Nucleotide Sequence and Expression in Escherichia coli of the Lactococcus lactis Citrate Permease Gene

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers

Analysis of Diffusion of Ras2 in Saccharomyces cerevisiae Using Fluorescence Recovery after Photobleaching

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane localized fluorophore with the cytosol during Fluorescence Recovery after Photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma-membrane localized wild type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 micron x 1 micron bleach region-of-interest (ROI) and a 0.5 micron x 0.5 micron bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane associated fluorophores using FRAP on commercial confocal laser scanning microscopes.Comment: Accepted for publication in Physical Biology (2010). 28 pages, 7 figures, 3 table

Customer engagement in sales promotion

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Consumers Beware: Online Personalized Pricing in Action! How the Framing of a Mandated Discriminatory Pricing Disclosure Influences Intention to Purchase

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Nutritional status and structural brain changes in Alzheimer's disease: The NUDAD project

INTRODUCTION: Weight loss is associated with higher mortality and progression of cognitive decline, but its associations with magnetic resonance imaging (MRI) changes related to Alzheimer's disease (AD) are unknown. METHODS: We included 412 patients from the NUDAD project, comprising 129 with AD dementia, 107 with mild cognitive impairment (MCI), and 176 controls. Associations between nutritional status and MRI measures were analyzed using linear regression, adjusted for age, sex, education, cognitive functioning, and cardiovascular risk factors. RESULTS: Lower body mass index (BMI), fat mass (FM), and fat free mass index were associated with higher medial temporal atrophy (MTA) scores. Lower BMI, FM, and waist circumference were associated with more microbleeds. Stratification by diagnosis showed that the observed associations with microbleeds were only significant in MCI. DISCUSSION: Lower indicators of nutritional status were associated with more MTA and microbleeds, with largest effect sizes in MCI