Origin of apolipoprotein A-I polymorphism in plasma

The origin and the functional significance of apo-A-I polymorphism in man has been investigated. Together with proapo-A-I (identified as A-I1 of the polymorphic series), four other isoforms are found in human plasma, namely A-I2, A-I3, A-I4, and A-I5. A-I3 is the "mature" product of proapo-A-I conversion in plasma. In this study we provide evidence that the other, more acidic, mature apo-A-I isoproteins are derived from A-I3 by a stepwise deamidation process. This conclusion is based on the following observations. 1) Incubation of A-I3 or A-I4, either free or associated with high density lipoprotein, produces a series of more acidic isoproteins corresponding to the sequence found in plasma. The conversion process fits in well with a first order reaction, and A-I3 to A-I4 conversion occurs virtually at the same rate as A-I4 to A-I5 conversion. 2) A-I3 and A-I4 have the same NH2- and C-terminal residues. 3) Formation of apo-A-I acidic isoproteins is accompanied by liberation of ammonia. In order to investigate whether deamidation of apo-A-I results in the production of forms which have different catabolism, a series of turnover studies was carried out in normal volunteers. A-I3 and A-I4 residence times in plasma were, respectively, 3.50 +/- 0.16 and 3.00 +/- 0.10 days (mean +/- S.E.; n = 3). Degradation rate of A-I3 was 8.81 +/- 0.69 mg/kg/day and that of A-I4 was 1.66 +/- 0.15 mg/kg/day (mean +/- S.E.; n = 3). Conversion of A-I3 to A-I4 and A-I4 to A-I5 occurred at the same rate in vivo as that observed in vitro. These results are consistent with the concept that A-I3 is the precursor to the other mature apo-A-I isoforms in plasma. A-I3 is the major isoform through which apo-A-I is eliminated from plasma.     

Dietary cholesterol absorption, and sterol and bile acid excretion in hypercholesterolemia-resistant white rabbits

The New Zealand white (NZW) rabbit fed a 0.1% cholesterol-enriched diet (CD) typically responds (normoresponsive, NR) by quickly developing hypercholesterolemia. To study the underlying mechanisms responsible for the widespread phenomenon of inter-individual variability of response to dietary cholesterol, a unique hypercholesterolemia-resistant (RT) rabbit model was developed. These animals were utilized to investigate selected potential mechanisms that might enable the RT animal to compensate for dietary cholesterol overload. When rabbits were fed the low-cholesterol stock diet, there was no significant difference in the plasma cholesterol concentrations of the NR and the RT animals. However, a significant rise was observed in the NR rabbits within 1 month of their being placed on the cholesterol-enriched diet; the plasma cholesterol concentration of the RT animals was not affected. During consumption of the cholesterol diet the cholesterol absorption rate was somewhat greater in the NR rabbits (P less than 0.05), whereas intestinal transit times and the fecal excretion of neutral steroids were substantially the same in both groups. In contrast, the fecal bile acid excretion of the RT animals was more than twice as great (P less than 0.0001) as that of the NR animals. We conclude that the response to dietary cholesterol is a heritable trait in these rabbits and that, although less dietary cholesterol was absorbed by the RT animals, it appears that a major mechanism controlling plasma cholesterol levels involves the rate of conversion of cholesterol to bile acids and their subsequent excretion.     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.     

The primary structure of human plasma high density apolipoprotein glutamine I (ApoA-I). II. The amino acid sequence and alignment of cyanogen bromide fragments IV, III, and I.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is the major protein constituent of the human plasma high density lipoproteins. Cleavage of this protein with cyanogen bromide yields four fragments, designated in the order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the first paper in this series, the amino acid sequence of the NH2-terminal fragment, CNBr II, is reported. In the present study, CNBr IV, III, and I, containing, respectively, 25, 36, and 94 amino acids were sequenced by conventional means. To establish the alignment of the cyanogen bromide fragments, apoLP-Gln-I was digested with trypsin and two of the three methionine-containing tryptic peptides were isolated. The amino acid sequence of apoLP-Gln-I is as follows: (see article). With the complete amino acid sequence available, a CPK space-filling model of apoLP-Gln-I was constructed. The protein was placed into an alpha helical conformation wherever the primary structure permitted. Thirteen helical regions were identified. These regions account for 70% of the amino acid residues of the protein. Each helix is characterized as having two faces (amphipathic). One is a polar face that occupies approximately 180 degrees of the surface of the helix and the other is a hydrophobic face that occupies the other 180 degrees of the helical surface. Similar amphipathic helices have been identified previously in the other lipoprotein-proteins that have known sequences. It is suggested that the amphipathic helical regions of apoLP-Gln-I are important in the binding of phospholipids in high density lipoproteins.     

