Cholesterol efflux from cultured adipose cells is mediated by LpAI particles but not by LpAI:AII particles

Cholesterol efflux was studied in cultured adipose cells after preloading with LDL cholesterol. Long-term exposure to LpAI and LpAI:AII particles isolated from the HDL fraction showed that LpAI particles only were able to promote cholesterol efflux. Liposomes containing different ApoAI/ApoAII molar ratios were tested: the larger the proportion of ApoAI, the faster the ability to remove cholesterol from Ob1771 cells. Dose-response curves showed that LpAI particles were active within a physiological range of concentrations, whereas LpAI:AII particles had no effect at all concentrations. The results are in favour of LpAI particles being the active components of the HDL fraction for the promotion of cholesterol efflux and suggest that LpAI particles and LpAI:AII particles represent distinct metabolic entities.     

Acylgalactosylceramides in Developing Dysmyelinating Mutant Mice

Acylgalactosylceramides (AGC) from forebrains of normal and dysmyelinating (quaking and shiverer) mice were purified by Florisil column chromatography and preparative TLC. These procedures resolved the AGC on the basis of their R_f values into two main fractions which co-nigrate with their homologs from rat forebrains. In control animals, AGC were detectable in mouse forebrains from the eighth postnatal day and reached maximal values within 20 days. The same developmental pattern was obtained in dysmyelinating shiverer mice but the AGC content was reduced to approximately 30% of control values. In quaking mutants, the AGC were hardly detected. They were also present in sciatic nerve of normal mice and to a lesser extent in trembler mice. Gas chromatography-mass spectrometry analysis of both ester- and amide-linked fatty acids isolated from AGC of normal and shiverer mice shows that the shiverer mutant AGC display a chemical structure similar to that of normal AGC. AGC constituents of control myelin are reduced by approximately 70% in shiverer myelin, indicating that these molecules can be considered as early markers of oligodendrocyte differentiation. The early arrest of myelinogenesis in the quaking animals and the near absence of AGC are in good agreement with this proposal. Moreover, the reduced amount of AGC in the trembler PNS indicates that AGC could also be early markers for differentiation of the Schwann cell.     

The polymorphism ApoB/4311 in patients with myocardial infarction and controls: the ECTIM study

Summary The polymorphism affecting codon 4311 of the apolipoprotein B gene (ApoB/4311) was investigated in a large case-control study in two French and one Northern Irish geographically defined populations. Cases were recruited 3 to 9 months after a myocardial infarction (MI) and controls were randomly selected from the population. The polymorphism was assessed using allele-specific oligonucleotides (ASO). The genotype frequencies of the ApoB/4311 polymorphism did not differ in Northern Ireland and France and were in Hardy-Weinberg equilibrium in all groups; strong associations with three other polymorphisms of the ApoB gene (XbaI, EcoRI, VNTR(34 repeats)) were observed and it was possible to identify highly sensitive and specific markers of the ApoB/4311 rare variant. Homozygotes for the ApoB 4311 rare variant were slightly less frequent in cases than in controls: 22 (4.4%) and 35 (6.7%) respectively (population adjusted ²=3.3 P<0.07), especially in Belfast: 6 (3.1%) and 12 (7.6%), respectively (P<0.06). Several lipid and lipoprotein parameters were measured. Consistently among control groups, rare homozygotes had lower mean levels of ApoB (P<0.02), triglycerides (P<0.02), and lipoprotein particles containing ApoE and ApoB (LpE:B; P<0.001) and a higher mean level of lipoprotein particles containing ApoAI and not ApoAII (LpAI; P<0.02) than heterozygotes and frequent homozygotes combined. The strong association between the ApoB/4311 polymorphism and LpE:B was also observed in patients with MI. When present in the homozygous form, the ApoB/ 4311 AsnSer variant is associated with a lipoprotein profile that is apparently favourable.     

