Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.     

Mit den Waffen der Chemie gegen Insekten

Current industrialised farming applies multiple pesticides to fight against competitors of agricultural products. The consequences lead to direct and indirect detrimental effects on the environment. Neonicotinoids are particularly frequently applied to fight against pest insects but pollinating insects are also effected. These insecticides bind to the nicotinic acetylcholine receptors in the insect brain and interfere with neuronal processed underlying learning, memory formation and memory retrieval. Experience dependent behaviors are essential for effective pollination. Experimental studies with honeybees documented that even extremely small doses of neonicotinoids taken up with nectar and pollen massively interfere with learning, memory, navigation, social communication and foraging motivation. Besides these direct actions on non‐target insects neonicotinoids impoverish the environment, reduce the number of wild life and destroy the quality of the agricultural ground. The multiple effects are caused by the high water solubility and the persistence of the insecticides.     

Electrospray mass spectrometric investigation of the relative ligand properties of EPh3 ligands (E=P, As, Sb or Bi) towards Ag and Cu

Electrospray mass spectrometry (ESMS) has been used to investigate the relative ligand properties of the triphenylpnictogen ligands EPh₃ (E=P, As, Sb and Bi) towards silver(I) and copper(I) ions. It is found that the preferred species formed increase in coordination number from two for PPh₃ in [Ag(PPh₃)₂]⁺ to four for SbPh₃ in [Ag(SbPh₃)₄]⁺, consistent with the decreasing donor ligand ability and increasing metal –E bond length in the series PPh₃–AsPh₃–SbPh₃. With BiPh_3, the spectra were complex, suggesting considerable decomposition. These studies also suggest that silver(I) and copper(I) ions will have widespread utility in the characterisation of tertiary stibine ligands, as has been described previously for phosphines and arsines. These studies demonstrate the power of the ESMS technique in determining the donor properties of a related series of ligands, and this information is of significance in coordination chemistry.     

The intramammary inflammatory response of genetically resistant Merino ewes infected with Haemonchus contortus.

The mammary glands of 103 pasture-reared non-lactating, non-pregnant Merino ewes were infused via the teat canal with antigens prepared from the nematode Haemonchus contortus, and the inflammatory response to infusion assessed by washing the gland of its contents after 24 h and 14 days. The ewes were of two genotypes: one with proven high levels of resistance to infection with the nematode H. contortus, the other random-bred animals with relative susceptibility to infection. On day 0 of a H. contortus infection, one gland of the subgroups of both genotypes was infused with the antigen preparation. At the same time, the other gland of the random-bred ewes was infused with sterile physiological saline. A third group of infected random-bred ewes was infused with only sterile physiological saline. Similar infusions were performed on other subgroups on days 12, 21 and 35 of infection, which was then terminated with anthelmintic. A fourth group of uninfected random-bred control ewes was given both infusions 35 days after the other groups were infected. Sheep of the resistant genotype had lower worm egg counts and smaller reductions in blood packed cell volumes from day 21 of infection. Infusion of antigen had no effect on the course of infection and no effect on the response of the other gland, which had been infused with saline alone. The dominant leukocyte response from the antigen-infused gland was eosinophilia. On all days of infusion, and after both 24 h and 14 days, eosinophil counts from the resistant genotype were higher than those from their random-bred counterparts. The sheep mammary gland provides a source of eosinophils whose number is related to host genotype and stage of infection and may provide a model for the investigation of cellular responses in mucosal immunity to nematode infections.     

Inhibitory action of orotate, 2-thioorotate and isoorotate on nucleotide metabolism and nucleic acid synthesis in hepatoma cells.

1. The specificity of the action of orotate on hepatoma cells was investigated. 2. Orotic acid and its methyl ester had similar inhibitory effects on the incorporation of [3H]thymidine into DNA of hepatoma cells. 3. In contrast to previous studies in vivo, incubation of rat kidney cells with orotate caused an increase in the ratio of UTP/ATP concentrations that was similar to effects on hepatic cells. 4. Inhibitory effects of 2-thioorotate and isoorotate on metabolism were found to be less selective and required higher concentrations than with orotate.     

