Proposed selection strategy of surrogate matrix to quantify endogenous substances by Japan Bioanalysis Forum DG2015-15.

It is important to select an appropriate surrogate matrix for preparing calibration standards and quality control samples while quantitatively assaying for endogenous substances, because a blank matrix that does not contain the endogenous substance cannot be derived from the species from which the target study samples are collected. This is because the assay results might be affected, depending on the characteristics of the analyte in the surrogate matrix. Our discussion group that participated in the Japan Bioanalysis Forum discussed the recommended selection strategies, focusing on large and small molecules in ligand binding assays and LC-MS, respectively. We established an efficient selection strategy for a surrogate matrix, with simple compositions as the first candidates stated in this article.

Integrative genomic and proteomic analyses identifies glycerol-3-phosphate acyltransferase as a target of low-dose ionizing radiation in EBV infected-B cells.

PURPOSE: We sought to gain a better understanding of the low-dose ionizing radiation (LDIR)-induced molecular changes in transformed pre-malignant cells in their microenvironment. MATERIALS AND METHODS: The cellular response to LDIR was compared and contrasted using immortalized human Epstein-Barr virus-infected B-cells (EBV-B) in mono-culture, co-culture with human bone marrow derived stromal cells (MSC), or under the LDIR-induced bystander effect. The resulting alterations in protein and gene expression (including microRNA, miRNA) were evaluated by isobaric tags for relative and absolute quantification (iTRAQ) proteomics assay, western blot, cDNA array and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. RESULTS: The miRNAs let7a, miR-15b, miR-16, and miR-21, and a lipid metabolic miRNA hub miR-23b, were upregulated after LDIR exposure in the mono-cultured EBV-B cells, but were downregulated in EBV-B cells co-irradiated with MSC. A lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase, the common target of these miRNA, was downregulated at the level of protein and mRNA expression in the LDIR-exposed, mono-cultured EBV-B cells and upregulated MSC co-cultured EBV-B cells. CONCLUSIONS: These results suggest a putative miRNA regulatory mechanism controlling the LDIR-induced stress response, and illustrate that LDIR exposure, and the cell's microenvironment, can affect specific gene expression, both directly and indirectly, resulting in altered protein expression.

Mitsugumin 56 (hedgehog acyltransferase-like) is a sarcoplasmic reticulum-resident protein essential for postnatal muscle maturation.

Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation.

Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS.

Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids (GPs). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs.

Overexpression of autotaxin, a lysophosphatidic acid-producing enzyme, enhances cardia bifida induced by hypo-sphingosine-1-phosphate signaling in zebrafish embryo.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are second-generation lysophospholipid mediators that exert multiple biological functions through their own cognate receptors. They are both present in the blood stream, activate receptors with similar structures (endothelial differentiation gene receptors), have similar roles in the vasculature and are vasoactive. However, it is unclear whether these lysophospholipid mediators cross-talk downstream of each receptor. Here, we provide in vivo evidence that LPA signaling counteracted S1P signaling. When autotaxin (Atx), an LPA-producing enzyme, was overexpressed in zebrafish embryos by injecting atx mRNA, the embryos showed cardia bifida, a phenotype induced by down-regulation of S1P signaling. A similar cardiac phenotype was not induced when catalytically inactive Atx was introduced. The cardiac phenotype was synergistically enhanced when antisense morpholino oligonucleotides (MO) against S1P receptor (s1pr2/mil) or S1P transporter (spns2) was introduced together with atx mRNA. The Atx-induced cardia bifida was prominently suppressed when embryos were treated with an lpar1 receptor antagonist, Ki16425, or with MO against lpar1. These results provide the first in vivo evidence of cross-talk between LPA and S1P signaling.

Structure and function of Zucchini endoribonuclease in piRNA biogenesis.

