Correlation between haplotype of apolipoprotein B gene and natural longevity persons in Uygur Nationality

This paper investigated the correlation between polymorphisms and haplotypes in the apolipoprotein B (apoB) gene (SP-I/D, XbaI-RFLP, VNTR) and natural longevity persons among the Uygur people in Xin-jiang. For this purpose, 191 healthy Uygur individuals aged above 90 from Hetian area of Xinjiang were recruited, and another 53 persons aged 65—70 from the same nationality, the same region and with the same gender ratio, served as the control group. Genotyping was performed by PCR-SSP, PCR-RFLP and PCR-sequencing methods. Logistic regression analyses revealed that the frequencies of X X genotype, M and L alleles and the genetypes composed of M and L were significantly higher in the longevity group than in the control group. In haplotype analyses, we found that, in the long-lived people, the frequency of haplotypes composed of the X and M alleles was significantly higher whereas the frequency of haplotypes composed of the X- and S alleles was significantly lower (both P<0.05) I than those of their controls. These results indicated that the S allele, SS genotype and X -S, D-S, D-X -S haplotypes were the possible adverse factors, whereas the M, L alleles, X X , MM, ML, LL genotypes and I-X -M, X -M haplotypes were the possibe protective factors for the naturally long-lived Uygur people in China.     

Theoretical model of human apolipoprotein B100 tertiary structure

Low density lipoprotein (LDL) particles are the main cholesterol carriers in human plasma. The organization of the particle, composed of apolar lipids and phospholipid monolayer stabilized by apolipoprotein B100 (apoB), is highly complex and still unknown. ApoB is an extremely large protein (4563 amino acids) and very little is known about its structure. A 3D model of the N-terminal region has been recently proposed and has provided interesting insights about the physico-chemical properties of the protein and putative interaction zones with lipids. In the present article, we propose the first tentative 3D modelling for most remaining residues. All predicted features emerging from the models are confronted with agreement to experimental data available. Using different up-to-date prediction methods, we decomposed the protein into eight domains and predicted 3D structure for each of them. The analysis of hydrophobic patches, polar regions, coupled with functional predictions based on the 3D models, gives new clues to understanding of the functional role of apoB. We suggest precise regions putatively involved in the lipid interactions, and discuss the position of apoB on the LDL particle. Finally, we propose relative organization of the domains, providing a shape quite compatible with the low resolution electron microscopy map.     

Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.     

Can modulators of apolipoproteinB biogenesis serve as an alternate target for cholesterol-lowering drugs?

Understanding the molecular defects underlying cardiovascular disease is necessary for the development of therapeutics. The most common method to lower circulating lipids, which reduces the incidence of cardiovascular disease, is statins, but other drugs are now entering the clinic, some of which have been approved. Nevertheless, patients cannot tolerate some of these therapeutics, the drugs are costly, and/or the treatments are approved for only rare forms of disease. Efforts to find alternative treatments have focused on other factors, such as apolipoproteinB (apoB), which transports cholesterol in the blood stream. The levels of apoB are regulated by endoplasmic reticulum (ER) associated degradation as well as by a post ER degradation pathway in model systems, and we suggest that these events provide novel therapeutic targets. We discuss first how cardiovascular disease arises and how cholesterol is regulated, and then summarize the mechanisms of action of existing treatments for cardiovascular disease. We then review the apoB biosynthetic pathway, focusing on steps that might be amenable to therapeutic interventions.     

Intestine-specific expression of Apobec-1 rescues apolipoprotein B RNA editing and alters chylomicron production in Apobec1−/− mice

Intestinal apolipoprotein B (apoB) mRNA undergoes C-to-U editing, mediated by the catalytic deaminase apobec-1, which results in translation of apoB48. Apobec1^−/− mice produce only apoB100 and secrete larger chylomicron particles than those observed in wild-type (WT) mice. Here we show that transgenic rescue of intestinal apobec-1 expression (Apobec1^Int/O) restores C-to-U RNA editing of apoB mRNA in vivo, including the canonical site at position 6666 and also at approximately 20 other newly identified downstream sites present in WT mice. The small intestine of Apobec1^Int/O mice produces only apoB48, and the liver produces only apoB100. Serum chylomicron particles were smaller in Apobec1^Int/O mice compared with those from Apobec1^−/− mice, and the predominant fraction of serum apoB48 in Apobec1^Int/O mice migrated in lipoproteins smaller than chylomicrons, even when these mice were fed a high-fat diet. Because apoB48 arises exclusively from the intestine in Apobec1^Int/O mice and intestinal apoB48 synthesis and secretion rates were comparable to WT mice, we were able to infer the major sites of origin of serum apoB48 in WT mice. Our findings imply that less than 25% of serum apoB48 in WT mice arises from the intestine, with the majority originating from the liver.     

