IRIV-adjuvanted hepatitis A vaccine: in vivo absorption and biophysical characterization.

Immunopotentiating reconstituted influenza virosomes (IRIV) are 150-nm proteoliposomes composed of influenza surface glycoproteins and a mixture of natural and synthetic phospholipids. Due to size, structure and composition of the IRIVs, they serve as an antigen carrier system for efficacious vaccination, as was demonstrated for hepatitis A and influenza. This paper reviews the unique properties of IRIVs and describes the in vivo biodistribution of model antigens using 14C-labeled IRIVs and 125I-labeled streptavidin. IRIV formulated streptavidin induced a strong depot effect after intra muscular (i.m.) vaccination of mice, whereas soluble streptavidin was soon eliminated via the kidney of the animals. A mixture of antigen and IRIVs yielded higher antibody titers after i.m. inoculation than streptavidin alone. The highest immunostimulation was achieved by the binding of the antigen to the investigated adjuvant. The potential penetration of inactivated hepatitis A virions into lipid membranes was assessed by measuring the area increase of a lipid monolayer kept at a constant surface pressure corresponding to that of lipid bilayer vesicles. The monolayers were composed of phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) (75/25 mol/mol), thus resembling the lipid composition of the IRIV. The results suggested that the hepatitis A antigen may spontaneously bind to the reconstituted IRIV membranes.

Substance P and antagonists. Surface activity and molecular shapes.

The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met amide) and three of its antagonists were derived by measuring the Gibbs adsorption isotherm, providing information on the surface activity, the molecular shape, and the pK values of the different molecules. The following three antagonists were investigated: [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, ANT I; [D-Arg1,D-Trp7,9,Leu11]SP, ANT II and [D-Pro2,D-Trp7,9]SP, ANT III. SP is only moderately surface active. The amino acid substitutions lead, however, to an increased surface activity of the antagonists. From the concentration dependence of the surface activity it was possible to quantify the packing characteristics of the individual neuropeptides. SP shows cross-sectional areas of 300 +/- 5 A2 to 240 +/- 5 A2 (pH 5 to 8, 154 mM NaCl) at concentrations below 10(-5) M, i.e., in the physiological concentration range, indicating a folded SP conformation. Upon increasing the packing density to concentrations larger than 10(-5) M the surface area was only half as large (148 +/- 5 A2 to 124 +/- 3 A2) suggesting now a relatively extended conformation of the SP molecule with its long molecular axis perpendicular to the air/water interface. In contrast, the three antagonists were characterized by surface areas of 147 +/- 3 A2 to 126 +/- 3 A2 which were almost independent of concentration. The antagonists thus adopt a relatively extended conformation in the whole concentration range measured. This is further supported by computer modelling which shows that the antagonists are motionally restricted and can adopt neither a bent nor a alpha-helical conformation. The surface activity of the neuropeptides was dependent on the pH of the solution. At low peptide concentrations (about 10(-6) M) it was possible to resolve and determine the pK values of all individual charged amino acid side chains. The pK values observed for the neuropeptides were about two pK units lower than those of the free amino acids in solution. The pK shifts of the neuropeptides at the air/water interface are explained in terms of the Gouy-Chapman theory. SP and its antagonists bind to lipid bilayers in the order of their surface activity. While the binding of SP is mainly due to electrostatic interactions, hydrophobic peptide-lipid interactions contribute to the binding of the antagonists.

Local anesthetics and pressure: a comparison of dibucaine binding to lipid monolayers and bilayers.

