Separation of Sympathomimetic Amines of Abuse and Related Compounds by Micellar Electrokinetic Chromatography

Separation of twelve sympathomimetic amines and related compounds by micellar electrokinetic chromatography (MEKC) with UV absorbance detection is described. These amines were well separated within 25 min using 50 mM sodium tetraborate solution containing 15 mM sodium dodecylsulfate (SDS) of pH 9.3 as a running solution and detected at 210 nm. MEKC was performed with an applied voltage of 13 kV at 25 °C using a fused-silica capillary (50 cm×75 mm i.d.) with effective length of 37.5 cm. The detection limits of these compounds were in the range from 4 to 97 fmol/injection at a signal-to-noise ratio (S/N) of 3. The reproducibility of the method expressed as relative standard deviation (RSD) for within-day (n=6) and between-day (n=5) assays was less than 4.8 and 8.8%, respectively. The proposed method could be applied to the determination of an anorectic drug, phentermine, in Chinese tea with a detection limit of 99 μg/g (105 fmol/injection, S/N=3)

ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells

<p>Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted <i>ATG5</i>, an autophagy-related gene, in <i>Physcomitrella</i>, and confirmed that <i>atg5</i> mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, <i>atg5</i> colonies turned yellow earlier than the wild-type (WT) colonies, showing that <i>Physcomitrella atg5</i> mutants, like yeast and <i>Arabidopsis</i>, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in <i>atg5</i> colonies than WT colonies. The sugar content was almost the same between WT and <i>atg5</i> colonies, indicating that the early-senescence phenotype of <i>atg5</i> is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between <i>atg5</i> and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the <i>atg5</i> mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in <i>atg5</i> mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in <i>Physcomitrella</i>.</p