Convenient and rapid removal of detergent from glycolipids in detergent-resistant membrane microdomains

Although detergents are often essential in protocols, they are usually incompatible with further biochemical analysis. There are several methods for detergent removal, but the procedures are complicated or suffer from sample loss. Here, we describe a convenient and rapid method for detergent removal from sialic acid-containing glycosphingolipids (gangliosides) and neutral glycolipids in detergent-resistant membrane (DRM) microdomain. It is based on selective detergent extraction, in which the sample is dried on a glass tube, followed by washing with organic solvent. We investigated 18 organic solvents and used high performance thin-layer chromatography (HPTLC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) to confirm that dichloroethane (DCE) was the most suitable solvent and completely removed the nonionic detergent Triton X-100. Furthermore, DCE extraction effectively removed interference caused by other nonionic, zwitterionic, or ionic detergents in MALDI-QIT-TOF MS analysis.     

Detecting amplicons of loop‐mediated isothermal amplification

Loop‐mediated isothermal amplification (LAMP) assays are used to detect diverse pathogens. Initially, LAMP amplicons were detected using electrophoresis; later, real‐time monitoring based on turbidity was developed to overcome the problem of contamination with environmental DNA. Recently, real‐time monitoring of fluorescence signals using a quenching primer and probe has improved the reliability of amplification signals. Here, methods of detecting LAMP amplicons are reviewed.     

Selective regulation of interleukin-10 production via Janus kinase pathway in murine conventional dendritic cells

In this study, we examined the role of JAKs in regulation of inflammatory versus anti-inflammatory cytokine balance in murine conventional dendritic cells (DCs). Highly purified lipopolysaccharide (upLPS) combined with imiquimod (IQ) synergistically induced IL-10 production by DCs, while each ligand alone showed a slight effect on the IL-10 production. Marked phosphorylation of JAK2, STAT1 and STAT3 was detected in DCs following upLPS plus IQ stimulation. Blocking the JAK pathway by JAK inhibitor I (JAKi) resulted in significant inhibition of IL-10 production by the DCs. However, JAKi showed negligible effect on the DC production of IL-12, IL-6 and TNF-α. JAKi completely blocked the TLR-mediated STATs activation, and attenuated the activation of Akt, a downstream effector of PI3K, in DCs stimulated by upLPS plus IQ. LY294002, a specific inhibitor of PI3K, markedly inhibited the DC production of IL-10. Thus, JAK-PI3K axis appeared to be responsible for the IL-10 production by DCs.     

Enzymatic characterization of peroxisomal and cytosolic betaine aldehyde dehydrogenases in barley

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is an important enzyme that catalyzes the last step in the synthesis of glycine betaine, a compatible solute accumulated by many plants under various abiotic stresses. In barley ( Hordeum vulgare L.), we reported previously the existence of two BADH genes ( BBD1 and BBD2 ) and their corresponding proteins, peroxisomal BADH (BBD1) and cytosolic BADH (BBD2). To investigate their enzymatic properties, we expressed them in Escherichia coli and purified both proteins. Enzymatic analysis indicated that the affinity of BBD2 for betaine aldehyde was reasonable as other plant BADHs, but BBD1 showed extremely low affinity for betaine aldehyde with apparent K_m of 18.9 μ M and 19.9 m M , respectively. In addition, V_max/K_m with betaine aldehyde of BBD2 was about 2000-fold higher than that of BBD1, suggesting that BBD2 plays a main role in glycine betaine synthesis in barley plants. However, BBD1 catalyzed the oxidation of ω-aminoaldehydes such as 4-aminobutyraldehyde and 3-aminopropionaldehyde as efficiently as BBD2. We also found that both BBDs oxidized 4- N -trimethylaminobutyraldehyde and 3- N -trimethylaminopropionaldehyde.     

Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography

An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 μl of plasma and 240 μl of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 μM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects.     

Rhodanobacter sp. strain BPC1 in a benzo[a]pyrene-mineralizing bacterial consortium

A bacterial consortium which rapidly mineralizes benzo[a]pyrene when it is grown on a high-boiling-point diesel fuel distillate (HBD) was recovered from soil and maintained for approximately 3 years. Previous studies have shown that mobilization of benzo[a]pyrene into the supernatant liquid precedes mineralization of this compound (R. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, Appl. Environ. Microbiol. 66:4205-4211, 2000). In the present study, we found that sterilized supernatant liquid filtrate (SSLF) obtained from the growing consortium stimulated mineralization of benzo[a]pyrene when it was readministered to a consortium inoculum without HBD. Following this observation, eight bacterial strains were isolated from the consortium, and SSLF of each of them was assayed for the ability to stimulate benzo[a]pyrene mineralization by the original consortium. The SSLF obtained from one strain, designated BPC1, most vigorously stimulated benzo[a]pyrene mineralization by the original consortium; its effect was more than twofold greater than the effect of the SSLF obtained from the original consortium. A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genus Rhodanobacter, whose type strain, Rhodanobacter lindaniclasticus RP5557, which was isolated for its ability to grow on the pesticide lindane, is not extant. Strain BPC1 could not grow on lindane, benzo[a]pyrene, simple hydrocarbons, and HBD in pure culture. In contrast, a competitive PCR assay indicated that strain BPC1 grew in the consortium fed only HBD and benzo[a]pyrene. This growth of BPC1 was concomitant with growth of the total bacterial consortium and preceded the initiation of benzo[a]pyrene mineralization. These results suggest that strain BPC1 has a specialized niche in the benzo[a]pyrene-mineralizing consortium; namely, it grows on metabolites produced by fellow members and contributes to benzo[a]pyrene mineralization by increasing the bioavailability of this compound.     

