Isolation of apolipoprotein(a) from lipoprotein(a)

An easy method was developed for the rapid and selective isolation of apo(a) from human plasma Lp(a). This procedure was applied to a "low density" Lp(a) subspecies (usually found in the density interval 1.050 to 1.070 g/ml) from a single individual whose apo(a) was of a size smaller than apoB-100. After reduction with 0.01 M dithiothreitol, apo(a) was separated from the Lp(a) particle by rate zonal centrifugation on a 7.5-30% NaBr density gradient. Two completely water-soluble products were recovered: apo(a), which contained less than 1% each of phospholipid and cholesterol, remained at the bottom of the gradient, and a lipid-rich floating LDL-like particle which contained apoB but not apo(a) and which we referred to as Lp(a-). The separation of these two components was also achieved by subjecting reduced Lp(a) to electrophoresis on 2.5-16% polyacrylamide gradient gels. However, dissociation of reduced Lp(a) could not be achieved by gel filtration in either low or high salt solutions. These observations indicate that apo(a) is associated to Lp(a) by non-covalent interactions in addition to its disulfide linkage to apoB. The latter is sensitive to chemical reduction whereas the former are broken through the action of a gravitational or electrical field.     

Evaluation of a new apolipoprotein(a) isoform-independent assay for serum Lipoprotein(a)

The risk factor, Lipoprotein(a), [(Lp(a)], has been measured in numerous clinical studies by a variety of immunochemical assay methods. It is becoming apparent that for many of these assays antibody specificity towards the apolipoprotein(a) [apo(a)] repetitive component [the kringle 4 - type 2 repeats] and apo(a) size heterogeneity can significantly affect the accuracy of serum Lp(a) measurements. To address this issue, we investigated whether our current in house Lp(a) [Mercodia] assay showed such bias compared to a recently available assay [Apo-Tek], claiming to possess superior capability for isoform-independent measurement of Lp(a). Levels of Lipoprotein(a) by both Apo-Tek and Mercodia assays correlated inversely with apo(a) isoform sizes. No significant differences were observed between assays in ranges of Lp(a) concentration within each isoform group. The Mercodia assay exhibited similar isoform-independent behaviour to that of Apo-Tek for e quantitation of serum Lipoprotein(a). Essentially identical results were obtained by the two methods, suggesting that Mercodia assay's capture monoclonal antibody also (as is the case for Apo-Tek) does not recognize the kringle 4-type 2 repetitive domain of apo(a). Correlation of Lp(a) concentrations in patient specimens between Apo-Tek and Mercodia assays showed good agreement, although an overall higher degree of imprecision and non-linearity was noted for the Apo-Tek procedure. A change-over to the Apo-Tek assay would therefore not improve on our current assessment of risk contribution from Lp(a) for atherosclerotic vascular disease in individuals with measurable levels of circulating Lipoprotein(a).     

Cardiac glycosides in a scale insect (Aspidiotus), a ladybird (Coccinella) and a lacewing (Chrysopa)

The presence of cardiac glycosides in the body tissues of three species of insects from three different orders, all feeding on Nerium oleander L., is recorded.     

人血浆载脂蛋白（a）的分离及纯化

本文报道从人血浆脂蛋白Ｌｐ（ａ）中,分离纯化载脂蛋白（ａ）。收集富含Ｌｐ（ａ）的混合血浆,超离心,获密度１．０５ｇ／ｍｌ至１．０８ｇ／ｍｌ的粗制Ｌｐ（ａ）,经过Ｂｉｏ－Ｇｅｌ Ａ５ｍ层析后,证明纯化后的Ｌｐ（ａ）仅与ａｐｏ（ａ）抗血清反应,经ＤＴＴ处理过的Ｌｐ（ａ）,在琼脂糖电泳中的泳动率由胶β位移到β位,在印迹免疫反应中,对ａｐｏ（ａ）的抗血清反应依然显示在前β位,ＳＤＳ聚丙烯凝胶电脉的迁移率慢     

Assembly of lipoprotein (a) in transgenic rabbits expressing human apolipoprotein (a)

The study of human lipoprotein (a) [Lp(a)] has been hampered due to the lack of appropriate animal models since apolipoprotein (a) [apo(a)] is found only in primates and humans. In addition, human apo(a) in transgenic mice can not bind to murine apoB to form Lp(a) particles. In this study, we generated three independent transgenic rabbits expressing human apo(a) in their plasma at 1.8-4.5 mg/dl. In the plasma of transgenic rabbits, unlike the plasma of transgenic mice, about 80% of the apo(a) was covalently associated with rabbit apo-B and was contained in the fractions with density 1.02-1.10 g/ml, indicating the formation of Lp(a). These results suggest that transgenic rabbits expressing human apo(a) exhibit efficient assembly of Lp(a) and can be used as an animal model for the study of human Lp(a).     

Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a)

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp^55/57→Ala^55/57 substitution of kringle (K)IV₁₀)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV₁₀ LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp⁵⁷→Ala⁵⁷ substitution in KIV₁₀ that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV₁₀ LBS and is unique to human apo(a), which may explain Lp(a)’s pro-atherogenic potential.     

