Lipid metabolites and nitric oxide production in the cerebrospinal fluid and plasma of dogs with meningoencephalitis of unknown origin and idiopathic epilepsy: a pilot study.

Introduction: Idiopathic epilepsy (IE) and meningoencephalomyelitis of unknown origin (MUO) are common causes of brain diseases leading to seizures in dogs. In this study, the concentrations of 196 lipid metabolites and nitrogen oxide (NO) production in the cerebrospinal fluid (CSF) and plasma of dogs with MUO or IE were measured using a LC-MS/MS and a NOx analyzer, respectively. Methods: Nine clinically healthy dogs and 11 and 12 dogs with IE and MUO, respectively, were included in the study. Results: Lipid analysis revealed variations in the levels of four and six lipid metabolites in CSF and plasma, respectively, between the groups. The levels of 6-keto-prostaglandin (PG) F1α (PGF1α), 20-carboxy arachidonic acid (20-carboxy-AA), 9-hydroxyoctadecadienoic acid, and lyso-platelet-activating factor were high in the CSF of dogs with MUO. In addition, the plasma levels of 11,12-dihydroxyeicosatrienoic acid, 20-carboxy-AA, and oleoylethanolamide were high in dogs with IE, and those of PGF1α were high in dogs with MUO. NO production levels were high in CSF but not in plasma in dogs with MUO or IE. Discussion: It remains unknown whether these changes represent the cause or effect of diseases of the central nervous system; however, lipid metabolites and NO production in CSF and plasma may be used as diagnostic biomarkers and could be exploited for treating idiopathic or inflammatory epilepsy in dogs.

Bisecting-GlcNAc on Asn388 is characteristic to ERC/mesothelin expressed on epithelioid mesothelioma cells.

Mesothelioma is a highly aggressive tumour associated with asbestos exposure and is histologically classified into three types: epithelioid-type, sarcomatoid-type and biphasic-type. The prognosis of mesothelioma patients is poor and there is no effective molecular-targeting therapy as yet. ERC/mesothelin is a glycoprotein that is highly expressed on several types of cancers including epithelioid mesothelioma, but also expressed on normal mesothelial cells. This is a predicted reason why there is no clinically approved therapeutic antibody targeting ERC/mesothelin. In the present study, we focussed on the differential glycosylation between ERC/mesothelin present on epithelioid mesothelioma and that on normal mesothelial cells and aimed to reveal a distinct feature of epithelioid mesothelioma cells. Lectin microarray analysis of ERC/mesothelin using cells and patient specimens showed significantly stronger binding of PHA-E4 lectin, which recognizes complex-type N-glycans having a so-called bisecting-GlcNAc structure, to ERC/mesothelin from epithelioid mesothelioma cells than that from normal mesothelial cells. Further, liquid chromatography/mass spectrometry analysis on ERC/mesothelin from epithelioid mesothelioma cells confirmed the presence of a bisecting-GlcNAc attached to Asn388 of ERC/mesothelin. These results suggest that this glycoproteome could serve as a potential target for the generation of a highly selective and safe therapeutic antibody for epithelioid mesothelioma.

Identification of Poly-N-Acetyllactosamine-Carrying Glycoproteins from HL-60 Human Promyelocytic Leukemia Cells Using a Site-Specific Glycome Analysis Method, Glyco-RIDGE.

To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. Therefore, we developed a liquid chromatography/mass spectrometry (MS)-based glycopeptide analysis method to reveal the site-specific glycome (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile, Glyco-RIDGE). This method used accurate masses and retention times of glycopeptides, without requiring MS2, and could be applied to complex mixtures. To increase the number of identified peptide, fractionation of sample glycopeptides for reduction of sample complexity is required. Therefore, in this study, glycopeptides were fractionated into four fractions by hydrophilic interaction chromatography, and each fraction was analyzed using the Glyco-RIDGE method. As a result, many glycopeptides having long glycans were enriched in the highest hydrophilic fraction. Based on the monosaccharide composition, these glycans were thought to be poly-N-acetyllactosamine (polylactosamine [pLN]), and 31 pLN-carrier proteins were identified in HL-60 cells. Gene ontology enrichment analysis revealed that pLN carriers included many molecules related to signal transduction, receptors, and cell adhesion. Thus, these findings provided important insights into the analysis of the glycoproteome using our novel Glyco-RIDGE method. Graphical Abstract ᅟ.