Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Abeta(1-40) peptide in solid-supported lipid bilayers

We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.     

Selectivity, cooperativity, and reciprocity in the interactions between the delta-opioid receptor, its ligands, and G-proteins

A better understanding of signal transduction mechanisms is of critical importance. Methodologies that allow studies to be done while receptors are incorporated into lipid bilayers are advantageous. One such technique is plasmon-waveguide resonance (PWR) spectroscopy, which can follow changes in conformation accompanying protein-ligand, protein-protein, and protein-lipid interactions occurring in G-protein-coupled receptors in real time with high sensitivity and without the need for molecular labeling. Here we investigated several aspects of human delta-opioid receptor (hDOR)-G-protein interactions: 1) the effect of different types of agonists on the interaction with individual G-protein subtypes; 2) the affinities of the separate G-protein alpha and betagamma subunits to different ligand-occupied states of the receptor; and 3) the effect of the presence of the G-protein on the interactions of the ligand with the receptor. To accomplish this we have incorporated the receptor into a solid supported lipid bilayer in the presence of ligand or G-protein and monitored the PWR spectral changes induced by the reciprocal G-protein or ligand interactions. We found a high degree of selectivity in the interactions of different agonist-bound states of the receptor with the different G-protein subtypes. This has important implications for agonist-directed trafficking and selective drug design. Studies with the separated alpha and betagamma subunits show that cooperativity exists in these interactions. The high affinities of the separated subunits to the receptor point to the possibility of independent promotion of specific signaling events. The presence of G-proteins increased the affinity of agonists to the hDOR, and caused faster binding kinetics and different ligand-induced conformational changes. Because ligand also influences G-protein binding, reciprocity exists between these two binding processes.     

Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor

Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that can be used to probe the molecular interactions occurring within anisotropic proteolipid membranes in real time without requiring molecular labeling. This method directly monitors mass density, conformation, and molecular orientation changes occurring in such systems and allows determination of protein-ligand binding constants and binding kinetics. In the present study, PWR has been used to monitor the incorporation of the human beta(2)-adrenergic receptor into a solid-supported egg phosphatidylcholine lipid bilayer and to follow the binding of full agonists (isoproterenol, epinephrine), a partial agonist (dobutamine), an antagonist (alprenolol), and an inverse agonist (ICI-118,551) to the receptor. The combination of differences in binding kinetics and the PWR spectral changes point to the occurrence of multiple conformations that are characteristic of the type of ligand, reflecting differences in the receptor structural states produced by the binding process. These results provide new evidence for the conformational heterogeneity of the liganded states formed by the beta(2)-adrenergic receptor.     

Plasmon-waveguide resonance and impedance spectroscopy studies of the interaction between penetratin and supported lipid bilayer membranes

The interaction between the cell-penetrating peptide, penetratin, and solid-supported lipid bilayer membranes consisting of either egg phosphatidylcholine (PC) or a 75/25 mol% mixture of egg PC and palmitoyloleylphosphatidylglycerol has been studied by simultaneously measuring plasmon-waveguide resonance (PWR) spectra and impedance spectra of lipid-peptide mixtures. When penetratin was incorporated into an egg PC + palmitoyloleylphosphatidylglycerol bilayer, PWR measurements showed a hyperbolic increase in the average refractive index and the refractive index anisotropy, with no change in membrane thickness, over a concentration range between 0 and 2 micro M peptide. In the case of an egg PC bilayer, a biphasic dependence was observed, with a decrease in average refractive index and anisotropy and no thickness change occurring between 0 and 5 micro M peptide, and an increase in membrane thickness occurring between 5 and 15 micro M peptide with no further change in the refractive index parameters. For both membranes, the impedance spectroscopy measurements demonstrated that the electrical resistance was not altered by peptide incorporation, whereas a decrease in membrane capacitance occurred with the same concentration dependence as observed in the PWR experiments, although for the PC membrane no further changes in electrical properties were observed in the higher concentration range. A structural interpretation of these results is described, in which the peptide binds electrostatically within the headgroup region of the bilayer and influences the headgroup conformation, amount of bound water, and the lipid-packing density, without perturbing the hydrocarbon core of the bilayer.     

Plasmon-waveguide resonance spectroscopy: a new tool for investigating signal transduction by G-protein coupled receptors

Plasmon-waveguide resonance (PWR) spectroscopy provides a highly sensitive method for characterizing the kinetics, affinities and conformational changes involved in ligand binding to G-protein coupled receptors, without the need for radioactive or other labeling strategies. In the case of the cloned delta-opioid receptor from human brain incorporated into a lipid bilayer, we have shown that affinities determined in this way are consistent with those measured by standard binding procedures using membranes or whole cells containing the receptors, and that the spectral and kinetic properties of the binding processes allow facile distinction between agonist, inverse agonist, and antagonist ligands. We have also shown by direct measurements that G-protein binding affinities and the ability to undergo GTP/GDP exchange are dependent upon the type of ligand pre-bound to the receptor. PWR spectroscopy thus provides a powerful new approach to investigating signal transduction in biological membrane systems.     