Electroelution of lipoprotein(a) [Lp(a)] from native polyacrylamide gels: a new, simple method to purify Lp(a)

Lipoprotein(a), Lp(a), is an atherogenic lipoprotein consisting of an LDL like core particle and a covalently linked glycoprotein of variable size. Lp(a), isolated from serum always contains LDL and HDL(2) as contaminants since Lp(a) floats in the density range 1.05-1.12 g/ml which overlaps that of LDL and HDL(2). Purified Lp(a) is increasingly needed as a standard to overcome various problems in the standardization of Lp(a) measurements and for in vitro biological studies. Problems inherent to the purification of Lp(a) include the aggregation of Lp(a) with LDL, overlapping size distribution and the inability of some fractions to bind to affinity columns. Here, we describe the development of a new method to purify Lp(a) from contaminating LDL and HDL(2) particles. Lp(a) was isolated from serum by sequential ultracentrifugation, resolved by native polyacrylamide gel electrophoresis and the gel segments were electroeluted to obtain pure Lp(a). l-Proline was added to the sample to a final concentration of 0.1 M to prevent the aggregation of Lp(a) with LDL.     

Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 (congruent to 450,000). The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.     

Recovery of a sooty shearwater (

We describe the recovery of an 88-m² area of sooty shearwater breeding habitat on Northeast Island, The Snares, New Zealand, during the eight breeding seasons after it was completely destroyed by excavation in December 1996. Burrow entrance density did not differ between the destroyed site and three comparison sites one year after the event. We detected breeding attempts of shearwaters in the season following disturbance, but burrow occupant density recovered more slowly, perhaps because the overall population density was declining over the same period. Burrow occupant density did not differ between the destroyed site and comparison sites within five breeding seasons of disturbance.     

Sulfhydryl Compounds in Melanocytes of Yellow (Ay/a ), Nonagouti (a/a), and Agouti (A/A) Mice

CLEFFMANN (1953, 1963a,b) has reported that yellow but not black melanocytes of agouti (A/A) rabbits contained reducing sulfhydryl compounds. We have attempted to repeat CLEFFMANN's observations in mouse melanocytes of the lethal yellow (Ay/a), nonagouti (a/a) and agouti (A/A) genotypes. Our results contradict those of CLEFFMANN and reveal that yellow and black melanocytes, regardless of genotype, possess equivalent amounts of histochemically detectable sulfhydryl compounds. These results do not support the hypothesis that agouti-locus genes act by controlling the sulfhydryl metabolism of pigment cells.     

Energetics of offspring production: a comparison of a marsupial (Monodelphis domestica) and a eutherian (Mesocricetus auratus)

This study compares the energetic cost of reproduction during gestation and lactation of a eutherian, the golden hamster (Mesocricetus auratus), and a similar-sized (60–120 g) marsupial, the gray short-tailed opossum (Monodelphis domestica). Food consumption was monitored in 20 reproductively active (RA) opossums and 16 RA hamsters from conception to weaning and at equivalent intervals in 19 non-reproductive (NR) opossums and 21 NR hamsters, all maintained within their zone of thermoneutrality (30 °C). Total energy assimilated from conception to weaning [opossums: 1261.3 ± 28.0 Kcal (1 Kcal = 4.1868 J) and hamsters: 1647.5 ± 60.6 Kcal] was positively correlated with litter size and mass per young in both species. Maternal mass-specific assimilated energy was significantly greater in hamsters than in opossums during gestation (P < 0.001), but not during lactation or from conception to weaning (P > 0.05). Efficiency of offspring production (energy stored in young/incremental energy in RA females) was higher in hamsters than in opossums and, in both species, it was higher during lactation than in gestation. The energetic cost of reproduction (per young per day) was higher in hamsters than in opossums. The marsupial mode of reproduction, as seen in opossums, yields young at lower cost but requires a longer reproductive period than is the case for a similar-sized eutherian. Accepted: 8 September 1998     

Influence of dietary fat type on benzo(a)pyrene [B(a)P] biotransformation in a B(a)P-induced mouse model of colon cancer

In the US alone, around 60,000 lives/year are lost due to colon cancer. Diet and environment have been implicated in the development of sporadic colon tumors. The objective of this study was to determine how dietary fat potentiates the development of colon tumors through altered B(a)P biotransformation, using the Adenomatous polyposis coli with Multiple intestinal neoplasia mouse model. Benzo(a)pyrene was administered to mice through tricaprylin, and unsaturated (USF; peanut oil) and saturated (SF; coconut oil) fats at doses of 50 and 100 μg/kg via oral gavage over a 60-day period. Blood, colon, and liver were collected at the end of exposure period. The expression of B(a)P biotransformation enzymes [cytochrome P450 (CYP)1A1, CYP1B1 and glutathione-S-transferase] in liver and colon were assayed at the level of protein, mRNA and activities. Plasma and tissue samples were analyzed by reverse phase high-performance liquid chromatography for B(a)P metabolites. Additionally, DNA isolated from colon and liver tissues was analyzed for B(a)P-induced DNA adducts by the ³²P-postlabeling method using a thin-layer chromatography system. Benzo(a)pyrene exposure through dietary fat altered its metabolic fate in a dose-dependent manner, with 100 μg/kg dose group registering an elevated expression of B(a)P biotransformation enzymes, and greater concentration of B(a)P metabolites, compared to the 50 μg/kg dose group (P<.05). This effect was more pronounced for SF group compared to USF group (P<.05). These findings establish that SF causes sustained induction of B(a)P biotransformation enzymes and extensive metabolism of this toxicant. As a consequence, B(a)P metabolites were generated to a greater extent in colon and liver, whose concentrations also registered a dose-dependent increase. These metabolites were found to bind with DNA and form B(a)P-DNA adducts, which may have contributed to colon tumors in a subchronic exposure regimen.