The binding of the local anesthetic dibucaine to monolayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was studied with a Langmuir trough at pH 5.5 (22 degrees C, 0.1 M NaCl). At this pH value only the charged form of the local anesthetic exists in solution. Charged dibucaine was found to be surface active and to penetrate into the lipid monolayer, with the hydrophobic part of the molecule being accommodated between the fatty acyl chains of the lipid. The dibucaine intercalation could be quantitated by measuring the expansion of the film area, delta A, at constant surface pressure, pi. At a given surface pressure, delta A increased with increasing dibucaine in the buffer phase. On the other hand, keeping the dibucaine concentration constant, the area increase, delta A, was strongly dependent on the surface pressure. The area increase, delta A, was large at low surface pressure and decreased with increasing surface pressure. A plot of the relative change in surface area, delta A/A, versus the surface pressure yielded straight lines in the pressure range of 25-36 mN/m for five different concentrations. The delta A/A vs. pi isotherms intersected at pi = 39.5 +/- 1 mN/m with delta A = O, indicating that charged dibucaine apparently can no longer penetrate into the monolayer film. By making judicial assumptions about the area requirement of dibucaine the monolayer expansion curves could be transformed into true binding isotherms. Dibucaine binding isotherms were constructed for different monolayer pressures and were compared to a bilayer binding isotherm measured under similar conditions with ultraviolet spectroscopy. The best agreement between monolayer and bilayer binding data was obtained for a monolayer held at a pressure of 30.7 to 32.5 mN/m, which can thus be considered as the bilayer-monolayer equivalence pressure. It is further suggested from this analogy that the binding of dibucaine does not change the internal pressure in the bilayer phase, at least not in the concentration range of physiological interest (0-2 mM dibucaine) but induces a lateral expansion. At higher molar ratios of cationic dibucaine to lipid, chi b, in the monolayer (chi b greater than 0.20) the area increase is larger than would be expected from the molecular dimensions of dibucaine. This is probably due to charge repulsion effects, which at still higher molar ratios (chi b greater than 0.6) lead to a micellisation. The pressure dependence of the intercalation of cationic dibucaine into lipid membranes may also be of relevance for the phenomenon of pressure reversal in anesthesia.

Phospholipid composition and organization of cytochrome c oxidase preparations as determined by 31P-nuclear magnetic resonance.

The molecular organization as well as the composition of the phospholipids in cytochrome c oxidase preparations (bovine heart) were investigated by 31P-nuclear magnetic resonance. In the so-called 'lipid-rich' preparation the lipids were found to form a fluid bilayer around the enzyme since the 31P-NMR spectrum was characteristic of a fast, axially symmetric motion of the phosphate groups with a chemical shift anisotropy of delta sigma = -45 ppm. In contrast, the 'lipid-depleted' cytochrome c oxidase gave rise to a broader spectrum where the motion of the phospholipids was no longer axially symmetric. Nevertheless, the total width of the spectrum was still considerably narrower than observed for immobilized phospholipids in solid crystals. Both enzyme preparations were dissolved in 1% detergent solution and used for high-resolution 31P-NMR spectroscopy. Narrow lines of about 20 Hz linewidth were obtained for both types of enzyme preparations, and well-resolved resonances could be assigned to cardiolipin, phosphatidylethanolamin and phosphatidylcholine. The major differences between lipid-rich and lipid-depleted cytochrome c oxidase were the absolute amount of phospholipid associated with the protein and the relative contribution of the individual lipid classes to the 31P-NMR spectrum. For lipid-rich cytochrome c oxidase about 130 molecules phospholipid were bound per enzyme (approx. 11 cardiolipins, 54 phosphatidylethanolamines and 64 phosphatidylcholines). For lipid-depleted cytochrome c oxidase only 6-18 lipids were bound per enzyme (1 or 2 cardiolipins, 3-8 phosphatidylethanolamines and 2-8 phosphatidylcholines). In contrast to earlier suggestions that cardiolipin is the only remaining lipid in lipid-depleted cytochrome c oxidase, the 31P-NMR studies demonstrate that all three lipids remain associated with the protein.

Bilayers of dipalmitoyl-3-sn-phosphatidylcholine. Conformational differences between the fatty acyl chains.

Dipalmitoyl-3-sn-phosphatidylcholine is specifically deuterated at the C-2 position of the fatty acyl chains. Using deuterium magnetic resonance it is then possible to probe the chain conformation in the vicinity of the polar head groups. Three separate quadrupole splittings are observed for bilayers of 1,2[2'-2H2]palmitoyl-3-sn-phosphatidylcholine, indicating that the two chains behave differently. The synthesis of phosphatidylcholines each deuterated in only one chain allows the assignment of the three resonances. It is concluded that the beginnings of the two chains have orientations parallel and perpendicular to the bilayer normal. The data further suggest the possibility of two long-lived conformations of the glycerol constituent.

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group.

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 +/- 80 M-1 and 11 +/- 2 M-1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a 'molecular electrometer' with respect to surface charges.

Sequence and genomic organization of the Drosophila proteasome PROS-Dm25 gene.

