractions by ultracentrifugation and assayed for Ann. by fluorimetry. The HDL plasma cholesterol:triglyceride concentration ratio was significantly decreased following 18 h of DTNB pretreatment compared to untreated plasma controls. No significant differences in LDL/VLDL plasma cholesterol:triglyceride concentration ratio following 18 h of DTNB pretreatment was observed. An increased number of discoidal HDL particles were observed following 18 h of DTNB pretreatment. When Ann. was incubated in plasma pretreated with DTNB for 18 h the percentage of Ann. recovered in the HDL, LDL, and VLDL fractions significantly increased. However, the percentage of Ann. recovered within the LPDP fraction was significantly decreased. When LAnn was incubated in plasma pretreated with DTNB for 18 h the percentage of Ann. recovered in the HDL fraction significantly decreased. The percentage of Ann. recovered in the LPDP fraction significantly increased when LAnn was incubated in plasma pretreated with DTNB for 18 h. No significant differences in Ann. lipoprotein distribution were observed when Ann. and LAnn were co-incubated with DTNB in plasma for 1 h. These findings suggest that the cholesterol:triglyceride concentration ratio and physical structure of HDL maybe important in defining the capacity of HDL to sequester Ann.     

Acid lipase inhibitor in chicken plasma identified as apolipoprotein A-I

We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895-898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken. The purified inhibitor migrated with the same mobility on SDS-PAGE as apo A-I, and had a molecular weight of 27,000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.     

Secretion and processing of apolipoprotein A-I in the avian sciatic nerve during development

Apolipoprotein A-I (apo A-I), a major apolipoprotein synthesized by liver and intestine to facilitate transport of plasma lipids as lipoproteins, has been detected also in the avian sciatic nerve. The mRNA and protein levels of apo A-I have been shown to increase during the period of rapid myelination (LeBlanc et al.: J Cell Biol 109:1245-1256, 1989). In order to assess the synthesis of apo A-I protein and the processing of apo A-I isoforms during development, endoneurial slices of avian sciatic nerves from chicks during active myelination at 15 and 17 days embryonic and 1 day posthatch age were incubated with [35]S-methionine. The incubations were fractionated into secreted and intracellular fractions, and incorporation of the label was assessed for apo A-I protein. The pattern of labeling of Po protein, as a marker of myelination, was also determined in the intracellular and compact myelin fractions. Methionine incorporation into Po protein was highest in the intracellular compartment at the 15-day embryonic stage and decreased thereafter, with a corresponding increase in the myelin fraction. During these developmental periods, the levels of nascent apo A-I increased in both the secreted and intracellular fractions. The synthesis of apo A-I specifically increases in the secreted fraction compared with total protein synthesis. The processing of the pro-apo A-I is also developmentally regulated. In the intracellular compartment, there are approximately equal proportions of the acidic and basic isoforms. However, with increasing age, a higher proportion of the apo A-I is secreted as acidic isoforms. It is concluded that the secretion and processing of apo A-I is developmentally regulated in the chick sciatic nerve, in parallel with the process of active myelination.     

Lipoproteins containing apolipoprotein A-IV but not apolipoprotein A-I take up and esterify cell-derived cholesterol in plasma

Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) identifies distinct apoA-I-or apoE-containing subclasses of high-density lipoproteins (HDLs), each of which plays a different role in reverse cholesterol transport. In this study we used 2D-PAGGE to investigate the role of apoA-IV-containing lipoproteins in reverse cholesterol transport in native plasma. Incubation of 2D electrophoretograms with anti-apoA-IV antibodies identified up to three subclasses of particles. The smaller particle subclasses, LpA-IV-1 and LpA-IV-2, were found in every plasma sample. The largest particle subclass, LpA-IV-3, was observed in fewer than 10% of the plasmas analyzed. 2D-PAGGE of apoA-I-deficient plasma and apoA-I-depleted plasma and anti-apoA-I immunosubtracting 2D-PAGGE of normal plasma revealed that LpA-IV-1 and LpA-IV-2 do not contain apoA-I. The importance of LpA-IV-1 and LpA-IV-2 for uptake and esterification of cell-derived cholesterol was investigated using pulse-chase incubations of plasma with [3H]cholesterol-labeled fibroblasts followed by anti-apoA-I immunosubtracting 2D-PAGGE. During 1-minute pulse incubation with cells, [3H]cholesterol was taken up by gamma-LpE > LpA-IV-1 > pre-beta 1-LpA-I > LpA-IV-2 (">" denotes "more than"). During subsequent chase incubation without cells, proportionately less radioactivity disappeared from LpA-IV-1 and LpA-IV-2 than from pre-beta 1-LpA-I and gamma-LpE. During 5-minute pulse incubations, radioactive cholesteryl esters were formed in pre-beta 3-LpA-I > alpha-LpA-I > LpA-IV-1 > LpA-IV-2. The fractional estertification rate was highest in pre-beta 2-LpA-I and lowest in alpha-LpA-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of apolipoprotein CIII in triglyceride-rich lipoprotein metabolism

Fibroblast growth factor (FGF) has been reported to increase the volume of callus in a fracture model of rats. There are, however, no reports of successful repair of segmental bony defects by application of an FGF solution. In this study, the effects of basic FGF on the repair of segmental bony defects in the rabbit femur were examined. Minipellet, a new drug delivery system using atelocollagen, was employed to ensure effective delivery of FGF. Segmental bony defects (10 mm in length) were created in the right femurs of 19 rabbits. In pilot studies, no defects of this size healed spontaneously within 6 weeks. Bones were stabilized with miniexternal fixators. Minipellets containing basic FGF were implanted between fragments so as to bridge the two fragments. The healing processes were monitored radiographically and studied histologically. In rabbits in which FGF was added to the defect site at doses of 1.4 microgram or higher, approximately 90% of the defects were filled with new bone and cartilage within 6 weeks after minipellet implantation. In rabbits receiving placebo minipellets, however, approximately 15% of the defects were filled by callus within 6 weeks. Furthermore, this callus did not change into mature bone. An injection of 2 microgram of FGF solution to bony defects had no effect on the repair of segmental bony defects. These findings suggest that FGF plays a role in the production of adequate volumes of callus particularly in the initial stages of fracture healing and that sustained local release enables FGF to be effective at a low dose. In summary, large segmental bony defects healed after insertion of low-dose FGF minipellets. An adequate dose of FGF and an appropriate delivery system are required for successful healing of large bony defects. These findings imply the potential value of FGF minipellets in clinical practice.     

Centripetal cholesterol flux to the liver is dictated by events in the peripheral organs and not by the plasma high density lipoprotein or apolipoprotein A-I concentration

The major net flux of cholesterol in the intact animal or human is from the peripheral organs to the liver. This flux is made up of cholesterol that is either synthesized in these peripheral tissues or taken up as lipoprotein cholesterol. This study investigates whether it is the concentration of apolipoprotein (apo) A-I or high density lipoprotein in the plasma that determines the magnitude of this flux or, alternatively, whether events within the peripheral cells themselves regulate this important process. In mice that lack apoA-I and have very low concentrations of circulating high density lipoprotein, it was found that there was no accumulation of cholesterol in any peripheral organ so that the mean sterol concentration in these tissues was the same (2208 +/- 29 mg/kg body weight) as in control mice (2176 +/- 50 mg/kg). Furthermore, by measuring the rates of net cholesterol acquisition in the peripheral organs from de novo synthesis and uptake of low density lipoprotein, it was demonstrated that the magnitude of centripetal sterol movement from the peripheral organs to the liver was virtually identical in control animals (78 +/- 5 mg/day per kg) and in those lacking apoA-I (72 +/- 4 mg/day per kg). These studies indicate that the magnitude of net sterol flux through the body is not related to the concentration of high density lipoprotein or apolipoprotein A-I in the plasma, but is probably determined by intracellular processes in the peripheral organs that dictate the rate of movement of cholesterol from the endoplasmic reticulum to the plasma membrane.