Regulation of calcium homeostasis and flux between the endoplasmic reticulum and the cytosol

The concentration of Ca²⁺ in the endoplasmic reticulum (ER) is critically important for maintaining its oxidizing environment as well as for maintaining luminal ATP levels required for chaperone activity. Therefore, local luminal Ca²⁺ concentrations and the dynamic Ca²⁺ flux between the different subcellular compartments are tightly controlled. Influx of Ca²⁺ into the ER is enabled by a reductive shift, which opens the sarcoendoplasmic reticulum calcium transport ATPase pump, building the Ca²⁺ gradient across the ER membrane required for ATP import. Meanwhile, Ca²⁺ leakage from the ER has been reported to occur via the Sec61 translocon following protein translocation. In this review, we provide an overview of the complex regulation of Ca²⁺ homeostasis, Ca²⁺ flux between subcellular compartments, and the cellular stress response (the unfolded protein response) induced upon dysregulated luminal Ca²⁺ metabolism. We also provide insight into the structure and gating mechanism at the Sec61 translocon and examine the role of ER-resident cochaperones in assisting the central ER-resident chaperone BiP in the control of luminal Ca²⁺ concentrations.     

Photorespiratory metabolism of glyoxylate and formate in glycine-accumulating mutants of barley and Amaranthus edulis

Glycine-accumulating mutants of barley (Hordeum vulgare L.) and Amaranthus edulis (Speg.), which lack the ability to decarboxylate glycine by glycine decarboxylase (GDC; EC 2.1.2.10), were used to study the significance of an alternative photorespiratory pathway of serine formation. In the normal photorespiratory pathway, 5,10-methylenetetrahydrofolate is formed in the reaction catalysed by GDC and transferred to serine by serine hydroxymethyltransferase. In an alternative pathway, glyoxylate could be decarboxylated to formate and formate could be converted into 5,10-methylenetetrahydrofolate in the C1-tetrahydrofolate synthase pathway. In contrast to wild-type plants, the mutants showed a light-dependent accumulation of glyoxylate and formate, which was suppressed by elevated (0.7%) CO₂ concentrations. After growth in air, the activity and amount of 10-formyltetrahydrofolate synthetase (FTHF synthetase; EC 6.3.4.4), the first enzyme of the conversion of formate into 5,10-methylenetetrahydrofolate, were increased in the mutants compared to the wild types. A similar increase in FTHF synthetase could be induced by incubating leaves of wild-type plants with glycine under illumination, but not in the dark. Experiments with ¹⁴C showed that the barley mutants incorporated [¹⁴C]formate and [2-¹⁴C]glycollate into serine. Together, the accumulation of glyoxylate and formate under photorespiratory conditions, the increase in FTHF synthetase and the ability to utilise formate and glycollate for the formation of serine indicate that the mutants are able partially to compensate for the lack of GDC activity by bypassing the normal photorespiratory pathway. Received: 14 August 1998 / Accepted: 30 September 1998     

Molecular dynamics simulation of α-melanocyte stimulating hormone in a water-membrane model interface

The conformation of the tridecapeptide α-melanocyte stimulating hormone in the presence of a double water-membrane interface was studied by molecular dynamics simulation, using the computational package THOR. In this program the solvent is represented by a continuous medium with dielectric constant ɛ, and the interface between different media is simulated by a surface of discontinuity of the dielectric constant. The electrostatic image method was used to write down the terms, added to the force field, that describe the polarisation effects induced in the interface by the atomic charges. The program was further improved by the introduction of a second surface, parallel to the first one, to mimic the membrane. A conformational search using the software Prelude was employed to find an initial geometry for the peptide in water. The molecular dynamics simulation performed during 10 ns showed that the peptide structure is flexible in water, without stabilisation of any preferential conformation. In the presence of the model membrane, the peptide moved to the medium representing the interior of the membrane. Inside the low dielectric constant medium, the structure of the peptide showed a turn in the central sequence of amino acids and a packed conformation remained stabilised during more than 7.0 ns of simulation. Received: 27 November 1998 / Revised version: 11 March 1999 / Accepted: 8 April 1999