Molecular Characterization and Growth Association of Two Apolipoprotein A-Ib Genes in Common Carp (Cyprinus carpio)

Apolipoprotein A-I (ApoA-I) is functionally involved in the transportation and metabolism of lipids in vertebrates. In this study, two isoforms of apoA-Ib in common carp (Cyprinus carpio L.) were characterized. Sequence comparison and phylogenetic analysis showed that C. carpio ApoA-Ib is relatively conserved within cyprinid fishes. During embryonic development, C. carpio apoA-Ib was first expressed at the stage of multi-cells, and the highest mRNA level was observed at the stage of optic vesicle. A ubiquitous expression pattern was detected in various tissues with extreme predominance in the liver. Significantly different expression levels were observed between light and heavy body weight groups and also in the compensatory growth test. Seventeen and eight single-nucleotide polymorphisms (SNPs) were identified in matured mRNA of the C. carpio apoA-Ib.1 and apoA-Ib.2, respectively. Two of these SNPs (apoA-Ib.2-g.183A>T and apoA-Ib.2-g.1753C>T) were significantly associated with body weight and body length in two populations of common carp. These results indicate that apoA-Ib may play an important role in the modulation of growth and development in common carp.     

Mass spectrometric immunoassay and MRM as targeted MS-based quantitative approaches in biomarker development: Potential applications to cardiovascular disease and diabetes

Type 2 diabetes mellitus (T2DM) is an important risk factor for cardiovascular disease (CVD)—the leading cause of death in the United States. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed toward treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients. Thus newer targets and markers are needed for the development and testing of novel therapies. Herein we review two complementary MS-based approaches—mass spectrometric immunoassay (MSIA) and MS/MS as MRM—for the analysis of plasma proteins and PTMs of relevance to T2DM and CVD. Together, these complementary approaches allow for high-throughput monitoring of many PTMs and the absolute quantification of proteins near the low picomolar range. In this review article, we discuss the clinical relevance of the high density lipoprotein (HDL) proteome and Apolipoprotein A-I PTMs to T2DM and CVD as well as provide illustrative MSIA and MRM data on HDL proteins from T2DM patients to provide examples of how these MS approaches can be applied to gain new insight regarding cardiovascular risk factors. Also discussed are the reproducibility, interpretation, and limitations of each technique with an emphasis on their capacities to facilitate the translation of new biomarkers into clinical practice.     

On the Aggregation of Apolipoprotein A-I

In vivo, apolipoprotein A-I (ApoA-I) is commonly found together with lipids in so-called lipoprotein particles. The protein has also been associated with several diseases—such as atherosclerosis and amyloidosis—where insoluble aggregates containing ApoA-I are deposited in various organs or arteries. The deposited ApoA-I has been found in the form of amyloid fibrils, suggesting that amyloid formation may be involved in the development of these diseases. In the present study we investigated ApoA-I aggregation into amyloid fibrils and other aggregate morphologies. We studied the aggregation of wildtype ApoA-I as well as a disease-associated mutant, ApoA-I K107Δ, under different solution conditions. The aggregation was followed using thioflavin T fluorescence intensity. For selected samples the aggregates formed were characterized in terms of size, secondary structure content, and morphology using circular dichroism spectroscopy, dynamic light scattering, atomic force microscopy and cryo transmission electron microscopy. We find that ApoA-I may form globular protein-only condensates, in which the α-helical conformation of the protein is retained. The protein in its unmodified form appears resistant to amyloid formation; however, the conversion into amyloid fibrils rich in β-sheet is facilitated by oxidation or mutation. In particular, the K107Δ mutant shows higher amyloid formation propensity, and the end state appears to be a co-existence of β-sheet rich amyloid fibrils and α-helix-rich condensates.     

Characterization and Purification of Polydisperse Reconstituted Lipoproteins and Nanolipoprotein Particles

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results—indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 μm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles.     

