Genome-Wide Identification of the AP2/ERF Gene Family Involved in Active Constituent Biosynthesis in.

Tanshinones and phenolic acids are the major bioactive constituents in the traditional medicinal crop ; however, transcription factors (TFs) are seldom investigated with regard to their regulation of the biosynthesis of these compounds. Here a complete overview of the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor family in is provided, including phylogeny, gene structure, conserved motifs, and gene expression profiles of different organs (root, stem, leaf, flower) and root tissues (periderm, phloem, xylem). In total, 170 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (25 genes), DREB (61 genes), ethylene responsive factor (ERF; 79 genes), RAV (4 genes), and Soloist (1 gene). According to the distribution of bioactive constituents and the expression patterns of AP2/ERF genes in different organs and root tissues, the genes related to the biosynthesis of bioactive constituents were selected. On the basis of quantitative real-time polymerase chain reaction (qRT-PCR) analysis, coexpression analysis, and the prediction of -regulatory elements in the promoters, we propose that two genes ( and ) regulate tanshinone biosynthesis and two genes ( and ) participate in controlling phenolic acid biosynthesis. The genes related to tanshinone biosynthesis belong to the ERF-B3 subgroup. In contrast, the genes predicted to regulate phenolic acid biosynthesis belong to the ERF-B1 and ERF-B4 subgroups. These results provide a foundation for future functional characterization of AP2/ERF genes to enhance the biosynthesis of the bioactive compounds of .

A new gas chromatography-mass spectrometry method for simultaneous determination of total and free trans-3'-hydroxycotinine and cotinine in the urine of subjects receiving transdermal nicotine.

trans-3'-Hydroxycotinine (THOC) has been recognized as the most abundant metabolite of nicotine. In an attempt to assess THOC and cotinine (COT) concentrations during nicotine transdermal therapy, we developed a new quantitative gas chromatography-mass spectrometry (GC-MS) method for simultaneous determination of total and free THOC and COT in human urine. The method utilizes the following: (a) hydrolysis of conjugated THOC and COT by beta-glucuronidase; (b) basic extraction of THOC and COT with mixed dichloromethane and n-butyl acetate; (c) derivatization of THOC with bis(trimethylflurosilyl)acetamide; and (d) separation and identification by GC-MS with selective ion monitoring. Lower limits of quantification for the assay were 50 and 20 microg/L for THOC and COT, respectively. The intra- and interassay CVs were 4.4% and 11% for THOC, and 3.9% and 10% for COT at 1000 microg/L. The results from six consecutive 24-h urine collections in 71 subjects administered daily transdermal nicotine doses of 11, 22, and 44 mg showed that, on average, free THOC was 76% of total THOC and free COT was 48% of total COT in all subjects. THOC is the major metabolite of nicotine and constitutes 20% of total nicotine intake at steady state, whereas urinary nicotine and COT excretion were 8% and 17%, respectively. The method is useful for simultaneous determination of free and total THOCand COT and can be used to assess the urinary excretion of these metabolites during transdermal nicotine therapy.

Separation of urinary estrogens by micellar electrokinetic chromatography.

Urinary estrogen levels are important for monitoring the normal pregnancy process as well as for the diagnosis of reproductive diseases. 17 beta-Estradiol and estrone are maintained at very low concentrations in urine and, therefore, are difficult to determine using standard chromatography with UV detection. In the present study, we describe a potential method for the determination of urinary estrogens (estrone, estradiol and estriol) using a solid-phase extraction and rapid capillary electrophoretic (CE) separations. Micellar electrokinetic chromatographic (MEKC) analysis was optimized by evaluating the number of surfactants in a 5 mM borate-5 mM phosphate separation buffer, of which sodium cholate (75-90 mM) was found to be optimal. Changing the hydrophobic character of the separation buffer with organic additives had a significant effect on the resolution of the three estrogens and an internal standard (d-equilenin). The addition of an organic additive (20% acetonitrile) was found to be necessary for the resolution of all components of the mixture. Substitution with 20% methanol provided a similar separation with better resolution but at the cost of increased analysis time. Analysis of two extracted urine samples from 18-weeks and 21-weeks pregnant women showed that, with the present technology, CE can provide adequate resolution and superior speed, but the sensitivity limits attainable with the existing technology may limit its utility to the measurement of estriol and estrone.

Determination of total serum sulfite by HPLC with fluorescence detection.

An estimated 500,000 individuals in the US, mostly steroid-dependent asthmatics, suffer severe adverse reactions to sulfites in foods, beverages, and pharmaceutical products. In an attempt to understand the pathogenesis of sulfite hypersensitivity, we have developed an assay for the determination of total serum sulfite by utilizing: (a) reductive release of serum protein-bound sulfite; (b) derivatization of free sulfite with monobromobimane; (c) separation of sulfite-bimane from thiol-bimanes by reversed-phase HPLC; and (d) quantitation of sulfite-bimane by fluorescence detection. The detection limit of this assay was 0.44 mumol/L serum sulfite. The intra- and interassay CVs for total serum sulfite at 5.4 mumol/L were 8.1% and 22.0%, respectively. The standard addition method was used to determine total serum sulfite in normal subjects. More than 70 samples were prepared in 2-3 h, followed by automated overnight analysis. The mean concentrations (+/- SD) of total serum sulfite in female (n = 41) and male (n = 35) donors were 4.63 +/- 2.33 and 5.16 +/- 2.68 mumol/L, respectively (not statistically significant: P = 0.368). The combined mean concentration of total sulfite in both sexes was 4.87 +/- 2.49 mumol/L. There was no correlation between total serum sulfite and total serum cysteine, cysteinylglycine, homocysteine, subject age, serum cobalamin, or serum folic acid. The reference range (mean +/- 2 SD) for total serum sulfite in normal subjects is 0-9.85 mumol/L.