Novel IgE crosslinking-induced luciferase expression method using human-rat chimeric IgE receptor-carrying mast cells.

BACKGROUND: The measurement of antigen-specific serum IgE is common in clinical assessments of type I allergies. However, the interaction between antigens and IgE won't invariably trigger mast cell activation. We previously developed the IgE crosslinking-induced luciferase expression (EXiLE) method using the RS-ATL8 mast cell line; however, the method may not be sensitive enough in some cases. METHODS: In this study, we introduced an NF-AT-regulated luciferase reporter gene into the RBL-2H3 rat mast cell line and expressed a chimeric high-affinity IgE receptor (FcεRI) α chain gene, comprising an extracellular domain from humans and transmembrane/intracellular domains from rats. RESULTS: We generated multiple clones expressing the chimeric receptor. Based on their responsiveness and proliferation, we selected the HuRa-40 clone. This cell line exhibited significantly elevated human α chain expression compared to RS-ATL8 cells, demonstrating a 10-fold enhancement of antigen-specific reactivity. Reproducibility across different batches and operators was excellent. Moreover, we observed a detectable response inhibition by an anti-allergy drugs (omalizumab and cyclosporin A). CONCLUSIONS: HuRa-40 cells-which carry the human-rat chimeric IgE receptor-comprise a valuable reporter cell line for the EXiLE method. Their versatility extends to various applications and facilitates high-throughput screening of anti-allergy drugs.

Determination of (R)-xanthoanthrafil, a phosphodiesterase-5 inhibitor, in a dietary supplement promoted for sexual enhancement.

We describe here the first case of the finding of xanthoanthrafil, a phosphodiesterase-5 inhibitor, in a dietary supplement. A methanol extract of the supplement product was first analyzed by TLC and HPLC. The results indicated that the extract contained an unknown compound. The molecular weight of the compound was 389 and the accurate mass showed its elemental composition to be C(19)H(23)N(3)O(6). Combined with this data, NMR analysis revealed the planar structure of the unknown compound to be N-(3,4-dimethoxybenzyl)-2-(1-hydroxypropan-2-ylamino)-5-nitrobenzamide. The R-configuration of this compound had been synthesized as a phosphodiesterase-5 inhibitor, formerly reported as FR226807 by Fujisawa Pharmaceutical Co., Ltd. The absolute configuration of the isolated compound was estimated to have R-configuration by its optical rotation. Considering its general properties, this compound is renamed as (R)-xanthoanthrafil with the agreement of Astellas Pharma Inc. which is the successor of Fujisawa Pharmaceutical Co., Ltd. Quantitative analysis revealed that the content of (R)-xanthoanthrafil in the product was about 31 mg/capsule.

Rapid and simple determination of epoxyeicosatrienoic acids in rabbit renal artery by reversed-phase HPLC with fluorescence detection.

A liquid chromatographic method with fluorescence detection coupled with a solid-phase extraction was applied to the rapid determination of epoxyeicosatrienoic acids (EETs) in the rabbit renal artery. The EETs were extracted with an acetonitrile from renal artery homogenate and concentrated by a solid-phase extraction method. The concentrated EETs were reacted directly with a 6, 7-dimethoxy-1-methyl-2 (1H)-quinoxalinone-3-propionyl-carboxylic acid (DMEQ) hydrazide and separated by a reversed-phase HPLC with eluting a combination of a step-wise and a gradient of a mixture of methanol and water. The content of EETs in the renal arteries was significantly greater in the 0.5% cholesterol fed rabbits than in control rabbits. It is suggested that hyperchlesterolemia increases the production of EETs in the rabbit renal artery.

Hypothyroidism changes adrenoceptor- and muscarinic receptor-mediated blood pressure responses.

Hypothyroidism was induced by the administration of 0.03% methimazole to drinking water for 1, 2 or 6 weeks to study whether there is a change in adrenoceptor- and muscarinic receptor-mediated blood pressure responses in hypothyroid rats. After 1, 2 and 6 weeks of treatment, the pressor response to norepinephrine was progressively suppressed, and after 6 weeks a significant suppression was observed as compared to control. The depressor response induced by isoprenaline, acetylcholine or sodium nitroprusside was not significantly different between control and hypothyroid rats at any time. The pressor response induced by N(G)-nitro-L-arginine (L-NOARG), an inhibitor of nitric oxide (NO) synthase, was significantly reduced in hypothyroid rats after 1, 2 or 6 weeks of treatment, and the magnitude of the reduction was almost the same for three groups. These results indicated that hypothyroidism causes a time-dependent decrease in pressor responses mediated by alpha-adrenoceptors, but a time-independent decrease in those induced by L-NOARG, and suggest that a progressive decrease in alpha-adrenoceptor-mediated pressor responses occurs in hypothyroidism; however, the decrease in basal NO production and/or release in the peripheral vasculature already occurs in hypothyroid rats at an early stage of the disease.

[Analysis of the oral hypoglycemic agent, glibenclamide, in a health food].

Glibenclamide, a sulfonylurea derivative (SU) antidiabetic agent was detected in a health food by three different methods: TLC, HPLC, and liquid chromatography-mass spectrometry (LC-MS). For analysis of SU antidiabetics, the sample was extracted with acetone as a sample solution. TLC analysis of the sample solution showed a specific spot that had the same characteristics as those of glibenclamide standard solution. HPLC analysis monitored using a photo-diode array detector showed that the sample solution had a peak with a unique UV spectrum, with coincided with that of standard glibenclamide. In sample solution, LC-MS analysis in positive and negative modes indicated that the (M + H)+ and (M - H)- ions occurred at m/z 494 and m/z 492, respectively. These results indicate that the monoisotopic mass is 493, coincident with that of glibenclamide. Quantitative HPLC analysis showed that the glibenclamide content in the health food was 0.78 mg/capsule (1.55 mg/g of sample contents). Because the initial dosage of glibenclamide for diabetics is 1.25-2.5 mg per day, this health food has sufficient medicinal effect and also has the potential to cause adverse effects.