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.     

Altered peptide ligands inhibit arthritis induced by glucose-6-phosphate isomerase peptide

Introduction

Immunosuppressants, including anti-TNFα antibodies, have remarkable effects in rheumatoid arthritis; however, they increase infectious events. The present study was designed to examine the effects and immunological change of action of altered peptide ligands (APLs) on glucose-6-phosphate isomerase (GPI) peptide-induced arthritis.

Methods

DBA/1 mice were immunized with hGPI_325-339, and cells of draining lymph node (DLN) were stimulated with hGPI_325-339 to investigate the T-cell receptor (TCR) repertoire of antigen-specific CD4⁺ T cells by flow cytometry. Twenty types of APLs with one amino acid substitution at a TCR contact site of hGPI_325-339 were synthesized. CD4⁺ T cells primed with human GPI and antigen-presenting cells were co-cultured with each APL and cytokine production was measured by ELISA to identify antagonistic APLs. Antagonistic APLs were co-immunized with hGPI_325-339 to investigate whether arthritis could be antigen-specifically inhibited by APL. After co-immunization, DLN cells were stimulated with hGPI_325-339 or APL to investigate Th17 and regulatory T-cell population by flow cytometry, and anti-mouse GPI antibodies were measured by ELISA.

Results

Human GPI_325-339-specific Th17 cells showed predominant usage of TCRVβ8.1 8.2. Among the 20 synthesized APLs, four (APL 6; N329S, APL 7; N329T, APL 12; G332A, APL 13; G332V) significantly reduced IL-17 production by CD4⁺ T cells in the presence of hGPI_325-339. Co-immunization with each antagonistic APL markedly prevented the development of arthritis, especially APL 13 (G332V). Although co-immunization with APL did not affect the population of Th17 and regulatory T cells, the titers of anti-mouse GPI antibodies in mice co-immunized with APL were significantly lower than in those without APL.

Conclusions

We prepared antagonistic APLs that antigen-specifically inhibited the development of experimental arthritis. Understanding the inhibitory mechanisms of APLs may pave the way for the development of novel therapies for arthritis induced by autoimmune responses to ubiquitous antigens.     

Analysis of antioxidant activities contained in the Boesenbergia pandurata Schult. Rhizome

The rhizome of Boesenbergia pandurata Schult. was found to possess potent antioxidant activity in a rat brain homogenate model. Bioassay-guided isolation of the active compounds from a CH2Cl2-MeOH (1:1) extract led to the isolation of 5-hydroxy-7-methoxyflavanone, panduratin A, 5,7-dihydroxyflavanone, 2',6'-dihydroxy-4'-methoxychalcone, 2',4'-dihydroxy-6'-methoxychalcone, and 4-hydroxypanduratin A. Panduratin A, 4-hydroxypanduratin A, and 2',6'-dihydroxy-4'-methoxychalcone were also found to exert neuroprotective effects.     

N-acetyl-d-glucosamine induces the expression of multidrug exporter genes, mdtEF, via catabolite activation in Escherichia coli

The expression of MdtEF, a multidrug exporter in Escherichia coli, is positively controlled through multiple signaling pathways, but little is known about signals that induce MdtEF expression. In this study, we investigated compounds that induce the expression of the mdtEF genes and found that out of 20 drug exporter genes in E. coli, the expression of mdtEF is greatly induced by N-acetyl-d-glucosamine (GlcNAc). The induction of mdtEF by GlcNAc is not mediated by the evgSA, ydeO, gadX, and rpoS signaling pathways that have been known to regulate mdtEF expression. On the other hand, deletion of the nagE gene, encoding the phosphotransferase (PTS) system for GlcNAc, prevented induction by GlcNAc. The induction of mdtEF by GlcNAc was also greatly inhibited by the addition of cyclic AMP (cAMP) and completely abolished upon deletion of the cAMP receptor protein gene (crp). Other PTS sugars, glucose and d-glucosamine, also induced mdtEF gene expression. These results suggest that mdtEF expression is stimulated through catabolite control.     

Lysophosphatidic receptor, LPA3, is positively and negatively regulated by progesterone and estrogen in the mouse uterus

Reciprocal interactions between blastocysts and receptive uteri are essential for successful implantation. This process is regulated by the timely interplay of two ovarian hormones, progesterone and estrogen. However, the molecular targets of these hormones are largely unknown. We showed recently that a small bioactive lysophospholipid, lysophosphatidic acid, plays a pivotal role in the establishment of implantation via its cellular receptor, LPA(3). Here we demonstrate that LPA(3) expression is positively and negatively regulated by steroid hormones in mouse uteri. The LPA(3) mRNA level in the uteri increased during early pseudopregnancy, peaking around 3.5 days post coitus (3.5 d.p.c.), then, decreased to the basal level on 4.5 d.p.c. LPA(3) expression remained at a low level in ovariectomized mice, and administration of progesterone to ovariectomized mice up-regulated LPA(3) mRNA expression. In addition, simultaneous administration of estrogen counteracted the effect of progesterone. These results show that progesterone and estrogen cooperatively regulate LPA(3) expression, thereby contributing to the receptivity of uteri during early pregnancy.     

5-O-glucosyldihydroflavones from the leaves of Helicia cochinchinensis

From the leaves of Helicia cochinchinensis, collected on Okinawa Island, seven phenolic glucosides and two terpenic glucosides were isolated. Five of the phenolic glucosides were previously known, being identified with p-coumaric and ferulic acids glucosyl esters, rhodioloside, helicidiol, and naringenin 5-O-beta-D-glucopyranoside. The structures of two other phenolic glucosides, named heliciosides A and B, were elucidated to be 5-O-beta-D-glucosides of 3-hydroxyflavanone, namely aromadendrin and taxifolin, by means of spectroscopic analyses. The two terpenic glucosides were identified with ampelopsisionoside and icariside C1.