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.     

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.     

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.     

Toll-like receptor 3 and STAT-1 contribute to double-stranded RNA+ interferon-gamma-induced apoptosis in primary pancreatic beta-cells

Viral infections and local production of cytokines probably contribute to the pathogenesis of Type 1 diabetes. The viral replicative intermediate double-stranded RNA (dsRNA, tested in the form of polyinosinic-polycytidylic acid, PIC), in combination with the cytokine interferon-gamma (IFN-gamma), triggers beta-cell apoptosis. We have previously observed by microarray analysis that PIC induces expression of several mRNAs encoding for genes downstream of Toll-like receptor 3 (TLR3) signaling pathway. In this report, we show that exposure of beta-cells to dsRNA in combination with IFN-alpha, -beta, or -gamma significantly increases apoptosis. Moreover, dsRNA induces TLR3 mRNA expression and activates NF-kappaB and the IFN-beta promoter in a TRIF-dependent manner. dsRNA also induces an early (1 h) and sustained increase in IFN-beta mRNA expression, and blocking IFN-beta with a specific antibody partially prevents PIC plus IFN-gamma-induced beta-cell death. On the other hand, dsRNA plus IFN-gamma does not induce apoptosis in INS-1E cells, and expression of TLR3 and type I IFNs mRNAs is not detected in these cells. Of note, disruption of the STAT-1 signaling pathway protects beta-cells against dsRNA plus IFN-gamma-induced beta-cell apoptosis. This study suggests that dsRNA plus IFN-gamma triggers beta-cell apoptosis by two complementary pathways, namely TLR3-TRIF-NF-kappaB and STAT-1.     

Hybridization and genetic variation in Danish populations of European crab apple (Malus sylvestris)

The aim of the present study was to investigate the genetic variation in Danish populations of the endangered European crab apple (Malus sylvestris). Special emphasis was given to hybridization between the wild species and its cultivated relative Malus ×domestica. A total of 178 wild individuals from four Danish populations were studied along with a reference sample of 29 old cultivars. The genetic variation within and among samples was studied at ten microsatellite marker loci. Additionally, a morphological analysis was carried out to identify hybrids and test for correspondence between phenotypic and genotypic indices of hybridization. From application of ordination and a model-based cluster analysis to the molecular data, two clusters were identified consisting of wild and cultivated individuals, respectively. This indicates that pronounced admixture between the two species is not present. At the population level, a high correspondence was found between geographic isolation from M. ×domestica and genotypic and morphological indices of hybridization. As expected, isolated populations appeared less affected by hybridization than poorly isolated populations. Isolated “pure” M. sylvestris populations could thus be identified. However, morphological and molecular evidences of hybridization were found to be divergent at the individual level. This is suggestive of some historical introgression into the M. sylvestris gene pool and indicates that relying exclusively on either morphological or molecular characters as diagnostic markers in studies of hybridization between M. ×domestica and M. sylvestris might lead to fallible results. Combined application of genetic and morphological markers is therefore recommended.     

Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.