The genomic region encoding the Drosophila proteasome alpha-type subunit Dm25 has been isolated and analysed with regard to its nucleotide sequence and structure. Our data show that the Dm25 coding region is interrupted by four introns and that the 5' upstream region contains no sequence motifs common with the two previously described proteasome genes.

Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans.

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at -40 degrees C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.

20 S proteasomes are assembled via distinct precursor complexes. Processing of LMP2 and LMP7 proproteins takes place in 13-16 S preproteasome complexes.

The non-essential mouse proteasome beta-type subunits LMP2 and LMP7 are thought to connect proteasomes to the MHC class I antigen processing pathway. Both subunits are synthesized as proproteins. We have studied the processing of both subunits, correlated with the maturation of 20 S proteasomes in mouse T cells. Our data show that proteasome assembly occurs via 13-16 S precursor complexes which possess a protein pattern distinct from that of 20 S proteasomes. Both LMP2 and LMP7 proproteins are processed within these preproteasome complexes and only their processed forms become part of active 20 S proteasomes. Our data show that the maturation and assembly of 20 S proteasomes via precursor particles is a translation-dependent gradual process, that processing of subunit proproteins takes place in these 13-16 S complexes and that subunit processing and proteasome formation occur together.

Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide.

To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.

Inhibitors of multidrug efflux transporters: their membrane and protein interactions.

Modulators and inhibitors of multidrug efflux transporters, like P-glycoprotein, are used to reduce or inhibit multidrug resistance, MDR, which leads to a failure of the chemotherapy of e.g. cancers, epilepsy, bacterial, parasitic, and fungal diseases. Binding and transport of first-, second-, and third-generation modulators and inhibitors of P-glycoprotein are discussed, taking into account the properties of the drug (H-bonding potential, dimensions, and pK(a) values) as well as the properties of the membrane.

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

OBJECTIVE: Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. METHODS: We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. RESULTS: Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. CONCLUSIONS: We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties - in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.

Long-term resuscitation of hemorrhage/reperfusion injury (H/R) stimulates renal PGE2 release.

This study examines the hypothesis that long-term resuscitation with hyperalimentation (TPN) following acute hemorrhage/reperfusion (H/R) injury stimulates renal release of PGE2. Male Sprague-Dawley rats were anesthetized and subjected to sham or hemorrhage to 30 mmHg for 30 min followed by reperfusion. All rats were placed on TPN for 5 days, then underwent laparotomy for in vivo renal artery and aortic blood flow for 60 min. The kidney was perfused in vitro with Krebs-Henseleit buffer at 3 ml/min (pH 7.4, 37 degrees C) and venous effluent was collected for analysis of PGE2, 6-keto-PGF1 alpha and thromboxane B2 by EIA. Hemorrhage/reperfusion followed by TPN for 5 days increased renal PGE2 2-fold and decreased in vivo renal artery blood flow by 50% compared to the sham group. Hemorrhage/reperfusion followed by TPN did not alter release of the other eicosanoids measured. These data suggest that the kidney has a limited capacity to maintain renal blood flow by increasing release of PGE2 when the animal is subjected to long-term resuscitation with TPN following mild hemorrhage/reperfusion injury.

Structure-activity relationship of P-glycoprotein substrates and modifiers.

The air-water partition coefficients, K(aw), highly correlated with the corresponding lipid-water partition coefficients, K(lw), and the critical micelle concentrations, CMC, were measured for 11 compounds for which the kinetic parameters of P-glycoprotein ATPase activation (Michaelis-Menten constant, K(m), and maximal velocity, V(max)) had been determined previously in inside-out vesicles of CR1R12 Chinese hamster ovary cells. In addition, the hydrogen bond donor patterns (type I and type II) relevant for substrate recognition by P-glycoprotein were determined from the energy-minimized three-dimensional structure of these compounds. A linear relation between the air-water partition coefficient, K(aw), and the inverse of the Michaelis-Menten constant, K(m), was observed such that K(m) x K(aw) approximately = 1. The maximal velocity, V(max), was shown to decrease with the number and strength of electron donor (hydrogen bond acceptor) groups in recognition patterns. If two substrates are applied simultaneously to P-glycoprotein the compound with the higher potential to form hydrogen bonds generally acts as an inhibitor. We conclude that partitioning into the lipid membrane is the rate-limiting step for the interaction of a substrate with P-glycoprotein and that dissociation of the P-glycoprotein-substrate complex is determined by the number and strength of the hydrogen bonds formed between the substrate and the transporter.