PIWI-interacting RNAs (piRNAs) silence transposons to maintain genome integrity in animal germ lines. piRNAs are classified as primary and secondary piRNAs, depending on their biogenesis machinery. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters in the genome through the primary processing pathway. Although the existence of a ribonuclease participating in this pathway has been predicted, its molecular identity remained unknown. Here we show that Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily member, is an endoribonuclease essential for primary piRNA biogenesis. We solved the crystal structure of Drosophila melanogaster Zuc (DmZuc) at 1.75 Å resolution. The structure revealed that DmZuc has a positively charged, narrow catalytic groove at the dimer interface, which could accommodate a single-stranded, but not a double-stranded, RNA. DmZuc and the mouse homologue MmZuc (also known as Pld6 and MitoPLD) showed endoribonuclease activity for single-stranded RNAs in vitro. The RNA cleavage products bear a 5'-monophosphate group, a hallmark of mature piRNAs. Mutational analyses revealed that the conserved active-site residues of DmZuc are critical for the ribonuclease activity in vitro, and for piRNA maturation and transposon silencing in vivo. We propose a model for piRNA biogenesis in animal germ lines, in which the Zuc endoribonuclease has a key role in primary piRNA maturation.

Autotaxin regulates vascular development via multiple lysophosphatidic acid (LPA) receptors in zebrafish.

Autotaxin (ATX) is a multifunctional ecto-type phosphodiesterase that converts lysophospholipids, such as lysophosphatidylcholine, to lysophosphatidic acid (LPA) by its lysophospholipase D activity. LPA is a lipid mediator with diverse biological functions, most of which are mediated by G protein-coupled receptors specific to LPA (LPA1-6). Recent studies on ATX knock-out mice revealed that ATX has an essential role in embryonic blood vessel formation. However, the underlying molecular mechanisms remain to be solved. A data base search revealed that ATX and LPA receptors are conserved in wide range of vertebrates from fishes to mammals. Here we analyzed zebrafish ATX (zATX) and LPA receptors both biochemically and functionally. zATX, like mammalian ATX, showed lysophospholipase D activity to produce LPA. In addition, all zebrafish LPA receptors except for LPA5a and LPA5b were found to respond to LPA. Knockdown of zATX in zebrafish embryos by injecting morpholino antisense oligonucleotides (MOs) specific to zATX caused abnormal blood vessel formation, which has not been observed in other morphant embryos or mutants with vascular defects reported previously. In ATX morphant embryos, the segmental arteries sprouted normally from the dorsal aorta but stalled in midcourse, resulting in aberrant vascular connection around the horizontal myoseptum. Similar vascular defects were not observed in embryos in which each single LPA receptor was attenuated by using MOs. Interestingly, similar vascular defects were observed when both LPA1 and LPA4 functions were attenuated by using MOs and/or a selective LPA receptor antagonist, Ki16425. These results demonstrate that the ATX-LPA-LPAR axis is a critical regulator of embryonic vascular development that is conserved in vertebrates.

Autotaxin--an LPA producing enzyme with diverse functions.

Autotaxin (ATX) is an ecto-enzyme responsible for lysophosphatidic acid (LPA) production in blood. ATX is present in various biological fluids such as cerebrospinal and seminal fluids and accounts for bulk LPA production in these fluids. ATX is a member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family and was originally isolated from conditioned medium of melanoma cells as an autocrine motility stimulating factor. LPA, a second-generation lipid mediator, binds to its cognate G protein-coupled receptors through which it exerts a number of biological functions including influencing cell motility and proliferation stimulating activity. Some of the biological roles of LPA can be mediated by ATX. However, there are other LPA-producing pathways independent of ATX. The accumulating evidences for physiological and pathological functions of ATX strongly support that ATX is an important therapeutic target. This review summarizes the historical aspects, structural basis, pathophysiological functions identified in mice studies and clinical relevance discovered by measuring the blood ATX level in human. The general features and functions of each NPP family member will be also briefly reviewed. The presence of the ATX gene in other model organisms and recently developed ATX inhibitors, both of which will be definitely useful for further functional analysis of ATX, will also be mentioned.