Studies on scavenger receptor inhibitors. Part 1: synthesis and structure-activity relationships of novel derivatives of sulfatides

Scavenger receptors have been proven to be implicated in the formation of atherosclerotic lesions. A series of novel derivatives of sulfatides were synthesized, and their inhibitory activities against incorporation of DiI-acetyl-LDL into macrophages were evaluated in order to clarify the structure-activity relationships of sulfatides as a scavenger receptor inhibitor and find out novel inhibitors with synthetic easiness. The chemical modification of the substructures of sulfatides led to the establishment of the following structure-activity relationships; (1) the ceramide moiety can be replaced with another structure bearing two long chains, (2) the galactose moiety can be replaced with another structure or be deleted without a large decrease in the inhibitory activity, (3) the sulfate moiety was crucial, and it was the most preferable functional group for a potent inhibitory activity. The inhibitory activity of (S)-2-octadecanoylamino-2-tetradecylcarbamoyl)ethyl sulfate sodium salt (3a) against incorporation of DiI-acetyl-LDL into macrophages was proven to be based on the inhibition against the binding of acetyl-LDL to the surface of macrophages. We discovered novel scavenger receptor inhibitors with synthetic easiness, such as (S)-2-octadecanoylamino-2-(tetradecylcarbamoyl)ethyl sulfate sodium salt (3a) and 2-octadecanoylamino-1-(octadecanoylaminomethyl)ethyl sulfate sodium salt (13q).     

Changes in Flavor Components of Onion by γ-Irradiation

The effect of γ-irradiation on the flavor of Onions (Senshu Yellow) associated with sprout-inhibition has been investigated. The relative amounts of propionaldehyde, n-propyl mercaptan and di-n-propyl disulfide in onions stored for 0, 5, 10, 20 and 30 days, respectively, after irradiation were estimated by measurement of peak areas of gas chromatograms. It was observed that the lachrymatory character and the pungent flavor had been decreased by γ-irradiation (remarkably at 70 and 700 Krads). In this connection, the amounts of propionaldehyde and di-n-propyl disulfide were decreased with increasing radiation dose and storage period at room temperature (20 to 25°C) and at low temperature (4°C). Moreover, it was observed that the sweetness had been decreased by γ-irradiation, but the amount of n-propyl mercaptan was increased with radiation dose and storage period. Therefore it seems that there is no correlation between the sweetness of onion and the amount of n-propyl mercaptan.     

Carbendazim-induced abnormal development of the acrosome during early phases of spermiogenesis in the rat testis

Effects of a single, high dose of orally administered carbendazim (100 mg/kg) on acrosome formation in the early phases of spermiogenesis were examined by electron microscopy and immunocytochemistry up to day 7.5 post-treatment. No obvious abnormality of acrosome development was noted in the Golgi phase spermatids on day 1.5 post-treatment. On day 3, step 1 spermatids were seen in stage III seminiferous tubules. In stage V tubules at this post-treatment interval, direct connections between the trans-side saccules of the Golgi stacks and the outer acrosomic membranes were observed in step 5 spermatids. Similar direct connections between these two organelles were also observed in the advanced round spermatids in later stages at days 4.5 and 7.5. On day 4.5, step 1 and 3 spermatids were seen in stage V tubules. On day 7.5, round spermatids with various abnormalities of acrosome development were observed in stage VII tubules, in addition to the discontinuous and granular acrosomes reported previously. These features were not observed in testes of control animals. In the immunocytochemical analysis using an antibody mMN7 that recognizes a protein delivered from the Golgi apparatus to the acrosome, spermatids exposed to carbendazim showed various abnormal immunostaining patterns in the acrosomes. On the other hand, strong immunoreactivity was observed in the Golgi saccules connecting to the acrosomes. These results suggest that in testis treated with carbendazim acrosome development is impaired during the early phases of spermiogenesis, and material supply from the Golgi apparatus to the acrosome is perturbed, which is a possible cause of the abnormal development. Received: 31 March 1998 / Accepted: 28 May 1998