Ceramide acyl chain length markedly influences miscibility with palmitoyl sphingomyelin in bilayer membranes

Ceramides are precursors of major sphingolipids and can be important cellular effectors. The biological effects of ceramides have been suggested to stem from their biophysical effects on membrane structure affecting the lateral and transbilayer organization of other membrane components. In this study we investigated the effect of acyl chain composition in ceramides (C4-C24:1) on their miscibility with N-palmitoyl-sphingomyelin (PSM) using differential scanning calorimetry. We found that short-chain (C4 and C8) ceramides induced phase separation and lowered the T _m and enthalpy of the PSM endotherm. We conclude that short-chain ceramides were more miscible in the fluid-phase than in the gel-phase PSM bilayers. Long-chain ceramides induced apparent heterogeneity in the bilayers. The main PSM endotherm decreased in cooperativity and enthalpy with increasing ceramide concentration. New ceramide-enriched components could be seen in the thermograms at all ceramide concentrations above X _Cer = 0.05. These broad components had higher T _m values than pure PSM. C24:1 ceramide exhibited complex behavior in the PSM bilayers. The miscibility of C24:1 ceramide with PSM at low (X _Cer = 0.05–0.10) concentrations was exceptionally good according to the cooperativity of the transition. At higher concentrations, multiple components were detected, which might have arisen from interdigitated gel-phases formed by this very asymmetric ceramide. The results of this study indicate that short-chain and long-chain ceramides have very different effects on the sphingomyelin bilayers. There also seems to be a correlation between their miscibility in binary systems and the effect of ceramides of different hydrophobic length on sphingomyelin-rich domains in multicomponent membranes.     

Differences in the domain forming properties of N-palmitoylated neutral glycosphingolipids in bilayer membranes

We have compared the domain forming properties of three neutral acyl chain defined glycosphingolipids differing in their head group structures. The aim of the study was to explore if glycosphingolipids and sterols exist in the same lateral domains in bilayer membranes and how the structure of the head group influences the capacity of the glycosphingolipids to colocalize with cholesterol. The glycosphingolipids used in the study were galactosyl-, glucosyl- and lactosylceramides with a palmitic acid in the N-linked position. Domain formation in mixed bilayer vesicles was examined using fluorescent reporter molecules associating with ordered domains, together with a fluorescence quencher lipid in the disordered membrane phase. Our results show that the glycosphingolipids studied were poor in forming sterol-enriched domains compared to palmitoyl-sphingomyelin as detected by cholestatrienol quenching. However, the tendency to associate with cholesterol was clearly dependent on the carbohydrate structure of the glycosphingolipids, also when two glycosphingolipids with different head groups were mixed in the bilayer. All palmitoylated glycosphingolipids associated with palmitoyl-sphingomyelin/cholesterol domains. Our results show that the head group structures of neutral glycosphingolipids markedly affect their domain forming properties in bilayers both with and without cholesterol. The most striking observation being that large differences in domain forming properties were seen even between glucosylceramide and galactosylceramide, which differ only in the stereochemistry of one hydroxyl group in the carbohydrate head group.     

Cholesterol interactions with fluid-phase phospholipids: effect on the lateral organization of the bilayer

The lateral organization of lipids and proteins in cell membranes is recognized as an important factor in several cellular processes. Cholesterol is thought to function as a modulator of the lateral segregation of lipids into cholesterol-poor and cholesterol-rich domains. We investigated how the affinity of cholesterol for different phospholipids, as seen in cholesterol partitioning between methyl-β-cyclodextrin and large unilamellar vesicles, was reflected in the lateral organization of lipids in complex bilayers. We especially wanted to determine how the low-T_m lipid affected the lateral structure. Partition experiments showed that cholesterol had a higher affinity for N-oleoyl-sphingomyelin (OSM) than for palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers, but the highest preference was for N-palmitoyl-sphingomyelin (PSM)-containing bilayers. Partial phase diagrams of POPC/PSM/cholesterol and OSM/PSM/cholesterol bilayers at 23°C and 37°C were used to gain insight into the lateral organization of lipids in bilayers. Analysis of phase diagrams revealed that the phospholipid composition of cholesterol-poor and cholesterol-rich domains reflected the affinity that cholesterol exhibited toward bilayers composed of different lipids. Therefore, the determined affinity of cholesterol for different phospholipid bilayers was useful in predicting the cholesterol-induced lateral segregation of lipids in complex bilayers.     

