Localization of phosphatidylethanolamine in microsomal membranes and regulation of its distribution by the fatty acid composition

Rat liver microsomes were incubated with the monofunctional aminoreagent fluorescamine. Although the probe easily penetrated the membranes, two pools of phosphatidylethanolamine (PE) could be detected. The first pool rapidly reacted with the probe and comprised 80% of the total PE. The second pool exhibited a very slow interaction. The two pools showed differences in fatty acid composition as well as in their sites of attachment. In vivo labeling with ethanolamine, glycerol, and palmitic and stearic acid resulted in a higher specific activity in the first pool after 1 hr; equilibration with the second pool took about 3 hr. No equilibration between the pools could be detected under in vitro conditions. In vivo incorporation of labeled fatty acids showed that palmitic and stearic acids were mainly incorporated into phosphatidylethanolamine by de novo synthesis, while linoleic and arachidonic acids were introduced through deacylation-reacylation processes. Injection of liposomes consisting of labeled synthetic phosphatidylethanolamines into the portal vein was followed by uptake by the hepatocytes and incorporation of the lipids into the microsomal membranes. Depending on the fatty acid composition of the injected lipid, one of either of the two pools became labeled. It is suggested that the fatty acid composition of a given phospholipid molecule exerts a signal function directing the lipid to its final intramembranous location.     

Membrane lipid changes in root cells of rape (Brassica napus) as a function of water-deficit stress

Rape seedlings ( Brassica napus L. cv. Brink) were exposed to repeated water-deficit stress. The water-stress program started after 19 days of growth and consisted of three 24 h stress periods interspersed with 24 h rewatering periods. After the third stress period the seedlings were harvested and the membrane lipids of the roots were extracted, isolated and quantified. The stress caused an increased ratio of dry weight roots/shoot. Furthermore, the total amount of acyl lipids as well as phospholipids decreased drastically. However, the relative distribution of individual phospholipids was constant and independent of stress. Free and esterified sterols showed only a small decrease in response to water stress. As a consequence the ratio free sterols/phospholipids increased from 0.07 in the control root cells to 0.15 in the stressed cells. The lipid changes are discussed in relation to membrane activity.     

The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes

The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichyl phosphate on the structure and fluidity of model membranes was studied using 31P NMR, small-angle x-ray scattering, differential scanning calorimetry, and freeze-fracture electron microscopy. These studies suggest that dolichol and dolichol derivatives destabilize unsaturated phosphatidylethanolamine containing bilayer structures and promote hexagonal II phase formation; high concentrations of dolichol induce lipid structures characterized by "isotropic" 31P NMR and particulate fracture faces; dolichol, contrary to cholesterol, has no effect on the thermotropic behavior of membranes consisting of phosphatidylcholine, while dolichyl-P incorporation abolishes the transition from the gel to liquid crystalline phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine; both dolichol and dolichyl-P increase the fatty acid fluidity in phosphatidylethanolamine mixtures; the effect of dolichol on bilayer structure and fluidity is more pronounced with increasing number of isoprene residues; dolichol esters are only soluble to a limited extent in the bilayer and segregates into domains at low concentrations; the results are consistent with a localization of dolichyl-P in which the phosphate group is oriented to the water interphase. The induction of hexagonal II phase by dolichyl-P may elicit the transmembrane movement of glycosylated lipid intermediate.     

Polygyny and nest site selection in the pied flycatcher

Nest site choice, and its influence on mating and breeding success were studied in a population of individually marked pied flycatchers (Ficedula hypoleuca) for 7 years. Some nest boxes were used more frequently than expected from random choice of nest site. Flycatchers bred more successfully in such attractive boxes, irrespective of whether the brood was attended by the male or not. Arriving males and females settled at boxes in descending order of attractiveness. Boxes accepted by females were more attractive than alternative boxes advertised simultaneously by males. Secondary nest boxes of polygynous males tended to be more attractive than boxes at which monogamous males displayed, without success, for secondary mates. Returning males with local breeding experience started to sing at nest boxes earlier in spring than first season males, settled at more attractive boxes, and mated polygynously more often. The results indicate that male mating success should be closely dependent on the male's ability to monopolize attractive nest sites.     