Preformed PAF-acether and lyso PAF-acether are bound to blood lipoproteins

PAF-acether (PAF) is a newly formed mediator not normally present in circulating blood. A compound exhibiting all of its biological characteristics but coeluting with phosphatidylcholine (PC) in high-pressure liquid chromatography (HPLC) was unveiled ('peak X') in normal human plasma. A second HPLC run of peak X HPLC fractions revealed the presence of PAF itself with concomitant disappearance of peak X. Beside PAF, immunoreactive apolipoproteins A-I and E were found in peak X. Also lipoproteins (Ls) purified using either ultracentrifugation or immunoaffinity chromatography yielded peak X and, in a second HPLC run, authentic PAF. L-free plasma was devoid of peak X. Finally, after preincubation with plasma, labeled PAF was found associated with Ls. Thus in human blood preformed PAF is bound in high amounts to Ls, a result of interest given the role of Ls and platelets in vascular diseases and the present knowledge on PAF biosynthesis.     

Immunochemical characterization of two antigenic sites on human apolipoprotein A-I; localization and lipid modulation of these epitopes

Two monoclonal antibodies, A17 and A30, were raised against human apolipoprotein A-I (apo A-I). They were studied by competitive inhibition of 125I-labeled HDL3 with HDL subfractions, delipidated apo A-I, and complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I and apo A-II. Immunoblotting located the A17 antibody on CNBr fragment 4 of apo A-I and the A30 antibody on CNBr fragment 1. The A17 antigenic determinant was expressed identically in all HDL subclasses, on delipidated apo A-I as well as all on the DMPC-apo A-I and DMPC-apo A-I/apo A-II complexes. In contrast, the apparent affinity constant of the A30 antibody for delipidated apo A-I was about 30-times less than for HDL3 or for apo A-I/apo A-II-phospholipid complexes. These data suggest that the association of apo A-I with phospholipids improves the reactivity of the A30 monoclonal antibody towards apo A-I, and that this antigenic determinant has a different conformation in delipidated apo A-I compared to apo A-I complexed with phospholipids. Turbidimetric and fluorescence experiments monitoring the phospholipid-apo A-I association in the presence and in the absence of the A17 and A30 antibodies were consistent with the competition experiments carried out by solid phase radioimmunoassay (RIA). After reaction of apo A-I with the A30 antibody, we observed an enhancement of the degradation kinetics of large multilamellar vesicles (LMV), while the A17 antibody did not have a significant effect. Calcein leakage experiments carried out below the transition temperature of DPPC showed an enhancement of the degradation kinetics with both monoclonal antibodies, while the phase-transition release was independent of the reaction of apo A-I with the monoclonal antibodies. These data therefore suggest the existence of at least two different types of epitope on apo A-I, which might account for the differences in immunological reactivity of apo A-I that is either delipidated or present on HDL.     

Latex immunoassay of human serum Lp(a+) lipoprotein

A sensitive latex immunoassay for human serum lipoprotein Lp(a+) is based on direct agglutination by Lp(a+) of latex particles coated with specific antibody. The agglutination is quantified by turbidimetry using a photometer at 360 nm. The stabilization of antibody-coated latex particles by bovine serum albumin occurs under well-defined conditions (pH, concentration of bovine serum albumin, and antibody loading of latex particles). The standard curve of serum lipoprotein Lp(a+) ranges from 0.05 to 1.15 mg/l. Inter- and intra-assay coefficients of variation were less than 8% and 3%, respectively. Results were well correlated with those obtained by electroimmunodiffusion (r = 0.98, n = 108).     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Rat monoclonal antibodies to human apolipoprotein B: advantages and applications

Eight monoclonal antibodies (Mabs) to human serum low density lipoprotein (LDL) were derived from the fusion of spleen cells, from LOU rats immunized with human LDL, and the rat myeloma line IR983F. These Mabs were characterized in terms of isotype, specificity, and affinity. Competitive experiments indicated that the epitopes that were recognized could be grouped into three patterns depending on their apparent affinity for apoB-containing lipoprotein particles such as LDL, very low density lipoproteins (VLDL), or intermediate density lipoproteins (IDL). Six epitopes have been mapped in relation to elements of the sequence of apolipoprotein B-100 (apoB-100) and some have been assigned to the middle part of the median thrombolytic fragment T3, a region not yet well targeted by mouse Mabs. The presence of lipids for the expression of the epitopes was studied and confirmed a lipid dependence for epitopes that are close to the T2/T3 cleavage site. The capacity of binding to the LDL receptor was also tested; among the Mabs we described, one inhibited the uptake and degradation of LDL to HeLa cells receptor. Finally, some antibodies were able to precipitate LDL in gel.