Beneath the minerals, a layer of round lipid particles was identified to mediate collagen calcification in compact bone formation

Astronauts lose 1-2% of their bone minerals per month during space flights. A systematic search for a countermeasure relies on a good understanding of the mechanism of bone formation at the molecular level. How collagen fibers, the dominant matrix protein in bones, are mineralized remains mysterious. Atomic force microscopy was carried out, in combination with immunostaining and Western blotting, on bovine tibia to identify unrecognized building blocks involved in bone formation and for an elucidation of the process of collagen calcification in bone formation. Before demineralization, tiles of hydroxyapatite crystals were found stacked along bundles of collagen fibers. These tiles were homogeneous in size and shape with dimensions 0.69 x 0.77 x 0.2 micro m(3). Demineralization dissolved these tiles and revealed small spheres with an apparent diameter around 145 nm. These spheres appeared to be lipid particles since organic solvents dissolved them. The parallel collagen bundles had widths mostly <2 micro m. Composition analysis of compact bones indicated a high content of apolar lipids, including triglycerides and cholesterol esters. Apolar lipids are known to form lipid droplets or lipoproteins, and these spheres are unlikely to be matrix vesicles as reported for collagen calcification in epiphyseal cartilages. Results from this study suggest that the layer of round lipid particles on collagen fibers mediates the mineral deposition onto the fibers. The homogeneous size of these lipid particles and the presence of apolipoprotein in demineralized bone tissue suggest the possibility that these particles might be of lipoprotein origin. More studies are needed to verify the last claim and to exclude the possibility that they are secreted lipid droplets.     

Regulation of the apolipoprotein B in heterozygous hypobeta-lipoproteinemic knock-out mice expressing truncated apoB,B81. Low production and enhanced clearance of apoB cause low levels of apoB

Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined.The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2+ (determined as the ratio of SR Ca2+ ATPase with and without ionophore A23187) is the same in both MHS and MHR and did not change with ongoing time; (viii) No uncoupling of uptake from ATP hydrolysis occurred up to 4 h p.m., but the coupling differed between MHS and MHR for all time intervals with lower values for MHS pigs. The results suggest that the decreasing Ca2+ uptake rate of homogenates, sampled at different times p.m., is essentially caused by changes in the Ca2+ pump and not by changes in the CRC or an increased phospholipid membrane permeability to Ca2+.     

Surface study of apoB1694�C1880, a sequence that can anchor apoB to lipoproteins and make it nonexchangeable

Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein. During lipoprotein assembly, it recruits phospholipids and triacylglycerols (TAG) into TAG-rich lipoprotein particles. It remains bound to secreted lipoproteins during lipid metabolism in plasma. The β1 region (residues 827–1880) of apoB has a high amphipathic β strand (AβS) content and is proposed to be one region anchoring apoB to lipoproteins. The AβS-rich region between apoB37 and apoB41 (residues 1694–1880) was cloned, expressed, and purified. The interfacial properties were studied at the triolein/water (TO/W) and air/water (A/W) interfaces. ApoB[37–41] is surface-active and adsorbs to the TO/W interface. After adsorption the unbound apoB[37–41] was removed from the aqueous phase. Adsorbed apoB[37–41] did not desorb and could not be forced off by increasing the surface pressure up to 23 mN/m. ApoB[37–41] adsorbed on the TO/W interface was completely elastic when compressed and expanded by ±13% of its area. On an A/W interface, the apoB[37–41] monolayer became solid when compressed to 4 mN/m pressure indicating extended β-sheet formation. It could be reversibly compressed and expanded between low pressure and its collapse pressure (35 mN/m). Our studies confirm that the AβS structure of apoB[37–41] is a lipid-binding motif that can irreversibly anchor apoB to lipoproteins.     

Enhancing the contrast of ApoB to locate the surface components in the 3D density map of human LDL

A 26 Å resolution map of the structure of human low-density lipoprotein (LDL) was obtained from electron cryomicroscopy and single-particle image reconstruction. The structure showed a discoidal-shaped LDL particle with high-density regions mainly distributed at the edge of the particle and low-density regions at the flat surface that covers the core region. To determine the chemical components that correspond to these density regions and to delineate the distribution of protein and phospholipid located at the particle surface at the resolution of the map, we used Mono-Sulfo-NHS-Undecagold labeling to increase preferentially the contrast of the apolipoprotein B component on the LDL particle. In the three-dimensional map from the image reconstruction of the undecagold-labeled LDL particles, the high-density region from the undecagold label was distributed mainly at the edge of the particle, and lower density regions were found at the flat surfaces that cover the neutral lipid core. This suggests that apolipoprotein B mainly encircles LDL at the edge of the particle and the phospholipid monolayers are located at the flat surfaces, which are parallel to the cholesterol ester layers in the core and may interact with the core lipid layers through the acyl chains.