Enhanced catabolism of low density lipoproteins in rat by lactosaminated Fab fragment. A new carrier of macromolecules to the liver

Proteins conjugated with lactose residues exhibit enhanced hepatic uptake mediated by the galactose receptor. In this study, we demonstrate that lactosaminated Fab fragments (lac-Fab) of IgG can induce hepatic catabolism of specific antigens, especially low density lipoproteins (LDL). lac-Fab and human LDL-lac-Fab complex exhibited specific uptake in isolated rat hepatocytes. In vivo in the rat, lactosamination enhanced plasma clearance of Fab fragments 2-fold and hepatic localization 20-fold. Fab fragments retained their affinity after lactosamination. Hepatic uptake of rat 125I-IgG complexed in vitro with anti-rat lac-Fab was increased almost 5-fold, compared to rat 125I-IgG alone. Injection of rats with anti-LDL lac-Fab induced plasma clearance and hepatic uptake of tracer amounts of previously injected human 125I-LDL, which decreased 50% 10 min after injection of lac-Fab, with 30% present in the liver. Asialofetuin completely inhibited these processes. After a bolus of 6 mg of human LDL, administration of anti-LDL lac-Fab reduced the serum cholesterol of rats to basal values within 2.5 h. These findings suggest that lactosaminated Fab fragments of specific IgGs are effective reagents for inducing hepatic uptake of macromolecules through the galactose receptor. lac-Fab specific for LDL may be an effective hypocholesterolemic agent in vivo.     

Novel mutant of Anabaena sp. strain CA which growns on N2 but not on combined nitrogen.

A mutant has been isolated from Anabaena sp. strain CA by treatment with N-methyl-N'-nitro-N-nitrosoguanidine, which has the unusual phenotypic characteristic of growth only under N2-fixing conditions. Growth of the mutant was completely inhibited by NO3- or NH4+ at concentrations routinely used for growth of the wild type, and sensitivity to NH4+ was especially pronounced. The inhibitory effect of NH4+ could not be overcome by glutamine, glutamate, or casein hydrolysate. Ammonia had no immediate inhibitory effect on protein synthesis, CO2 fixation, or O2 evolution, and the gradual inhibition of C2H2 reduction activity by NH4+ resembled a repression phenomenon. The glutamine synthetase activity of N2-fixing cultures appeared normal, yet the mutant was incapable of utilizing exogenous NH4+ for growth. Preliminary evidence suggests a possible alteration of glutamine synthetase, which could result in sensitivity to exogenous NH4+ by progressive inactivation of the enzyme or repression of its synthesis.     

Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope

Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.     

Development and partial metabolic characterization of a dietary cholesterol-resistant colony of rabbits

A colony of New Zealand white rabbits has been developed which, when fed a cholesterol-supplemented diet, exhibit unusual resistance to hypercholesterolemia and atherosclerosis, disorders usually observed in normal cholesterol-fed rabbits. When resistant rabbits (RT) were fed a normal low cholesterol diet (ND), their plasma lipoprotein patterns were significantly different from those of normal rabbits (NR) fed the same diet. The low density lipoprotein cholesterol (LDL-c)/high density lipoprotein cholesterol (HDL-c) ratio and LDL-c/very low density lipoprotein cholesterol (VLDL-c) ratio were lower in the resistant rabbits. The hydrated density of HDL of the normal-responsive rabbits was greater than that of the resistant rabbits. LDL from resistant rabbits contained a lower proportion of esterified cholesterol and protein than LDL from normal rabbits. Peripheral mononuclear cells from resistant rabbits bound about 30% more 125I-labeled rabbit LDL than mononuclear cells from normal rabbits. These results demonstrate that the plasma cholesterol levels of these animals is at least partly under genetic control and that compositional differences exist between the major plasma lipoprotein classes of normal and resistant rabbits even during the ingestion of low-cholesterol diet. The results indicate that at least a part of the difference in the cholesterolemic responses between the two rabbit groups is due to an enhanced LDL uptake by the mononuclear cells, and presumably by other somatic cells of the resistant group.