Protein-DNA interaction and CpG methylation at rep*/vIL-10p of latent Epstein-Barr virus genomes in lymphoid cell lines.

The viral interleukin-10 promoter (vIL-10p), overlapping the rep* element in the Epstein-Barr virus (EBV) genome, is a promoter element active mostly in the late phase of the lytic cycle and immediately upon infection of B cells. rep* was, through transfection experiments with small plasmids, characterised as a cis element supporting oriP replicative function. In this study, in vivo protein binding and CpG methylation at rep*/vIL-10p were analysed in five cell lines that harbour strictly latent EBV genomes. Contrary to the invariably unmethylated dyad symmetry element (DS) of oriP, rep*/vIL-10p was highly methylated and showed only traces of protein binding in all examined cell lines. This result is in agreement with vIL-10p being an inactive promoter of EBV genomes, and makes it less likely that rep* functions as a replicative element of latent EBV genomes.     

To the problem of biologically active conformation of enkephalin

Summary A semi-rigid structural analog of [Leu⁵] enkephalin, possessing the azo-bridge between Tyr¹ and Phe⁴ residues, was synthesized, along with two other linear enkephalin analogs: [4′-amino Phe⁴] enkephalin and [4′hydroxyphenyl/-azo Phe⁴] enkephalin. The results of the determination of the analgesic activity of the synthesized compounds suggest that the biologically active conformation of the enkephalin molecule should be such that both aromatic rings, Tyr¹ and Phe⁴, are situated in close proximity.     

Fertility test of frozen boar semen.

The fertility results of two experiments are presented. In experiment 1, the semen was frozen in tris-fructose-EDTA or BF3 diluents at 0-25 X 10(9)/ml sperm concentration and extended after thawing with either seminal plasma (SP) or the freezing medium (FM) containing no cryoprotective agent. In the second experiment the semen was glycerolated by two methods, frozen at 1-0 X 10(9)/ml sperm concentration, and extended wtih FM before insemination. Fertility after double insemination within one oestrus with semen frozen in tris-fructose-EDTA or BF3 diluents varied depending on the medium used for extension of thawed semen. The farrowing rates for semen frozen in the former diluent with FM and SP post-thawing media were 4/8 and 1/8 respectively, and for semen frozen BF3 diluent with FM and SP post-thawing extenders 1/8 and 5/8. The mean farrowing for the 32 animals inseminasted was 34-4%. Pregnancies for semen frozen in tris-fructose-EDTA and glycerolated at 30 or 5 degrees C were 5/12 and 4/12 respectively, and for single and double inseminations 6/12 and 3/12 respectively. Of 24 animals inseminated 37-5% farrowed.     

Circulating miR-184 is a potential predictive biomarker of cardiac damage in Anderson–Fabry disease

Enzyme replacement therapy (ERT) is a mainstay of treatment for Anderson–Fabry disease (AFD), a pathology with negative effects on the heart and kidneys. However, no reliable biomarkers are available to monitor its efficacy. Therefore, we tested a panel of four microRNAs linked with cardiac and renal damage in order to identify a novel biomarker associated with AFD and modulated by ERT. To this end, 60 patients with a definite diagnosis of AFD and on chronic ERT, and 29 age- and sex-matched healthy individuals, were enrolled by two Italian university hospitals. Only miR-184 met both conditions: its level discriminated untreated AFD patients from healthy individuals (c-statistic = 0.7522), and it was upregulated upon ERT (P < 0.001). On multivariable analysis, miR-184 was independently and inversely associated with a higher risk of cardiac damage (odds ratio = 0.86; 95% confidence interval [CI] = 0.76–0.98; P = 0.026). Adding miR-184 to a comprehensive clinical model improved the prediction of cardiac damage in terms of global model fit, calibration, discrimination, and classification accuracy (continuous net reclassification improvement = 0.917, P < 0.001; integrated discrimination improvement [IDI] = 0.105, P = 0.017; relative IDI = 0.221, 95% CI = 0.002–0.356). Thus, miR-184 is a circulating biomarker of AFD that changes after ERT. Assessment of its level in plasma could be clinically valuable in improving the prediction of cardiac damage in AFD patients.Subject terms: Prognostic markers, Cardiovascular diseases     

Storage of ram semen

Storage of ram semen in liquid and frozen state, the diluents used for both methods, processing, cooling, freezing and thawing of semen are reviewed. Factors influencing the fertility of stored semen and methods used for improvement are discussed, and fertility results of long-term frozen stored ram semen are also given.