Spectroscopic evidence for a preferential location of lidocaine inside phospholipid bilayers

We examined the effect of uncharged lidocaine on the structure and dynamics of egg phosphatidylcholine (EPC) membranes at pH 10.5 in order to assess the location of this local anesthetic in the bilayer. Changes in the organization of small unilamellar vesicles were monitored either by electron paramagnetic resonance (EPR)-in the spectra of doxyl derivatives of stearic acid methyl esters labeled at different positions in the acyl chain (5-, 7-, 12- and 16-MeSL)-or by fluorescence, with pyrene fatty-acid (4-, 6-, 10- and 16-Py) probes. The largest effects were observed with labels located at the upper positions of the fatty-acid acyl-chain. Dynamic information was obtained by 1H-NMR. Lidocaine protons presented shorter longitudinal relaxation times (T(1)) values due to their binding, and consequent immobilization to the membrane. In the presence of lidocaine the mobility of all glycerol protons of EPC decreased, while the choline protons revealed a higher degree of mobility, indicating a reduced participation in lipid-lipid interactions. Two-dimensional Nuclear Overhauser Effect experiments detected contacts between aromatic lidocaine protons and the phospholipid-choline methyl group. Fourier-transform infrared spectroscopy spectra revealed that lidocaine changes the access of water to the glycerol region of the bilayer. A "transient site" model for lidocaine preferential location in EPC bilayers is proposed. The model is based on the consideration that insertion of the bulky aromatic ring of the anesthetic into the glycerol backbone region causes a decrease in the mobility of that EPC region (T(1) data) and an increased mobility of the acyl chains (EPR and fluorescence data).     

Inhibition of Membrane Lipid Peroxidation by a Radical Scavenging Mechanism: a Novel Function for Hydroxyl-Containing Ionophores

In the present study we show that K⁺/H⁺ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe²⁺-citrate and 2,2'-azobis(2-amidinopropane) di-hydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidyl-choline (PC) liposomes containing negatively charged lipids—dicetyl phosphate (DCP) or cardiolipin (CL)—and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na⁺ than for K⁺, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O₂ consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by buty-lated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, vali-nomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (S_eff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe²⁺-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe²⁺-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidyl-choline (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

Background  

Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).     

Heritabilities,social environment effects and genetic correlations of social behaviours in a cooperatively breeding vertebrate

Social animals interact frequently with conspecifics, and their behaviour is influenced by social context, environmental cues and the behaviours of interaction partners, allowing for adaptive, flexible adjustments to social encounters. This flexibility can be limited by part of the behavioural variation being genetically determined. Furthermore, behaviours can be genetically correlated, potentially constraining independent evolution. Understanding social behaviour thus requires carefully disentangling genetic, environmental, maternal and social sources of variations as well as the correlation structure between behaviours. Here, we assessed heritability, maternal, common environment and social effects of eight social behaviours in Neolamprologus pulcher, a cooperatively breeding cichlid. We bred wild‐caught fish in a paternal half‐sibling design and scored ability to defend a resource against conspecifics, to integrate into a group and the propensity to help defending the group territory (“helping behaviour”). We assessed genetic, social and phenotypic correlations within clusters of behaviours predicted to be functionally related, namely “competition,” “aggression,” “aggression‐sociability,” “integration” and “integration‐help.” Helping behaviour and two affiliative behaviours were heritable, whereas there was little evidence for a genetic basis in all other traits. Phenotypic social effects explained part of the variation in a sociable and a submissive behaviour, but there were no maternal or common environment effects. Genetic and phenotypic correlation within clusters was mostly positive. A group's social environment influenced covariances of social behaviours. Genetic correlations were similar in magnitude but usually exceeding the phenotypic ones, indicating that conclusions about the evolution of social behaviours in this species could be provisionally drawn from phenotypic data in cases where data for genetic analyses are unobtainable.     

β-Glutamate as a Substrate for Glutamine Synthetase

The conversion of β-glutamate to β-glutamine by archaeal and bacterial glutamine synthetase (GS) enzymes has been examined. The GS from Methanohalophilus portucalensis (which was partially purified) is capable of catalyzing the amidation of this substrate with a rate sevenfold less than the rate obtained with α-glutamate. Recombinant GS from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus were considerably more selective for α-glutamate than β-glutamate as a substrate. All the archaeal enzymes were much less selective than the two bacterial GS (from Escherichia coli and Bacillus subtilis), whose specific activities towards β-glutamate were much smaller than rates with the α-isomer. These results are discussed in light of the observation that β-glutamate is accumulated as an osmolyte in many archaea while β-glutamine (produced by glutamine synthetase) is used as an osmolyte only in M. portucalensis.     