Effect of the MTP –493 G/T polymorphism on the lipid profiles of the Guangxi Hei Yi Zhuang and Han populations

There are 56 nationalities in China. Han is the largest and Zhuang the second largest. Geographically and linguistically, Zhuang can be classified into 43 ethnic subgroups, in which Hei Yi (meaning black‐worship and black‐dressing) Zhuang is proved to be the most conservative group according to its unique culture and custom. Information on the genetic influence on the lipid profile is limited in this population. To obtain some of this information, a cross‐sectional survey of hyperlipidemia was carried out in 500 people of Hei Yi Zhuang and 500 people of Han in the same area. The genotypes and alleles of the microsomal triacylglycerol transfer protein (MTP –493 G/T) were also determined, by polymerase chain reaction combined with an allele‐specific oligonucleotide hybridization method, and then confirmed by direct sequencing. The frequencies of the G and T alleles were 0.74 and 0.26, respectively, in Hei Yi Zhuang, and 0.73 and 0.27 (p >0.05) in Han. The frequencies of the GG, GT and TT genotypes were 0.58, 0.33 and 0.09, respectively, in Hei Yi Zhuang, and 0.56, 0.34 and 0.09 (p >0.05) in Han. In the combined population of both Hei Yi Zhuang and Han, the levels of total cholesterol, triacylglycerol, low‐density lipoprotein cholesterol, and apolipoprotein B in the TT genotype were significantly higher than those in the GT (p <0.05 to <0.01) or GG genotypes (p <0.05 to <0.01), but there were no significant differences in their values between the GG and GT genotypes (p >0.05). The levels of high‐density lipoprotein cholesterol and apolipoprotein A1 were not significantly different among the three genotypes (p >0.05).     

Apolipoprotein A-I: Insights from redox proteomics for its role in neurodegeneration

Proteomics has a wide range of applications, including determination of differences in the proteome in terms of expression and post-translational protein modifications. Redox proteomics allows the identification of specific targets of protein oxidation in a biological sample. Using proteomic techniques, apolipoprotein A-I (ApoA-I) has been found at decreased levels in subjects with a variety of neurodegenerative disorders including in the serum and cerebrospinal fluid (CSF) of Alzheimer disease (AD), Parkinson disease (PD), and Down syndrome (DS) with gout subjects. ApoA-I plays roles in cholesterol transport and regulation of inflammation. Redox proteomics further showed ApoA-I to be highly oxidatively modified and particularly susceptible to modification by 4-hydroxy-2-trans-nonenal (HNE), a lipid peroxidation product. In the current review, we discuss the consequences of oxidation of ApoA-I in terms of neurodegeneration. ROS-associated chemotherapy related ApoA-I oxidation leads to elevation of peripheral levels of tumor necrosis factor-α (TNF-α) that can cross the blood-brain barrier (BBB) causing a signaling cascade that can contribute to neuronal death, likely a contributor to what patients refer to as “chemobrain.” Current evidence suggests ApoA-I to be a promising diagnostic marker as well as a potential target for therapeutic strategies in these neurodegenerative disorders.     

免疫散射比浊法测定人血清载脂蛋白AⅠ和B

研究了荧光分光光度计的免疫散射比浊法代替激光散射浊度仪来测定载脂蛋白AⅠ(ApoAⅠ)和载脂蛋白B(ApoB),同时测定正常人的ApoAⅠ和ApoB的参考值.结果表明:以荧光分光光度计进行免疫散射比浊法测定ApoAⅠ和ApoB,标准曲线线性、回收率和重复性良好,与激光散射浊度仪测定的值非常接近.ApoAⅠ的参考值分别为1 215±255 mg/L(男)和1 134±179 mg/L(女),ApoB的参考值分别为681±210 mg/L(男)和561±166 mg/L(女).荧光分光光度计可代替激光散射浊度仪来测定ApoAⅠ和ApoB.     

LXR Agonist Increases the Lymph HDL Transport in Rats by Promoting Reciprocally Intestinal ABCA1 and apo A-I mRNA Levels

Liver and intestine are major sites of apo A-I synthesis in mammals. ABCAI is reported to be involved in the secretion of nascent HDL from cultured intestinal cells. However, whether ABCA1 participates in the secretion of nascent HDL from the intestine has not been assessed directly in vivo. This study examined the effect of a synthetic LXR-agonist “TO” on the lymphatic transport of HDL in thoracic duct-cannulated rats. The feeding of a TO-containing diet resulted in an increased transport of cholesterol and apo A-I in the lymph d > 1.063 g/ml lipoprotein fraction than did the feeding of a control diet without TO. The transport of cholesterol in whole lymph was lower, whereas the transport of apo A-I was higher, in the TO group. The abundance of mRNAs for ABCAI and apo A-I in the intestine was increased in the TO group. Furthermore, although the TO-containing diet reportedly increased the serum HDL concentration in intact mice and rats, no such effect was observed in the cannulated rats. The LXR agonist stimulated in vivo the synthesis of nascent HDL by increasing reciprocally the mRNA for ABCAI and apo A-I in the intestine, thereby contributing to an increase in the circulating HDL.