Metabonomics and Transcriptomic Analysis of Free Fatty Acid Synthesis in Seedless and Tenera Oil Palm.

Oil palm, a tropical woody oil crop, is widely used in food, cosmetics, and pharmaceuticals due to its high production efficiency and economic value. Palm oil is rich in free fatty acids, polyphenols, vitamin E, and other nutrients, which are beneficial for human health when consumed appropriately. Therefore, investigating the dynamic changes in free fatty acid content at different stages of development and hypothesizing the influence of regulatory genes on free fatty acid metabolism is crucial for improving palm oil quality and accelerating industry growth. LC-MS/MS is used to analyze the composition and content of free fatty acids in the flesh after 95 days (MS1 and MT1), 125 days (MS2 and MT2), and 185 days (MS3 and MT3) of Seedless (MS) and Tenera (MT) oil palm species fruit pollination. RNA-Seq was used to analyze the expression of genes regulating free fatty acid synthesis and accumulation, with differences in genes and metabolites mapped to the KEGG pathway map using the KEGG (Kyoto encyclopedia of genes and genomes) enrichment analysis method. A metabolomics study identified 17 types of saturated and 13 types of unsaturated free fatty acids during the development of MS and MT. Transcriptomic research revealed that 10,804 significantly different expression genes were acquired in the set differential gene threshold between MS and MT. The results showed that FabB was positively correlated with the contents of three main free fatty acids (stearic acid, myristate acid, and palmitic acid) and negatively correlated with the contents of free palmitic acid in the flesh of MS and MT. ACSL and FATB were positively correlated with the contents of three main free fatty acids and negatively correlated with free myristate acid. The study reveals that the expression of key enzyme genes, FabB and FabF, may improve the synthesis of free myristate in oil palm flesh, while FabF, ACSL, and FATB genes may facilitate the production of free palmitoleic acid. These genes may also promote the synthesis of free stearic acid and palmitoleic acid in oil palm flesh. However, the FabB gene may inhibit stearic acid synthesis, while ACSL and FATB genes may hinder myristate acid production. This study provides a theoretical basis for improving palm oil quality.

A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors.

INTRODUCTION: Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. OBJECTIVE: To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. METHODS: The new TLC bioautographic assay was based on reaction of lipase with ß-naphthyl myristate and the subsequent formation of the purple dye between ß-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. RESULTS: The ß-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%). CONCLUSION: The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors.

Attenuation of 19-9 antigen secretion in human colorectal carcinoma cell cultures by transfection with cDNA encoding novel ADP-ribosylation factor-like proteins.

We have used cDNAs coding for novel ADP-ribosylation factor-like molecules (ARL184 and ARL184Delta) to alter 19-9 antigen glycoprotein secretion in cultured human colorectal carcinoma cells SW1116 by transfection and cloning. This ARL contains a lipophilic N-terminal with an isoleucyl and 3 leucyl residues, 4 functioning consensus sequence GTP binding sites, and 184 total aminoacyl residues. An ARL cDNA was also constructed deleting the codon for the N-terminal glycyl moiety. The resulting cell clones were shown by Northern blots to overexpress ARL mRNA. Electron microscopy-immunocytochemistry also indicated the overexpression of ARL granules subcellularly. Secretion of the tumor-associated 19-9 antigen into apical medium was decreased 3- to 5-fold and the secretion of TCA/PTA precipitable 3H-labeled glycoprotein was decreased by 34% in clone SW1116(ARL184)Delta. Western blot analyses of cell homogenates and media were in agreement with the secretion assays and showed a diminution of 170-200 kDa, 19-9, antigenicity in transfected cells and their media. Apical secretion of 19-9 antigen was diminished 14-fold in cells, SW1116 (ARL184)alpha, transfected with the complete ARL cDNA sequence, suggesting that the glycyl moiety may be required for maximal abatement. However, incorporation of label from [3H]myristate into 22-kDa bands of NP-40 extracts and ARL-antigenic molecules of parent cells was 3-fold greater than that in samples from the two transfectants; thus the transfected cells may not myristylate the overexpressed ARL efficiently. Notwithstanding the N-terminal glycyl moiety undergoing some other modification, we conclude that overexpression of this ARL is sufficient to generate a 19-9-deficient phenotype. These ARLs may eventually disrupt terminal oligosaccharide glycosylation, resulting in an apparent diminished exocytosis of 19-9 glycoprotein carriers by transfected and cloned cells.

Molecular cloning and functional reconstitution of a urate transporter/channel.