Regulation of type VI collagen synthesis in transformed mesenchymal cells.

We have analysed the effects of oncogenic transformation on the expression of type VI collagen in mesenchymal cells. Synthesis of type VI collagen was almost completely inhibited in fibroblasts transformed by DNA or RNA tumour viruses or in cells derived from spontaneous mesenchymal tumours. Inhibition of type VI collagen synthesis appears, therefore, to be a common phenomenon of transformed mesenchymal cells. When introduced into normal cells by viral vectors, the 'nuclear' oncogene v-myc had an inhibitory effect similar to that of the 'cytoplasmic' oncogene v-src. Fibroblasts infected with a temperature-sensitive strain of Rous sarcoma virus (NY68) produced type VI collagen at the restrictive, but not at the permissive temperature. If such cells were shifted from the permissive to the restrictive temperature, synthesis of the individual subunits of type VI collagen was co-ordinately induced. These results demonstrate that the activity of a single oncogene product is sufficient to inhibit type VI collagen expression.     

Artificial viral envelopes containing recombinant human immunodeficiency virus (HIV) gp160.

An artificial viral envelope was constructed, resembling the human immunodeficiency virus (HIV) envelope with respect to ultrastructure, size, phospholipid profile and lipid:cholesterol ratio. Recombinant HIV surface protein gp160 was anchored in the outer surface of the envelope membrane using a double detergent dialysis. The envelopes remained physically stable for several months. Immunolabeling with anti-gp160/41 monoclonal antibody revealed surface insertion and availability of gp160 for binding. Cell fusion and cytosolic transfer of the encapsulated fluorescent marker FITC-dextran was demonstrated. Flow cytometry indicated more efficient transfer of the fluorescent marker to cells which were approximately 60% CD4+ (REX-1B), relative to cells which were only approximately 18% CD4+ (KG-1). However, plain lipid envelopes without gp160 fused very efficiently with both cell types, indicating their potential usefulness as "fusogenic liposomes". Complete artificial viral envelopes may serve as subunit vaccines, and receptor-targeted delivery systems for drugs, toxins and genetic constructs.     

The fourth level of social structure in a multi‐level society: ecological and social functions of clans in hamadryas baboons

Hamadryas baboons are known for their complex, multi‐level social structure consisting of troops, bands, and one‐male units (OMUs) [Kummer, 1968. Social organization of hamadryas baboons. Chicago: The University of Chicago Press. 189p]. Abegglen [1984. On socialization in hamadryas baboons: a field study. Lewisburg, PA: Bucknell University Press. 207p.] observed a fourth level of social structure comprising several OMUs that rested near one another on sleeping cliffs, traveled most closely together during daily foraging, and sometimes traveled as subgroups independently from the rest of the band. Abegglen called these associations “clans” and suggested that they consisted of related males. Here we confirm the existence of clans in a second wild hamadryas population, a band of about 200 baboons at the Filoha site in lowland Ethiopia. During all‐day follows from December 1997 through September 1998 and March 2005 through February 2006, data were collected on activity patterns, social interactions, nearest neighbors, band fissions, and takeovers. Association indices were computed for each dyad of leader males, and results of cluster analyses indicated that in each of the two observation periods this band comprised two large clans ranging in size from 7 to 13 OMUs. All band fissions occurred along clan lines, and most takeovers involved the transfer of females within the same clan. Our results support the notion that clans provide an additional level of flexibility to deal with the sparse distribution of resources in hamadryas habitats. The large clan sizes at Filoha may simply be the largest size that the band can split into and still obtain enough food during periods of food scarcity. Our results also suggest that both male and female relationships play a role in the social cohesion of clans and that males exchange females within clans but not between them. Am. J. Primatol. 71:948–955, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: <Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> studies

Background  

The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats.     

Spin label study of detergents in the region of critical micelle concentration.

A series of spin probes was employed to examine the behavior of the detergent sodium dodecyl sulfate (SDS) at concentrations above and below the critical micelle concentration (cmc). The existence of detergent aggregates below the cmc was evidenced by the appearance of composite electron spin resonance (ESR) spectra for probes that have measurable solubility in water. The spectra were indicative of two probe populations: one in an aqueous environment and another in detergent aggregates. The ESR spectra of probes which are highly insoluble in water exhibited line broadening due to intermolecular spin exchange interactions, indicating that the probes were concentrated in detergent aggregates present below the experimental cmc. The results are discussed in terms of their significance for the study of the mechanisms of micelle formation and for the detection of detergent aggregates at very low concentrations.