Sterol content, fatty acid composition of phospholipids, and permeability of labeled ethylene glycols in relation to salt-tolerance of yeasts

Two yeasts, the salt-tolerant Debaryomyces hansenii and the non-tolerant Saccharomyces cerevisiae were grown in basal media (4 m M NaCl) and also a high salinities that produced a similar salt stress in the two species in terms of growth rate reduction (i.e., 1.4 M NaCl for S. cerevisae and 2.7 M NaCl for D. hansenii ). A study was made of the sterol content, the fatty acid composition of the phospholipids, and the permeation of a series of tritiated ethylene glycols of graded molecular weights. On the basis of cell dry weight the amount of total and free sterols increased in both species when cultured at high salinity. Irrespective of growth medium salinity, the molar ratio of free sterols to phospholipids was higher in D. hansenii than in S. cerevisiae . Increased salinity produced only minor changes in the fatty acid composition of the phospholipids in D. hansenii , whereas in S. cerevisiae there was a marked decrease of linolenic acid with a concomitant increase of linoleic acid.
In both yeasts there was an energy linked component in the uptake of ethylene glycol, which component could be inhibited by sodium azide and N -ethylmaleimide. The passive permeability for ethylene-, diethylene- and triethylene glycol increased for both species at increased salinity. This increase was more pronounced for S. cerevisiae than for D. hansenii . Polyethylene glycol of M , 200 as well as higher polyethylene glycols appeared to be excluded or very slowly admitted by the yeasts.     

Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells: new insights into the pathways involved

Cytokines released by islet-infiltrating immune cells play a crucial role in beta-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this beta-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 +/- 164 (pH 4-7) and 1641 +/- 73 (pH 6-9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p < or = 0.01) by a combination of interleukin-1beta and interferon-gamma, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e-05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a beta-cell dysfunction/apoptotic beta-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to beta-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of beta-cell loss in type 1 diabetes.     

Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts

In higher plants, photorespiratory Gly oxidation in leaf mitochondria yields ammonium in large amounts. Mitochondrial ammonium must somehow be recovered as glutamate in chloroplasts. As the first step in that recovery, we report glutamine synthetase (GS) activity in highly purified Arabidopsis thaliana mitochondria isolated from light-adapted leaf tissue. Leaf mitochondrial GS activity is further induced in response to either physiological CO(2) limitation or transient darkness. Historically, whether mitochondria are fully competent for oxidative phosphorylation in actively photorespiring leaves has remained uncertain. Here, we report that light-adapted, intact, leaf mitochondria supplied with Gly as sole energy source are fully competent for oxidative phosphorylation. Purified intact mitochondria efficiently use Gly oxidation (as sole energy, NH(3), and CO(2) source) to drive conversion of l-Orn to l-citrulline, an ATP-dependent process. An A. thaliana genome-wide search for nuclear gene(s) encoding mitochondrial GS activity yielded a single candidate, GLN2. Stably transgenic A. thaliana ecotype Columbia plants expressing a p35S::GLN2::green fluorescent protein (GFP) chimeric reporter were constructed. When observed by laser scanning confocal microscopy, leaf mesophyll and epidermal tissue of transgenic plants showed punctate GFP fluorescence that colocalized with mitochondria. In immunoblot experiments, a 41-kD chimeric GLN2::GFP protein was present in both leaf mitochondria and chloroplasts of these stably transgenic plants. Therefore, the GLN2 gene product, heretofore labeled plastidic GS-2, functions in both leaf mitochondria and chloroplasts to faciliate ammonium recovery during photorespiration.     

Mid-Tertiary dispersal, not Gondwanan vicariance explains distribution patterns in the wax palm subfamily (Ceroxyloideae: Arecaceae)

The Ceroxyloideae is a small but heterogeneous subfamily of palms (Arecaceae, Palmae). It includes a Caribbean lineage (tribe Cyclospathae), a southern hemisphere disjunction (tribe Ceroxyleae), and an amphi-Andean element (tribe Phytelepheae), until recently considered a distinct subfamily (Phytelephantoideae) due to its highly derived morphology. A variety of hypotheses have been proposed to account for the biogeography of the subfamily, involving Gondwanan vicariance, austral interplate dispersal from South America to Australia via Antarctica, Andean orogeny, and Pleistocene refuges. We assessed the systematic classification and biogeography of the group based on a densely sampled phylogeny using >5.5kb of DNA sequences from three plastid and two nuclear genomic regions. The subfamily and each of its three tribes were resolved as monophyletic with high support. Divergence time estimates based on penalized likelihood and Bayesian dating methods indicate that Gondwanan vicariance is highly unlikely as an explanation for basic disjunctions in tribe Ceroxyleae. Alternative explanations include a mid-Tertiary trans-Atlantic/trans-African dispersal track and the "lemurian stepping stones" hypothesis. Austral interplate dispersal of Oraniopsis to Australia could have occurred, but apparently only in the mid-Eocene/early Oligocene interval after global cooling had begun. Our data do not support Pleistocene climatic changes as drivers for speciation in the Andean-centered Phytelepheae as previously proposed. Radiation in this tribe coincides largely with the major uplift of the Andes, favoring Andean orogeny over Pleistocene climatic changes as a possible speciation-promoting factor in this tribe.