How does P-glycoprotein recognize its substrates?

P-glycoprotein actively transports a wide variety of chemically diverse compounds out of the cell. Based on a comparison of 100 compounds previously tested as P-glycoprotein substrates we suggest that a set of well-defined structural elements is required for an interaction with P-glycoprotein. The recognition elements are formed by 2 (type I unit) or 3 electron donor groups (type II unit) with a fixed spatial separation. Type I units consist of 2 electron donor groups with a spatial separation of 2.5 +/- 0.3 A. Type II units contain either 2 electron donor groups with a spatial separation of 4.6 +/- 0.6 A or 3 electron donor groups with a spatial separation of the outer 2 groups of 4.6 +/- 0.6 A. All molecules which contain at least 1 type I or 1 type II unit are predicted to be P-glycoprotein substrates. The binding to P-glycoprotein increases with the strength and the number of electron donor or hydrogen-bonding acceptor groups forming the type I and type II units. Correspondingly a high percentage of amino acids with hydrogen bonding donor side chains is found in the transmembrane sequences of P-glycoprotein relevant for substrate interaction. Molecules which minimally contain 1 type II unit are predicted to be inducers of P-glycoprotein overexpression.

Substrate recognition by P-glycoprotein and the multidrug resistance-associated protein MRP1: a comparison.

OBJECTIVES: It has recently been suggested that substrate recognition patterns for human P-glycoprotein encoded by mdr1 consist of two electron donor groups with a spatial separation of 2.5 +/- 0.3 A (type I units) or three electron donor groups with a spatial separation of the two outer groups of 4.6 +/- 0.6 A (type II units) [Seelig 1998]. Since P-gp and the multidrug resistance-associated protein (MRP1) have overlapping substrate specificity, we screened the chemical structures of 21 compounds, previously tested as MRP1 substrates, for electron donor units. In addition, we searched the putative transmembrane domains (TMD 1-12) of P-gp and (TMD 6-17) of MRP1 for amino acid side chains having the potential to interact with the respective substrates. METHODS: The three-dimensional structures of potential MRP1 substrates were modeled with a force-field approach and were then screened for electron donor units. Helical wheel projections of the 12 putative transmembrane domains of P-gp (1-12) and MRP (6-17) were analyzed for their content of amino acid residues with hydrogen bonding side chains, charged amino acid residues, and amino acid residues with pi-electron systems. RESULTS: MRP1 recognizes compounds with type I and type II units. At least one electrically neutral together with either one negatively charged type I unit or two electrically neutral type I units are required for the compound to be bound and transported. Transport increases with increasing number of electron donor units. Compounds which carry exclusively electrically neutral type I units (P-gp substrates) are transported only weakly by MRP1, and compounds with cationic type I units (P-gp substrates) are not transported at all. An analysis of the putative transmembrane alpha-helices of MRP1 and P-gp reveals that the amino acid residues with hydrogen-bond donor side chains are arranged preferentially on one side of the helix and amino acid residues with inert (non-hydrogen-bonding) side chains on the other side. In the case of MRP1, the hydrogen-bonding face also contains several cationic residues whereas, in the case of P-gp, it contains clusters of amino acid residues with beta-electron systems. CONCLUSIONS: We propose that P-gp and MRP1 recognize type I or type II units in chemical compounds having diverse structures, and that these transporters bind their substrates via hydrogen bond formation. Furthermore, we propose that transport of anionic substrates by MRP1 is facilitated by cationic amino acid residues present in the transmembrane helices of MRP1, whereas the transport of cationic substrates by P-gp is facilitated by a beta-electron slide guide.

Preparation of cycles for cryopreservation transfers using estradiol patches and Crinone 8% vaginal gel is effective and does not need any monitoring.

Supernumary pronucleated stage oocytes (PN) can be cryopreserved and later transferred in spontaneous, stimulated or artificial cycles. In this study, we re-evaluated 342 artificial cycles with a transdermal estradiol release system (Estraderm TTS 100) in combination with a vaginal progesterone delivery system (Crinone 8%). Endometrial thickness and serum estradiol on day 14 were correlated with clinical and ongoing pregnancy rates. Endometrial thickness between 7 and 15 mm did not relate to significantly different pregnancy rates. The estradiol serum level did not predict success. In conclusion, this method of endometrial preparation is comfortable for patients and monitoring is unnecessary.