Lipid-destabilizing properties of the hydrophobic helices H8 and H9 from colicin E1

Colicins are toxic proteins produced by Escherichia coli that must cross the membrane to exert their activity. The lipid insertion of their pf domain is linked to a conformational change which enables the penetration of a hydrophobic hairpin. They provide useful models to more generally study insertion of proteins, channel formation and protein translocation in and across membranes. In this paper, we study the lipid-destabilizing properties of helices H8 and H9 forming the hydrophobic hairpin of colicin E1. Modelling analysis suggests that those fragments behave like tilted peptides. The latter are characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. Fluorescence techniques using labelled liposomes clearly show that H9, and H8 to a lesser extent, destabilize lipid particles, by inducing fusion and leakage. AFM assays clearly indicate that H8 and especially H9 induce membrane fragilization. Holes in the membrane are even observed in the presence of H9. This behaviour is close to what is seen with viral fusion peptides. Those results suggest that the peptides could be involved in the toroidal pore formation of colicin E1, notably by disturbing the lipids and facilitating the insertion of the other, more hydrophilic, helices that will form the pore. Since tilted, lipid-destabilizing fragments are also common to membrane proteins and to signal sequences, we suggest that tilted peptides should have an ubiquitous role in the mechanism of insertion of proteins into membranes.     

Short-Term Flooding Effects on Gas Exchange and Quantum Yield of Rabbiteye Blueberry (Vaccinium ashei Reade)

Roots of 1.5-year-old `Woodard' rabbiteye blueberry plants (Vaccinium ashei Reade) were flooded in containers or maintained at container capacity over a 5-day period. Carbon assimilation, and stomatal and residual conductances were monitored on one fully expanded shoot/plant using an open flow gas analysis system. Quantum yield was calculated from light response curves. Carbon assimilation and quantum yield of flooded plants decreased to 64 and 41% of control values, respectively, after 1 day of flooding and continued decreasing to 38 and 27% after 4 days. Stomatal and residual conductances to CO₂ also decreased after 1 day of flooding compared with those of unflooded plants with residual conductance severely limiting carbon assimilation after 4 days of flooding. Stomatal opening occurred in 75 to 90 minutes and rate of opening was unaffected by flooding.     

Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.     

Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch

Genetic linkage maps have been increasingly developed for a wide variety of plants, using segregating populations such as F₂s or backcrosses between inbred lines. These pedigrees are rarely available in outbred species like forest trees which have long generation times. Thus genetic mapping studies have to use peculiar pedigrees and markers in appropriate configurations. We constructed single-tree genetic linkage maps of European larch (Larix decidua Mill.) and Japanese larch [Larix kaempferi (Lamb.) Carr.] using segregation data from 112 progeny individuals of an hybrid family. A total of 266 markers (114 AFLP, 149 RAPD and 3 ISSR loci) showing a testcross configuration, i.e.heterozygous in one parent and null in the other parent, were grouped at LOD 4.0, θ=0.3. The maternal parent map (L. decidua)consisted of 117 markers partitioned within 17 linkage groups (1152 cM) and the paternal parent map (L. kaempferi) had 125 markers assembled into 21 linkage groups (1206 cM). The map distance covered by markers was determined by adding a 34.7-cM independence distance at the end of each group and unlinked marker. It reached 2537 cM and 2997 cM respectively for European larch and Japanese larch, and represented respectively a 79.6% and 80.8% coverage of the overall genome. A few 3:1 segregating markers were used to identify homologous linkage groups between the European larch and the Japanese larch genetic maps. The PCR-based molecular markers allowed the construction of genetic maps, thus ensuring a good coverage of the larch genome for further QTL detection and mapping studies. Received: 15 March 1999 / Accepted: 29 March 1999     

Lipid-destabilizing properties of the hydrophobic helices H8 and H9 from colicin E1

