RESUMO
Valproic acid (VPA) is widely used to treat epilepsy and manic-depressive illness. Although VPA has been reported to exert a variety of biochemical effects, the exact mechanisms underlying its therapeutic effects remain elusive. To gain further insights into the molecular mechanisms of VPA action, a genetic screen for fission yeast mutants that show hypersensitivity to VPA was performed. One of the genes that we identified was vps45+, which encodes a member of the Sec1/Munc18 family that is implicated in membrane trafficking. Notably, several mutations affecting membrane trafficking also resulted in hypersensitivity to VPA. These include ypt3+ and ryh1+, both encoding a Rab family protein, and apm1+, encoding the mu1 subunit of the adaptor protein complex AP-1. More importantly, VPA caused vacuolar fragmentation and inhibited the glycosylation and the secretion of acid phosphatase in wild-type cells, suggesting that VPA affects membrane trafficking. Interestingly, the cell-wall-damaging agents such as micafungin or the inhibition of calcineurin dramatically enhanced the sensitivity of wild-type cells to VPA. Consistently, VPA treatment of wild-type cells enhanced their sensitivity to the cell-wall-digesting enzymes. Altogether, our results suggest that VPA affects membrane trafficking, which leads to the enhanced sensitivity to cell-wall damage in fission yeast.
Assuntos
Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Ácido Valproico/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Calcineurina/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , DNA Fúngico/genética , Farmacorresistência Fúngica/genética , Genes Fúngicos , Microscopia Eletrônica , Mutação , Schizosaccharomyces/genética , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
A collaborative study involving 8 laboratories was conducted to evaluate a method for the simultaneous determination of pesticide residues in 6 types of fruits and vegetables (spinach, tomato, apple, radish, cabbage and carrot). The method of analysis was the same as reported by Kakimoto et al. in 2003. One hundred and thirty-nine pesticides were spiked by each of 8 laboratories at levels of 0.1 microg/g (pesticides analyzed by GC/MS) or 0.5 microg/g (pesticides analyzed by HPLC) into the 6 kinds of samples. Statistical analysis showed that 111 pesticides could be analyzed with practical precision by this method. For screening purposes, the method could analyze 118 pesticides. The median values of the limits of detection were 0.001-0.041 microg/g. The calibration curves were linear in the range of 0.5-5 microg/mL for most pesticides with median correlation coefficients of 0.983-1.000.