Nano-multilamellar lipid vesicles (NMVs) enhance protective antibody responses against Shiga toxin (Stx2a) produced by enterohemorrhagic Escherichia coli strains (EHEC).

Microlipid vesicles (MLV) have a broad spectrum of applications for the delivery of molecules, ranging from chemical compounds to proteins, in both in vitro and in vivo conditions. In the present study, we developed a new set of nanosize multilayer lipid vesicles (NMVs) containing a unique combination of lipids. The NMVs enable the adsorption of histidine-tagged proteins at the vesicle surface and were demonstrated to be suitable for the in vivo delivery of antigens. The NMVs contained a combination of neutral (DOPC) and anionic (DPPG) lipids in the inner membrane and an external layer composed of DOPC, cholesterol, and a nickel-containing lipid (DGS-NTA [Ni]). NMVs combined with a recombinant form of the B subunit of the Shiga toxin (rStx2B) produced by certain enterohemorragic Escherichia coli (EHEC) strains enhanced the immunogenicity of the antigen after parenteral administration to mice. Mice immunized with rStx2B-loaded NMVs elicited serum antibodies capable of neutralizing the toxic activities of the native toxin; this result was demonstrated both in vitro and in vivo. Taken together, these results demonstrated that the proposed NMVs represent an alternative for the delivery of antigens, including recombinant proteins, generated in different expression systems.

Anxiolytic-like effect of Citrus limon (L. ) Burm f. essential oil inhalation on mice / Efeito ansiolítico da inalação do óleo essencial de Citrus limon (L. ) Burm f. em camundongos.

ABSTRACT Experimental in vivo study aimed to characterize the anxiolytic-like effect of the Citrus limon fruit peel’s essential oil (CLEO) in animal models of anxiety, besides evaluating the viability J774.A1 cells in vitro through the MTT reduction method at the concentrations of 10 and 100 µg/mL. The anxiolytic behavior was evaluated in Swiss mice (n = 8) using the methodology of Elevated Plus-Maze (EPM) and Open-Field (OF). CLEO was tested by inhalation at the doses of 100, 200, and 400 µL, and as control, animals were subjected to inhalation of the vehicle (saline solution 0.9% + Tween80®) and intraperitoneal administration of diazepam (1.5 mg/kg). In the cell viability assay, it was observed that none of the concentrations showed cytotoxicity. OF test showed significant anxiolytic activity at all tested doses of OECL, compared to the control group, without changing the motor performance of the animals. Corroborating OF data, the EPM test confirmed anxiolytic activity in at least two doses of the tested oil (200 and 400 µL), justified by the number of entries and increase in the percentage of time in the open arms. The data analysis of this study evidenced that inhalation of OECL was able to induce an anxiolytic behavior in mice; however, further studies are required to ensure its safe use by the population.

RESUMO O estudo experimental in vivo objetivou caracterizar o efeito ansiolítico do óleo essencial das cascas do fruto de Citrus limon (OECL) em modelos animais de ansiedade, além de avaliar in vitro a viabilidade de células J774.A1 através do método de redução do MTT nas concentrações de 10 e 100 µg/mL. O comportamento ansiolítico foi avaliado em camundongos Swiss (n = 8), utilizando a metodologia do Labirinto em Cruz Elevado (LCE) e do Campo Aberto (CA). O OECL foi testado por inalação nas doses de 100, 200 e 400 µL, e como controle, os grupos foram submetidos à inalação do veículo (Solução fisiológica 0,9% + Tween80®) e administração intraperitoneal de diazepam (1,5 mg/kg). No ensaio de viabilidade celular, observou-se que nenhuma das concentrações apresentou citotoxicidade. O teste de CA demonstrou atividade ansiolítica significativa em todas as doses testadas do OECL, comparada ao grupo controle, sem alterar a performance motora dos animais. Corroborando os dados do CA, o teste do LCE confirmou a atividade ansiolítica em pelo menos duas das doses do óleo testadas (200 e 400 µL), justificada pelo número de entradas e aumento na percentagem de tempo nos braços abertos. A análise dos dados deste estudo evidenciou que a inalação do OECL foi capaz de induzir um comportamento ansiolítico em camundongos; entretanto, outros estudos são necessários para garantir o seu uso seguro pela população.

Chain length effect on the binding of amphiphiles to serum albumin and to POPC bilayers.

The interaction of small molecules, such as drugs or metabolites, with proteins and biomembranes is of fundamental importance for their bioavailability. The systematic characterization of the binding affinity for structurally related ligands may provide rules that allow its prediction for any other relevant molecule. In this work we have studied a homologous series of fluorescent fatty amines with the fluorescent moiety 7-nitrobenz-2-oxa-1,3-diazol-4-yl covalently bound to the amine group (NBD-C(n); n = 4, 6, 8, 10, 12, 14, and 16) in aqueous solution and associated with BSA or lipid bilayers. We have found a linear dependence with the length of the alkyl chain, up to NBD-C(10), for the Gibb's free energy of partition between the aqueous solution and 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers equal to ΔΔG = -2.5 ± 0.3 kJ/mol per methylene group. Additionally, the amphiphiles interacted efficiently with bovine serum albumin, and it was inhibited by fatty acids indicating that binding occurs to the fatty acids highest affinity binding site. The association of the amphiphiles with BSA and POPC bilayers was performed at different temperatures (15-35 °C) allowing for the calculation of the enthalpic and entropic contributions. A value of ΔH = -15 ± 4 kJ/mol was obtained for all amphiphiles and binding agents. The entropy contribution was always positive and increased with the length of the alkyl chain. The location of the ligand in the biological membrane is also of high relevance, namely because this will determine its effect on biomembrane properties at high ligand concentrations. With this goal, we have measured some photophysical properties of the amphiphiles inserted in POPC bilayers, and we found no significant variation along the series, indicating that the NBD group is located in a region with similar properties regardless of the length of the nonpolar group. An exception was noted for the case of NBD-C(14) whose parameters were somewhat different from the trend observed.

Expression and preliminary X-ray diffraction studies of cytosolic Cu,Zn superoxide dismutase from Schistosoma mansoni.

Active cytosolic (CT) Cu,Zn superoxide dismutase from Schistosoma mansoni (SmCTSOD) was recovered after thrombin cleavage of a glutathione-S-transferase linked fusion protein (GST-SmCTSOD) expressed in the presence of the active-site metals. Crystals have been obtained in two space groups, P2(1)2(1)2(1) and P2(1). The former have unit-cell parameters a = 74.64, b = 78.24, c = 95.18 A and typically diffract to 2.2 A. The monoclinic crystals have unit-cell parameters a = 39.27, b = 95.08, c = 78.41 A, beta = 103.55 degrees and diffract to at least 1.55 A. The calculated solvent content of the crystals is compatible with two dimers of SmCTSOD in the asymmetric unit in both cases. Molecular-replacement solutions have been obtained for both crystal forms and show that slight distortions in the crystal packing relate one form to the other.

Dendritic degeneration and regrowth in the cerebral cortex of patients with Alzheimer's disease.

Biopsy fragments from the frontal cortex of three patients with Alzheimer's disease were studied by electron microscopy. Electron-dense degenerating dendrites were observed in all cortical layers, but more abundantly in layers 3 and 4. Dendrites with lipid-like droplets and others filled with mitochondria were also seen in these two layers. These findings are in keeping with the abnormalities found with Golgi techniques in the dendritic trees of patients with familial forms of Alzheimer's disease, and are similar to the alterations observed in other diseases accompanied by mental changes.