Molecular characterization,expression profile and association analysis with fat deposition traits of the porcine APOM gene

Apolipoprotein M (APOM), a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in plasma, is involved in lipid and lipoprotein metabolism. Through comparative mapping, we have mapped this gene to SSC7 p1.1 in which many QTLs affecting fat deposition traits have been reported. As a candidate gene for fat deposition traits, in this study, we obtained the 742-bp mRNA sequence of porcine APOM including the full coding region and encoding a protein of 188 amino acids. The sequence was deposited into the GenBank under the accession no. DQ329240. Semi-quantitative RT-PCR results showed that the porcine APOM gene is expressed predominantly in liver and kidney tissue. The genomic sequence of this gene which contains six exons and five introns, is 3,621 bp in length (DQ272488). Bioinformatic analysis of the 5′ regulatory region has revealed that classical TATA-box element and species conserved Hepatocyte nuclear factor-1a (HNF-1α) biding site were represented in this region. A G2289C single nucleotide polymorphism (SNP) in the intron 2 of porcine APOM gene detected as an Eco130I PCR–restriction fragment length polymorphism (PCR–RFLP) showed allele frequency differences among three purebreds. Association of the genotypes with fat deposition traits showed that different genotypes of porcine APOM gene were significantly associated with leaf fat weight (P < 0.05), backfat thickness at shoulder (P < 0.05), backfat thickness at thorax-waist (P < 0.05), backfat thickness at buttock (P < 0.01) and average backfat thickness over shoulder, thorax-waist and buttock (P < 0.01).     

No significant association between Fc receptor-like 3 gene polymorphisms and human leukocyte antigen-B27 positive ankylosing spondylitis in Han Chinese population

Ankylosing spondylitis (AS) is a rhematoid arthritis, which is a common autoimmune disease with a complex genetic etiology. Although HLA-B27 has been identified to be associated with AS, a number of other genes may also be involved in the disease. Fc receptor-like 3 (FCRL3) gene has been shown to be associated with rheumatoid arthritis in Japanese population. Here we aim to explore the association FCRL3 gene and susceptibility to human leukocyte antigen (HLA)-B27-positive AS in Han Chinese population. Among 169 AS patients, the frequencies of C and T (rs7522061) in FCRL3 gene were 38.7 and 61.3%, respectively; in 184 controls (HLA-B27-positive), the frequencies of C and T were 38.6 and 61.4%, respectively. The frequencies of alleles and genotype are not of statistically significant difference in two groups (χ² = 0.000, P = 0.983; χ² = 0.099, P = 0.952, respectively),but the distribution of HLA-B27 subtypes are statistically significant difference between cases and controls (χ² = 8.214, P = 0.042). Our data reveal that the FCRL3 gene does not appear associated with susceptibility to HLA-B27-positive AS in Han Chinese population.     

Lack of apoE causes alteration of cytokines expression in young mice liver

To investigate the effect of apolipoprotein E (apoE) on cytokine expression profile of the liver of young mice, quantitative RT-PCR (qRT-PCR) assay and cytokine antibody array for multiplex analysis of 62 cytokines have been used to analyze characteristics of expression of cytokines in the liver of 6-week-old apoE-null (apoE^−/−) mice. The levels of plasma cytokines were also analyzed. The mRNA level of IL-1β, IL-2, IL-6, ICAM-1, VCAM-1, MCP-1, NF-κB (p65), IFN-γ and IκB-α were increased significantly in apoE^−/− mice comparative to wild-type (WT) mice. IL-4, IL-10 and GM-CSF, however, were slightly decreased. Compared with WT, levels of 21 cytokines altered twofold or more in apoE^−/− mice, including 10 cytokines increased and 11 decreased. Expression patterns of IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and VCAM-1 showed identical trend between cytokine antibody array and qRT-PCR analysis. Moreover, levels of IL-1β, IFN-γ and IL-6 in the plasma were elevated, while IL-4 was lightly decreased in apoE^−/− mice compared to those in WT mice. These results implied that promotion of type I immune response in the liver of young apoE^−/− mice due to alteration of these cytokines, and the phenotypes may be caused by the regulation of NF-κB. The inflammation and lipid metabolism dysfunction in the liver cooperated in dysfunction of the liver in young apoE^−/− mice.     

Shorter GT repeat polymorphism in the heme oxygenase-1 gene promoter has protective effect on ischemic stroke in dyslipidemia patients

Background  

The microsatellite polymorphism of heme oxygenase (HO)-1 gene promoter has been shown to be associated with the susceptibility to ischemic event, including coronary artery disease (CAD), myocardial infarction, and peripheral vascular disease. We aimed to examine whether the length of (GT)_n repeats in HO-1 gene promoter is associated with ischemic stroke in people with CAD risk factors, especially low level of HDL.     

Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in Arabidopsis

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.     

Taurine protection of PC12 cells against endoplasmic reticulum stress induced by oxidative stress

Background

Taurine is a free amino acid present in high concentrations in a variety of organs of mammalians. As an antioxidant, taurine has been found to protect cells against oxidative stress, but the underlying mechanism is still unclear.

