Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2--galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.     

DBA/2J Genetic Background Exacerbates Spontaneous Lethal Seizures but Lessens Amyloid Deposition in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a leading cause of dementia in the elderly and is characterized by amyloid plaques, neurofibrillary tangles (NFTs) and neuronal dysfunction. Early onset AD (EOAD) is commonly caused by mutations in amyloid precursor protein (APP) or genes involved in the processing of APP including the presenilins (e.g. PSEN1 or PSEN2). In general, mouse models relevant to EOAD recapitulate amyloidosis, show only limited amounts of NFTs and neuronal cell dysfunction and low but significant levels of seizure susceptibility. To investigate the effect of genetic background on these phenotypes, we generated APP^swe and PSEN1^de9 transgenic mice on the seizure prone inbred strain background, DBA/2J. Previous studies show that the DBA/2J genetic background modifies plaque deposition in the presence of mutant APP but the impact of PSEN1^de9 has not been tested. Our study shows that DBA/2J.APP^swePSEN1^de9 mice are significantly more prone to premature lethality, likely to due to lethal seizures, compared to B6.APP^swePSEN1^de9 mice—70% of DBA/2J.APP^swePSEN1^de9 mice die between 2-3 months of age. Of the DBA/2J.APP^swePSEN1^de9 mice that survived to 6 months of age, plaque deposition was greatly reduced compared to age-matched B6.APP^swePSEN1^de9 mice. The reduction in plaque deposition appears to be independent of microglia numbers, reactive astrocytosis and complement C5 activity.     

Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O₂-evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two protein-translocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.     

mtDNA variation in Inuit populations of Greenland and Canada: migration history and population structure

We examined 395 mtDNA control-region sequences from Greenlandic Inuit and Canadian Kitikmeot Inuit with the aim of shedding light on the migration history that underlies the present geographic patterns of genetic variation at this locus in the Arctic. In line with previous studies, we found that Inuit populations carry only sequences belonging to haplotype clusters A2 and D3. However, a comparison of Arctic populations from Siberia, Canada, and Greenland revealed considerable differences in the frequencies of these haplotypes. Moreover, large sample sizes and regional information about birthplaces of maternal grandmothers permitted the detection of notable differences in the distribution of haplotypes among subpopulations within Greenland. Our results cast doubt on the prevailing hypothesis that contemporary Inuit trace their all of their ancestry to so-called Thule groups that expanded from Alaska about 800-1,000 years ago. In particular, discrepancies in mutational divergence between the Inuit populations and their putative source mtDNA pool in Siberia/Alaska for the two predominant haplotype clusters, A2a and A2b, are more consistent with the possibility that expanding Thule groups encountered and interbred with existing Dorset populations in Canada and Greenland.     

The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.     

High-selenium yeast supplementation in free-living North American men: no effect on thyroid hormone metabolism or body composition.

In a prior study, we observed decreased serum 3,3',5-triiodothyronine (T(3)), increased serum thyrotropin and increased body weight in five men fed 297 microg/d of selenium (Se) in foods naturally high in Se while confined in a metabolic research unit. In an attempt to replicate and confirm those observations, we conducted a randomized study of high-Se yeast supplements (300 microg/d) or placebo yeast administered to 42 healthy free-living men for 48 weeks. Serum thyroxine, T(3) and thyrotropin did not change in supplemented or control subjects. Body weight increased in both groups during the 48-week treatment period and remained elevated for the 48-week follow-up period. Body fat increased by 1.2 kg in both groups. Energy intake and voluntary activity levels were not different between the groups and remained unchanged during the treatment period. Dietary intakes of Se, macronutrients and micronutrients were not different between groups and remained unchanged during the treatment period. These results suggest that our previous observation of a hypothyroidal response to high-Se foods was confounded by some aspect of the particular foods used, or were merely chance observations. Because of the high dose and long administration period, the present study suggests that the effects of Se supplements on thyroid hormone metabolism and energy metabolism in healthy North American men with adequate Se status do not represent a significant risk for unhealthy weight gain.     

A conformationally mobile cysteine residue (Cys-561) modulates Na+ and H+ activation of human CNT3

In humans, the SLC28 concentrative nucleoside transporter (CNT) protein family is represented by three Na(+)-coupled members; human CNT1 (hCNT1) and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, whereas hCNT3 transports both purine and pyrimidine nucleosides and nucleoside drugs. Belonging to a phylogenetic CNT subfamily distinct from hCNT1/2, hCNT3 also mediates H(+)/nucleoside cotransport. Using heterologous expression in Xenopus oocytes, we have characterized a cysteineless version of hCNT3 (hCNT3C-). Processed normally to the cell surface, hCNT3C-exhibited hCNT3-like transport properties, but displayed a decrease in apparent affinity specific for Na(+) and not H(+). Site-directed mutagenesis experiments in wild-type and hCNT3C-backgrounds identified intramembranous Cys-561 as the residue responsible for this altered Na(+)-binding phenotype. Alanine at this position restored Na(+) binding affinity, whereas substitution with larger neutral amino acids (threonine, valine, and isoleucine) abolished hCNT3 H(+)-dependent nucleoside transport activity. Independent of these findings, we have established that Cys-561 is located in a mobile region of the hCNT3 translocation pore adjacent to the nucleoside binding pocket and that access of p-chloromercuribenzene sulfonate to this residue reports a specific H(+)-induced conformational state of the protein ( Slugoski, M. D., Ng, A. M. L., Yao, S. Y. M., Smith, K. M., Lin, C. C., Zhang, J., Karpinski, E., Cass, C. E., Baldwin, S. A., and Young, J. D. (2008) J. Biol. Chem. 283, 8496-8507 ). The present investigation validates hCNT3C- as a template for substituted cysteine accessibility method studies of CNTs and reveals a pivotal functional role for Cys-561 in Na(+)- as well as H(+)-coupled modes of hCNT3 nucleoside transport.     

Genetic differences among populations in sexual dimorphism: evidence for selection on males in a dioecious plant

Genetic variation among populations in the degree of sexual dimorphism may be a consequence of selection on one or both sexes. We analysed genetic parameters from crosses involving three populations of the dioecious plant Silene latifolia, which exhibits sexual dimorphism in flower size, to determine whether population differentiation was a result of selection on one or both sexes. We took the novel approach of comparing the ratio of population differentiation of a quantitative trait (Q(ST) ) to that of neutral genetic markers (F(ST) ) for males vs. females. We attributed 72.6% of calyx width variation in males to differences among populations vs. only 6.9% in females. The Q(ST) /F(ST) ratio was 4.2 for males vs. 0.4 for females, suggesting that selection on males is responsible for differentiation among populations in calyx width and its degree of sexual dimorphism. This selection may be indirect via genetic correlations with other morphological and physiological traits.