Revised method for routine determination of urinary dialkyl phosphates using gas chromatography–mass spectrometry

Among urinary organophosphorus pesticide (OP) metabolites, dialkyl phosphates (DAPs) have been most often measured as a sensitive biomarker in non-occupational and occupational OP exposure risk assessment. In our conventional method, we have employed a procedure including simple liquid–liquid extraction (diethyl ether/acetonitrile), derivatization (pentafluorobenzylbromide, PFBBr) and clean-up (multi-layer column) for gas chromatography–mass spectrometry (GC–MS) analysis starting from 5-mL urine samples. In this study, we introduce a revised analytical method for urinary DAPs; its main modification was aimed at improving the pre-derivatization dehydration procedure. The limits of detection were approximately 0.15 μg/L for dimethylphosphate (DMP), 0.07 μg/L for diethylphosphate (DEP), and 0.05 μg/L for both dimethylthiophosphate (DMTP) and diethylthiophosphate (DETP) in 2.5-mL human urine samples. Within-run precision (percent of relative standard deviation, %RSD) at the DAP levels varying in the range of 0.5–50 μg/L was 6.0–19.1% for DMP, 3.6–18.3% for DEP, 8.0–25.6% for DMTP and 9.6–27.8% for DETP. Between-run precision at 5 μg/L was below 15.7% for all DAPs. The revised method proved to be feasible to routine biological monitoring not only for occupational OP exposure but also for environmental background levels in the general population. Compared to our previous method, the revised method underscores the importance of adding pre-derivatization anhydration for higher sensitivity and precision.     

Distribution of In Vitro Fermentation Ability of Lacto-N-Biose I,a Major Building Block of Human Milk Oligosaccharides,in Bifidobacterial Strains

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudocatenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.Bifidobacteria are gram-positive anaerobic bacteria that naturally colonize the human intestinal tract and are believed to be beneficial to human health (21, 30). Breastfeeding has been shown to be associated with an infant fecal microbiota dominated by bifidobacteria, whereas the fecal microbiota of infants who are consuming alternative diets has been described as being mixed and adult-like (12, 21). It has been suggested that the selective growth of bifidobacteria observed in breast-fed newborns is related to the oligosaccharides and other factors that are contained in human milk (human milk oligosaccharides [HMOs]) (3, 4, 10, 11, 16, 17, 34). Kitaoka et al. (15) have recently found that bifidobacteria possess a unique metabolic pathway that is specific for lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc). LNB is a building block for the type 1 HMOs [such as lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and lacto-N-difucohexaose I (Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc)], and GNB is a core structure of the mucin sugar that is present in the human intestine and milk (18, 27). The GNB/LNB pathway, as previously illustrated by Wada et al. (33), involves proteins/enzymes that are required for the uptake and degradation of disaccharides such as the GNB/LNB transporter (29, 32), galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP; LnpA) (15, 24) (renamed from lacto-N-biose phosphorylase after the finding of phosphorylases specific to GNB [23] and LNB [22]), N-acetylhexosamine 1-kinase (NahK) (25), UDP-glucose-hexose 1-phosphate uridylyltransferase (GalT), and UDP-galactose epimerase (GalE). Some bifidobacteria have been demonstrated to be enzymatically equipped to release LNB from HMOs that have a type 1 structure (lacto-N biosidase; LnbB) (33) or GNB from the core 1-type O-glycans in mucin glycoproteins (endo-α-N-acetylgalatosaminidase) (6, 13, 14). It has been suggested that the presence of the LnbB and GNB/LNB pathways in some bifidobacterial strains could provide a nutritional advantage for these organisms, thereby increasing their populations within the ecosystem of these breast-fed newborns (33).The species that predominantly colonize the infant intestine are the bifidobacterial species B. breve, B. longum subsp. infantis, B. longum subsp. longum, and B. bifidum (21, 28). On the other hand, strains of B. adolescentis, B. catenulatum, B. pseudocatenulatum, and B. longum subsp. longum are frequently isolated from the adult intestine (19), and strains of B. animalis subsp. animalis, B. animalis subsp. lactis, B. thermophilum and B. pseudolongum have been shown to naturally colonize the guts of animals (1, 2, 7, 8). However, it is unclear whether there is a relationship between the differential colonization of the bifidobacterial species and the presence of the GNB/LNB pathway. In the present study, we investigated the ability of individual bifidobacterial strains in the in vitro fermentation of LNB and in addition, we also tried to determine whether or not the GLNBP gene (lnpA), which is a key enzyme of the GNB/LNB pathway, was present.     