Differential expression of cellular microRNAs in HPV-11 transfected cells. An analysis by three different array platforms and qRT-PCR

Human papillomavirus type 11 (HPV-11) infects the genital and the respiratory tract leading to condylomas and respiratory papillomatosis. HPV infections are restricted to epithelial tissue and the progression through the virus lifecycle is tightly coordinated to the differentiation of the host cell. The changes of cellular microRNAs by HPV-11 gene expression were investigated in a cell culture model of HaCaT cells transfected with HPV-11, with the goal of understanding which cellular processes were affected by the virus. Human microRNA profiling was conducted on three different array platform systems and because very few microRNAs (miR-663, -638, -149* and -92b*) were consistently found in all three array data sets we performed extensive statistical analyses of the array data and the qRT-PCR validation. We assume that the most reliable differentially expressed microRNAs are the ones identified by more than one array platform. We also show that TaqMan? qRT-PCR validation is of limited use for less abundant microRNAs.     

Estrogenic effect of propylparaben (propylhydroxybenzoate) in rainbow trout Oncorhynchus mykiss after exposure via food and water

The estrogenic effect of propylparaben was investigated in a rainbow trout Oncorhynchus mykiss test system. Propylparaben was administered orally to sexually immature rainbow trout every second day for up to 10 days in doses between 7 and 1830 mg kg(-1) 2 d(-1) and in the water at 50 and 225 microg l(-1) for 12 days. Plasma vitellogenin was measured before and during the exposures and the concentrations of propylparaben in liver and muscle were determined at the end of experiments. Increases in average plasma vitellogenin levels were seen at oral exposure to 33 mg propylparabenkg(-1) 2 d(-1); the most sensitive fish responded to 7 mg kg(-1). The ED(50) values for increase in vitellogenin synthesis were 35, 31 and 22 mg kg(-1) 2 d(-1) at day 3, 6 and 11, respectively. Exposure to 225 microg propylparabenl(-1) increased vitellogenin synthesis, but exposure to 50 microg l(-1) did not. Propylparaben showed little tendency to bioaccumulation in rainbow trout; less than 1 per thousand of the total amount of propylparaben administered orally at 1830 mg kg(-1) 2 d(-1) over the 10-d experimental period was retained in muscle and liver 24 h after the end of the experiment. Exposure to 225 microg propylparabenl(-1) for 12 d led to concentrations of 6700 and 870 microg propylparabenkg(-1) liver and muscle, respectively. Half lives for propylparaben were 8.6 h in liver and 1.5 h in muscle.     

"Ménage à trois": the presence/absence of thyme shapes the mutualistic interaction between the host plant Medicago truncatula (Fabaceae) and its symbiotic bacterium Sinorhizobium meliloti

The long-term maintenance of specialized mutualisms remains an evolutionary puzzle. Recent focus has been on factors governing the stability of these mutualisms, including sanctions by the host, partner choice, and coevolutionary constraint, that is, the genetic correlation (r(G)) between fitness of both partners. So far these studies have been typically carried out in a single environment. Here, we ask if the genetic correlation between fitness of the host plant Medicago truncatula (Fabaceae) and its bacterial symbiont Sinorhizobium meliloti is affected by the presence/absence of a monoterpene (carvacrol) leached into the soil by Thymus vulgaris-a common plant of the Mediterranean vegetation, often co-occuring with Medicago. We show that the presence of carvacrol in the soil dramatically affects fitness of the rhizobial partner and increases the magnitude of r(G) between plant and rhizobia fitness (r(G) = 0.02 ± 0.05 vs. r(G) = 0.57 ± 0.02). This finding emphasizes the importance of heterogeneity in the biotic environment for understanding the evolution of species interactions.     