Methods

In this report, we present evidence to support the conclusion that taurine exerts a protective function against endoplasmic reticulum (ER) stress induced by H₂O₂ in PC 12 cells. Oxidative stress was introduced by exposure of PC 12 cells to 250 uM H₂O₂ for 4 hours.

Results

It was found that the cell viability of PC 12 cells decreased with an increase of H₂O₂ concentration ranging from approximately 76% cell viability at 100 uM H₂O₂ down to 18% at 500 uM H₂O₂. At 250 uM H₂O₂, cell viability was restored to 80% by taurine at 25 mM. Furthermore, H₂O₂ treatment also caused a marked reduction in the expression of Bcl-2 while no significant change of Bax was observed. Treatment with taurine restored the reduced expression of Bcl-2 close to the control level without any obvious effect on Bax. Furthermore, taurine was also found to suppress up-regulation of GRP78, GADD153/CHOP and Bim induced by H₂O₂, suggesting that taurine may also exert a protective function against oxidative stress by reducing the ER stress.

Conclusion

In summary, taurine was shown to protect PC12 cells against oxidative stress induced by H₂O₂. ER stress was induced by oxidative stress and can be suppressed by taurine.

     

Wintersweet accumulates apoplastic chitinase with a dual role in petals

Most monocotyledons like cereals accumulate antifreeze proteins in the apoplast during cold acclimation, but it is still uncertain whether dicotyledons do. Here we report the isolation and characterisation of a 33-kD apoplastic chitinase extracted from the corolla of wintersweet (Chinmonanthus praecox communis L.), which was purified using successive column chromatography on Phenyl-Sepharose, DEAE-Sepharose, and CM-Sepharose. Antifreezing activity of chitinase was confirmed in terms of the formation of bipyramidal ice crystals and high thermal-hysteresis values. Interestingly, chitinase was also found to affect germination of fungal spores of four major plant pathogens. From these data, we hypothesize that, under natural conditions, wintersweet as one of the overwintering dicotyledons also accumulates apoplastic antifreeze proteins like monocotyledons. To our knowledge, this is the first report on the isolation of dicotyledon apoplastic chitinase with high-level antifreeze and antifungal activities.     

Structural requirements of the ASBT by 3D-QSAR analysis using aminopyridine conjugates of chenodeoxycholic acid

The human apical sodium-dependent bile acid transporter (ASBT) is a validated drug target and can be employed to increase oral bioavailability of various drug conjugates. The aim of the present study was to investigate the chemical space around the 24-position of bile acids that influences both inhibition and uptake by the transporter. A series of 27 aminopyridine and aminophenol conjugates of glutamyl-chenodeoxycholate were synthesized and their ASBT inhibition and transport kinetics (parametrized as K(i), K(t), and J(max)) measured using stably transfected ASBT-MDCK cells. All conjugates were potent ASBT inhibitors. Monoanionic conjugates exhibited higher inhibition potency than neutral conjugates. However, neutral conjugates and chloro-substituted monoanionic conjugates were not substrates, or at least not apparent substrates. Kinetic analysis of substrates indicated that similar values for K(i) and K(t) implicate substrate binding to ASBT as the rate-limiting step. Using 3D-QSAR, four inhibition models and one transport efficiency model were developed. Steric fields dominated in CoMFA models, whereas hydrophobic fields dominated CoMSIA models. The inhibition models showed that a hydrophobic or bulky substitute on the 2 or 6 position of a 3-aminopyridine ring enhanced activity, while a hydrophobic group on the 5 position was detrimental. Overall, steric and hydrophobic features around the 24 position of the sterol nucleus strongly influenced bile acid conjugate interaction with ASBT. The relative location of the pyridine nitrogen and substituent groups also modulated binding.     

Molecular cloning and expression of two HSP70 genes in the Wuchang bream (Megalobrama amblycephala Yih)

Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate 70 (HSC70) and inducible heat shock protein 70 (HSP70) were isolated from the liver of Wuchang bream (Megalobrama amblycephala Y.) using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly (A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kDa, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule whereas Ma-HSP70 gene did not contain any intron in the coding region. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates, while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinase II phosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins were α-helix and random coil. Fluorescent real-time quantitative RT-PCR was used to study the effects of heat shock (34 °C), crowding stress (100 g L^?1) and challenge with bacteria Aeromonas hydrophila on the mRNA expression of the two HSP70s in Wuchang bream liver. The results indicated that, during 24 h stress, Ma-HSC70 mRNA expression decreased at first and then rose to the level before stress under heat shock and crowding stress, but Ma-HSP70 mRNA expression increased at first and then decreased under heat stress, and appeared to increase continuously under crowding stress. After bacterial challenge, the mRNA levels of both Ma-HSC70 and Ma-HSP70 increased at first and then decreased. The cloning and expression analysis of the two HSP70s provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of Wuchang bream.