Colicins are toxic proteins produced by Escherichia coli that must cross the membrane to exert their activity. The lipid insertion of their pf domain is linked to a conformational change which enables the penetration of a hydrophobic hairpin. They provide useful models to more generally study insertion of proteins, channel formation and protein translocation in and across membranes. In this paper, we study the lipid-destabilizing properties of helices H8 and H9 forming the hydrophobic hairpin of colicin E1. Modelling analysis suggests that those fragments behave like tilted peptides. The latter are characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. Fluorescence techniques using labelled liposomes clearly show that H9, and H8 to a lesser extent, destabilize lipid particles, by inducing fusion and leakage. AFM assays clearly indicate that H8 and especially H9 induce membrane fragilization. Holes in the membrane are even observed in the presence of H9. This behaviour is close to what is seen with viral fusion peptides. Those results suggest that the peptides could be involved in the toroidal pore formation of colicin E1, notably by disturbing the lipids and facilitating the insertion of the other, more hydrophilic, helices that will form the pore. Since tilted, lipid-destabilizing fragments are also common to membrane proteins and to signal sequences, we suggest that tilted peptides should have an ubiquitous role in the mechanism of insertion of proteins into membranes.     

Degradation of the apical sodium-dependent bile acid transporter by the ubiquitin-proteasome pathway in cholangiocytes

To attenuate injury during cholestasis, adaptive changes in bile acid transporter expression in the liver provide alternative bile acid excretory pathways. Apical sodium-dependent bile acid transporter (ASBT) (SLC10A2), only expressed in the liver on the cholangiocyte apical membrane, is rapidly regulated in response to inflammation and bile acids. Here, we studied the mechanisms controlling ASBT protein levels in cholangiocytes to determine whether ASBT expression is regulated by ubiquitination and disposal through the proteasome. Protein turnover assays demonstrated that ASBT is an unstable and short-lived protein. Treatment with MG-132, a proteasome inhibitor, causes time-dependent increased ASBT levels and increased intracellular accumulation of ASBT. In cells cotransfected with green fluorescent protein-tagged ASBT and hemagglutinin-tagged ubiquitin, we demonstrated coimmunoprecipitation and colocalization of ASBT and ubiquitin. Interleukin-1beta (IL-1beta) induced down-regulation of ASBT is abrogated by a JNK inhibitor and is accompanied by an increase in ASBT polyubiquitin conjugates and a reduced ASBT half-life. In phosphorylation-deficient S335A and T339A mutants, the ASBT half-life is markedly prolonged, IL-1beta-induced ASBT ubiquitination is significantly reduced, and IL-1beta fails to increase ASBT turnover. These results indicate that ASBT undergoes ubiquitin-proteasome degradation under basal conditions and that ASBT proteasome disposal is increased by IL-1beta due to JNK-regulated serine/threonine phosphorylation of ASBT protein at both Ser-335 and Thr-339. These studies are the first report of regulation of a bile acid transporter expression by the ubiquitin-proteasome pathway.     

Trajectories of Self-Rated Health in People with Diabetes: Associations with Functioning in a Prospective Community Sample

Background

Self-rated health (SRH) is a single-item measure that is one of the most widely used measures of general health in population health research. Relatively little is known about changes and the trajectories of SRH in people with chronic medical conditions. The aims of the present study were to identify and describe longitudinal trajectories of self-rated health (SRH) status in people with diabetes.

Methods

A prospective community study was carried out between 2008 and 2011. SRH was assessed at baseline and yearly at follow-ups (n=1288). Analysis was carried out through trajectory modeling. The trajectory groups were subsequently compared at 4 years follow-up with respect to functioning.

Results

Four distinct trajectories of SRH were identified: 1) 72.2% of the participants were assigned to a persistently good SRH trajectory; 2) 10.1% were assigned to a persistently poor SRH trajectory; 3) mean SRH scores changed from good to poor for one group (7.3%); while 4) mean SRH scores changed from poor to medium/good for another group (10.4%). Those with a persistently poor perception of health status were at higher risk for poor functioning at 4 years follow-up than those whose SRH scores decreased from good to poor.

Conclusions

SRH is an important predictor for poor functioning in diabetes, but the trajectory of SRH seems to be even more important. Health professionals should pay attention to not only SRH per se, but also changes in SRH over time.     