Maintenance of urate homeostasis requires urate efflux from urate-producing cells with subsequent renal and gastrointestinal excretion. The molecular basis for urate transport, however, has not been identified. A novel full-length cDNA encoding a 322-amino acid protein, designated UAT (urate transporter), has been cloned from a rat renal cDNA library by antibody screening. UAT mRNA transcripts that approximate 1.55 kilobases are present, but differentially expressed in various rat tissues. Recombinant UAT protein that was expressed from the cloned cDNA in Escherichia coli and purified via immobilized metal affinity chromatography has been functionally reconstituted as a highly selective urate transporter/channel in planar lipid bilayers. The IgG fraction of the polyclonal antibody that was used to select the UAT clone from the cDNA library, but not nonimmune IgG, blocked urate channel activity. Based on the wide tissue distribution of the mRNA for UAT we propose that UAT provides the molecular basis for urate flux across cell membranes, allowing urate that is formed during purine metabolism to efflux from cells and serving as an electrogenic transporter that plays an important role in renal and gastrointestinal urate excretion.

Calcium-myristoyl protein switch.

Recoverin, a recently discovered member of the EF-hand superfamily of Ca(2+)-binding proteins, serves as a Ca2+ sensor in vision. The amino terminus of the protein from retinal rod cells contains a covalently attached myristoyl or related N-acyl group. We report here studies of unmyristoylated and myristoylated recombinant recoverin designed to delineate the biological role of this hydrophobic unit. Ca2+ induces the binding of both the unmyristoylated and myristoylated proteins to phenyl-agarose, a hydrophobic support. Binding was half-maximal at 1.1 and 1.0 microM Ca2+, respectively. The Hill coefficients of 1.8 and 1.7, respectively, indicate that binding was cooperative. In contrast, Ca2+ induced the binding of myristoylated but not of unmyristoylated recoverin to rod outer segment membranes. Binding to these membranes was half-maximal at 2.1 microM Ca2+, and the Hill coefficient was 2.4. Likewise, myristoylated but not unmyristoylated recoverin exhibited Ca(2+)-induced binding to phosphatidylcholine vesicles. These findings suggest that the binding of Ca2+ to recoverin has two effects: (i) hydrophobic surfaces are exposed, allowing the protein to interact with complementary nonpolar sites, such as the aromatic rings of phenyl-agarose; and (ii) the myristoyl group is extruded, enabling recoverin to insert into a lipid bilayer membrane. The myristoyl group is likely to be an active participant in Ca2+ signaling by recoverin and related EF-hand proteins such as visinin and neurocalcin.

Expression of a new tyrosine protein kinase is stimulated by retrovirus promoter insertion.

Tyrosine protein kinases are important both in the normal regulation of cellular proliferation and in the oncogenic transformation of cells by several tumour viruses. The LSTRA Moloney murine leukaemia virus (M-MuLV)-induced thymoma cell line contains approximately 20-fold more phosphotyrosine in protein than do typical haematopoietic cell lines; this seems to result from the expression of an abnormally high level of a cellular tyrosine protein kinase termed p56tck (refs 3, 4). This kinase is normally expressed at low levels in most, but not all, murine T cells. The elevated levels of p56tck could contribute to the malignant properties of LSTRA cells. Therefore, we have isolated cloned complementary DNAs encoding the whole of p56tck. Sequence analysis shows it to be a novel cellular tyrosine protein kinase which is distinct from all others described to date. p56tck is encoded in LSTRA cells by a hybrid messenger RNA; approximately 200 nucleotides at the 5' end of the mRNA are identical to the 5' end of the genome of M-MuLV. The three- to ninefold transcriptional activation of the gene therefore results from retroviral promoter insertion.

The effects of lipid phase transitions on the interaction of mitochondrial NADH--ubiquinone oxidoreductase with ubiquinol--cytochrome c oxidoreductase.

1. The endogenous phosphatidylcholine and phosphatidylethanolamine of Complexes I and III from bovine heart mitochondria may be completely replaced with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine with at least partial retention of activity. 2. The lipid-replaced enzymes associate in 1:1 molar ratio to give a Complex I--III unit catalysing NADH-cytochrome c oxidoreductase activity. 3. On increasing the concentration of ubiquinone-10 and the synthetic phospholipid, the lipid-replaced Complexes appear to operate independently of each other as in the natural membrane. Thus the lipid-replaced enzymes associate in exactly the same ways as the enzymes containing natural phospholipids. 4. Arrhenius plots of NADH--cytochrome c oxidoreductase activity reconstituted from lipid-replaced Complexes I and III exhibit changes in slope at 24 degrees C. When the concentrations of phospholipid and ubiquinone-10 are increased, the Arrhenius plots show discontinuities at 24 degrees C as well as changes in slope. 5. The kinetics of cytochrome b reduction by NADH were measured in mixtures containing 2 mol of Complex III/mol of Complex I. When the enzymes contained natural phospholipids. the reduction kinetics were biphasic. When the enzymes had been supplemented with further phospholipid and ubiquinone-10 the kinetics were monophasic. When lipid-replaced enzymes were supplemented with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine and ubiquinone-10, reduction of cytochrome b was monophasic above the phase-transition temperature of the lipid but biphasic below it. 6. These findings are interpreted in terms of the model for the interaction of Complexes in the natural membrane proposed by Heron, Ragan & Trum-power [(1978) Biochem. J. 174, 791--800].