Outer envelope membranes from chloroplasts are isolated as right-side-out vesicles

Outer envelope membranes were isolated from purified chloroplasts of pea leaves. The sidedness of the vesicles was analyzed by (i) aqueous polymer-two phase partitioning, (ii) the effect of limited proteolysis on the outer-envelope proteins (OEP) 86 and OEP 7 in intact organelles and isolated membranes, (iii) fluorescence-microscopy and finally (iv) binding of precursor polypeptides to isolated outer-membrane vesicles. The results demonstrate that purified outer envelope membranes occur largely (>90%) as right-side-out vesicles

Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts

Isolated outer envelope membrane from pea (Pisum sativum L.) chloroplasts can be used in vitro to study binding and partial translocation of precursor proteins destined for the inside of the organelle. Efficient binding to a receptor protein on the outside of the membrane vesicle and generation of a translocation intermediate depends strictly on the presence of ATP. Protease treatment of the translocation intermediate demonstrates its insertion into the membrane. The membraneinserted precursor protein cannot be extracted by 1 M NaCl and is also NaOH resistant to a large extent. Mild solubilization of outer envelope membranes by detergent resulted in the isolation of a complex which still contained the precursor protein. We have identified a constitutively expressed homologue hsc 70 as part of this membrane complex. Antibodies against hsp 70 (inducible heat shock protein 70) were able to immunoprecipitate the complex bound precursor protein. A second protein of 86 kDa molecular weight (OEP 86) from the outer envelope membrane was also identified as a major component of this complex

mHealth Series:mHealth project in Zhao County, rural China - Description of objectives, field site and methods

BACKGROUND: We set up a collaboration between researchers in China and the UK that aimed to explore the use of mHealth in China. This is the first paper in a series of papers on a large mHealth project part of this collaboration. This paper included the aims and objectives of the mHealth project, our field site, and the detailed methods of two studies. FIELD SITE: The field site for this mHealth project was Zhao County, which lies 280 km south of Beijing in Hebei Province, China. METHODS: We described the methodology of two studies: (i) a mixed methods study exploring factors influencing sample size calculations for mHealth–based health surveys and (ii) a cross–over study determining validity of an mHealth text messaging data collection tool. The first study used mixed methods, both quantitative and qualitative, including: (i) two surveys with caregivers of young children, (ii) interviews with caregivers, village doctors and participants of the cross–over study, and (iii) researchers’ views. We combined data from caregivers, village doctors and researchers to provide an in–depth understanding of factors influencing sample size calculations for mHealth–based health surveys. The second study, a cross–over study, used a randomised cross–over study design to compare the traditional face–to–face survey method to the new text messaging survey method. We assessed data equivalence (intrarater agreement), the amount of information in responses, reasons for giving different responses, the response rate, characteristics of non–responders, and the error rate. CONCLUSIONS: This paper described the objectives, field site and methods of a large mHealth project part of a collaboration between researchers in China and the UK. The mixed methods study evaluating factors that influence sample size calculations could help future studies with estimating reliable sample sizes. The cross–over study comparing face–to–face and text message survey data collection could help future studies with developing their mHealth tools