Stimulation of the locus coeruleus elicits noradrenaline and dopamine release in the medial prefrontal and parietal cortex

Our previous studies have suggested that dopamine and noradrenaline may be coreleased from noradrenergic nerve terminals in the cerebral cortex. To further clarify this issue, the effect of electrical stimulation of the locus coeruleus on extracellular noradrenaline, dopamine and DOPAC in the medial prefrontal cortex, parietal cortex and caudate nucleus was analysed by microdialysis in freely moving rats. Stimulation of the locus coeruleus for 20 min with evenly spaced pulses at 1 Hz failed to modify cortical catecholamines and DOPAC levels. Stimulation with bursts of pulses at 12 and 24 Hz increased, in a frequency-related manner, not only noradrenaline but also dopamine and DOPAC in the two cortices. In both cortices noradrenaline returned to baseline within 20 min of stimulation, irrespective of the stimulation frequency, whereas dopamine returned to normal within 20 and 60 min in the medial prefrontal cortex and within 60 and 80 min in the parietal cortex after 12 and 24 Hz stimulation, respectively. DOPAC remained elevated throughout the experimental period. Phasic stimulation of the locus coeruleus at 12 Hz increased noradrenaline in the caudate nucleus as in the cerebral cortices but was totally ineffective on dopamine and DOPAC. Tetrodotoxin perfusion into the medial prefrontal cortex dramatically reduced noradrenaline and dopamine levels and suppressed the effect of electrical stimulation. These results indicate that electrical stimulation-induced increase of dopamine is a nerve impulse exocytotic process and suggest that cortical dopamine and noradrenaline may be coreleased from noradrenergic terminals.     

Flooding, gas exchange and hydraulic root conductivity of highbush blueberry

Highbush blueberry plants ( Vaccinium corymbosum L. cv. Bluecrop) growing in containers were flooded in the laboratory for various durations to determine the effect of flooding on carbon assimilation, photosynthetic response to varying CO₂ and O₂ concentrations and apparent quantum yield as measured in an open flow gas analysis system. Hydraulic conductivity of the root was also measured using a pressure chamber. Root conductivity was lower and the effect of increasing CO₂ levels on carbon assimilation less for flooded than unflooded plants after short-(i-2 days), intermediate-(10–14 days) and long-term (35–40 days) flooding. A reduction in O₂ levels surrounding the leaves from 21 to 2% for unflooded plants increased carbon assimilation by 33% and carboxylation efficiency from 0.012 to 0.021 mol CO₂ fixed (mol CO₂)⁻¹. Carboxylation efficiency of flooded plants, however, was unaffected by a decrease in percentage O₂, averaging 0.005 mol CO₂ fixed (mol CO₂)⁻¹. Apparent quantum yield decreased from 2.2 × 10⁻¹ mol of CO₂ fixed (mol light)⁻¹ for unflooded plants to 2.0 × 10⁻³ and 9.0 × 10⁻⁴ for intermediate- and long-term flooding durations, respectively. Shortterm flooding reduced carbon assimilation via a decrease in stomatal conductance, while longer flooding durations also decreased the carboxylation efficiency of the leaf.     

Maternal stress alters endocrine function of the feto-placental unit in rats

Prenatal stress (PS) can cause early and long-term developmental effects resulting in part from altered maternal and/or fetal glucocorticoid exposure. The aim of the present study was to assess the impact of chronic restraint stress during late gestation on feto-placental unit physiology and function in embryonic (E) day 21 male rat fetuses. Chronic stress decreased body weight gain and food intake of the dams and increased their adrenal weight. In the placenta of PS rats, the expression of glucose transporter type 1 (GLUT1) was decreased, whereas GLUT3 and GLUT4 were slightly increased. Moreover, placental expression and activity of the glucocorticoid "barrier" enzyme 11beta-hydroxysteroid dehydrogenase type 2 was strongly reduced. At E21, PS fetuses exhibited decreased body, adrenal pancreas, and testis weights. These alterations were associated with reduced pancreatic beta-cell mass, plasma levels of glucose, growth hormone, and ACTH, whereas corticosterone, insulin, IGF-1, and CBG levels were unaffected. These data emphasize the impact of PS on both fetal growth and endocrine function as well as on placental physiology, suggesting that PS could program processes implied in adult biology and pathophysiology.