Low-dose inhalation of an endothelin-A receptor antagonist in experimental acute lung injury: ET-1 plasma concentration and pulmonary inflammation

Inhalation of endothelin (ET)-A receptor antagonists has been shown to improve gas exchange in experimental acute lung injury (ALI) but may induce side effects by increasing circulating ET-1 levels. We investigated whether the inhaled ET(A) receptor antagonist, LU-135252, at low doses, improves gas exchange without affecting ET-1 plasma concentrations and lung injury in an animal model of ALI. Twenty-two piglets were examined in a prospective, randomized, controlled study. In anesthetized animals, ALI was induced by surfactant depletion. Animals received either LU-135252 at a dose of 0.3 mg/kg during 20 mins (LU group; n = 11), or nebulization of saline buffer (control group; n = 11). The Mann-Whitney U test was used to compare groups (P < 0.05). In the LU group, arterial partial pressure of oxygen (PaO2) and mean pulmonary artery pressure (MPAP) improved compared with the control group (PaO2, 319 +/- 44 mm Hg vs. 57 +/- 3 mm Hg; MPAP, 32 +/- 2 mm Hg vs. 41 +/- 2 mm Hg; values at 6 hrs after induction of ALI; P < 0.05). Mean arterial pressure and cardiac output were not different between groups. ET-1 plasma concentrations increased from 0.96 +/- 0.06 fmol/ml after induction of ALI to a maximum of 1.17 +/- 0.09 fmol/ml at 3 hrs after ALI onset in the LU group and did not differ significantly from the control group (1.21 +/- 0.08 fmol/ml, not significant). On histologic examination, we found no differences in total lung injury score between groups. However, the LU group revealed significantly reduced interstitial inflammation and hemorrhage (P < 0.05 vs. control group). In this animal model of ALI, inhalation of LU-135252 at a dose of 0.3 mg/kg induced a significant and sustained improvement in gas exchange, whereas there were no changes in ET-1 plasma concentrations. Furthermore, our data indicate a trend toward decreased pulmonary inflammation in the group receiving the inhaled ET(A) receptor antagonist.     

Cyanogenic glycosides: a case study for evolution and application of cytochromes P450

Cyanogenic glycosides are ancient biomolecules found in more than 2,650 higher plant species as well as in a few arthropod species. Cyanogenic glycosides are amino acid-derived β-glycosides of α-hydroxynitriles. In analogy to cyanogenic plants, cyanogenic arthropods may use cyanogenic glycosides as defence compounds. Many of these arthropod species have been shown to de novo synthesize cyanogenic glycosides by biochemical pathways that involve identical intermediates to those known from plants, while the ability to sequester cyanogenic glycosides appears to be restricted to Lepidopteran species. In plants, two atypical multifunctional cytochromes P450 and a soluble family 1 glycosyltransferase form a metabolon to facilitate channelling of the otherwise toxic and reactive intermediates to the end product in the pathway, the cyanogenic glycoside. The glucosinolate pathway present in Brassicales and the pathway for cyanoalk(en)yl glucoside synthesis such as rhodiocyanosides A and D in Lotus japonicus exemplify how cytochromes P450 in the course of evolution may be recruited for novel pathways. The use of metabolic engineering using cytochromes P450 involved in biosynthesis of cyanogenic glycosides allows for the generation of acyanogenic cassava plants or cyanogenic Arabidopsis thaliana plants as well as L. japonicus and A. thaliana plants with altered cyanogenic, cyanoalkenyl or glucosinolate profiles.