Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics

The moss Physcomitrella patens is increasingly being used as a model for plant systems biology studies. While genomic and transcriptomic resources are in place, tools and experimental conditions for proteomic studies need to be developed. In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema. Routinely, 60–100 μg mitochondrial and 3–5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue. Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed. This isolation protocol and these validated compartment markers may serve as basis for sub-cellular proteomics in P. patens and other mosses

Pharmacokinetic and behavioral effects of allopregnanolone in healthy women

Rationale  The behavioral effects of allopregnanolone (3α-hydroxy-5α-pregnan-20-one) in women are not known. Objective  Allopregnanolone, a neuroactive steroid secreted by the mammalian ovary, exerts its anesthetic, anxiolytic, and sedative/hypnotic effects through potentiation of GABAA receptors. The purpose of this study was to evaluate the behavioral effects of allopregnanolone in healthy women. Methods  Ten healthy women were given three increasing intravenous doses of allopregnanolone in the follicular phase of the menstrual cycle. Saccadic eye movement parameters and visual analogue scales of sedation were used to evaluate the behavioral response of allopregnanolone. Repeated blood samples for analyses of allopregnanolone were drawn throughout the study day. Results  Exogenously administered allopregnanolone decreases saccadic eye movement parameters and increases subjective ratings of sedation that correlate with increased serum concentrations of this neuroactive steroid. Conclusion  The behavioral effects of allopregnanolone are similar to that of its 5β-stereoisomer, pregnanolone (3α-hydroxy-5β-pregnan-20-one). Apart from fatigue and mild nausea, allopregnanolone given in a cumulative dose of 0.09 mg/kg did not have any adverse effects