Circulating muscle-specific microRNA, miR-206, as a potential diagnostic marker for rhabdomyosarcoma

Presently there is no serum biomarker of rhabdomyosarcoma (RMS). Several studies have shown that profiles of microRNA (miRNA) expression differ among tumor types. Here we evaluated the feasibility of using muscle-specific miRNAs (miR-1, -133a, -133b and -206) as biomarkers of RMS. Expression of muscle-specific miRNAs, especially miR-206, was significantly higher in RMS cell lines than in other tumor cell lines, as well as in RMS tumor specimens. Further, serum levels of muscle-specific miRNAs were significantly higher in patients with RMS tumors than in patients with non-RMS tumors. Normalized serum miR-206 expression level could be used to differentiate between RMS and non-RMS tumors, with sensitivity of 1.0 and specificity of 0.913. These results raise the possibility of using circulating muscle-specific miRNAs, especially miR-206, as landmark biomarkers for RMS.     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

Identification of a novel type of CA19-9 carrier in human bile and sera of cancer patients: an implication of the involvement in nonsecretory exocytosis

Carbohydrate antigen 19-9 (CA19-9) is a well-known tumor marker for pancreatic cancer. Although the CA19-9 level is measured using anti-sialyl Lewis A antibodies, it remains unknown which molecules carry CA19-9 other than mucins. Here we report the identification and characterization of a novel type of CA19-9 carrier, BGM (bile globular membrane), which is thought to exist in normal bile and to be secreted into sera of patients with pancreatic cancer. We purified the BGM from bile juice using a β-casein column because surface plasmon resonance analysis could detect such carrier vesicles binding to β-casein in sera of patients with pancreatic cancer. We identified characteristic molecules for BGM such as AHNAK (desmoykoin) and a novel golgin family member, CABIN (CAsein Binding domain integral protein with golgIN motif) by mass spectrometry analysis. BGM was detected in the sera of patients with pancreatic cancer as well as athymic mice with transplanted pancreatic cancer cells. Down regulation of CABIN inhibited the secretion of CA19-9 on BGM in pancreatic cancer cell lines. We measured and visualized BGM in sera of patients with cancer. Thus, BGM might be another CA19-9 carrier (glyco-lipids on membrane vesicles) other than mucins and could be applied to the diagnosis of pancreatic cancer.     

Partial Characterization of a 17 kDa Acidic Protein, EFP, Induced by Thiocarbamate in the Early Flowering Phase in Asparagus Seedlings

Asparagus officinalis seedlings treated with either thiocarbamatesor carbamates are induced to flower within 2-3 weeks of application.SDS-PAGE and 2-D PAGE resolutions of total soluble proteinsof treated seedlings showed the accumulation of an acidic 17kDa polypeptide. Partialmino acid sequence of the acidic proteinshowed homology to the wound-responsive A. officinalisPR 1,AoPRl. We named this protein EFP, early flowering protein, owingto its advanced appearance during the early transition fromvegetative to floral phase. Carbamates and thiocarbamates thatinduced more than 80% flowering rates corresponded with increasedlevel of EFP while compounds that are weak flower inducers showedreduced amount of EFP. The observation thus denned the correlationshipof EFP to variable flowering rates. (Received May 27, 1996; Accepted July 11, 1996)