Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles

Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50 degrees C. This effect becomes negligible at temperatures higher that ca. 60 degrees C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis. Published by Elsevier B.V.

Effect of the thermostat in the molecular dynamics simulation on the folding of the model protein chignolin

Molecular dynamics simulations of the model protein chignolin with explicit solvent were carried out, in order to analyze the influence of the Berendsen thermostat on the evolution and folding of the peptide. The dependence of the peptide behavior on temperature was tested with the commonly employed thermostat scheme consisting of one thermostat for the protein and another for the solvent. The thermostat coupling time of the protein was increased to infinity, when the protein is not in direct contact with the thermal bath, a situation known as minimally invasive thermostat. In agreement with other works, it was observed that only in the last situation the instantaneous temperature of the model protein obeys a canonical distribution. As for the folding studies, it was shown that, in the applications of the commonly utilized thermostat schemes, the systems are trapped in local minima regions from which it has difficulty escaping. With the minimally invasive thermostat the time that the protein needs to fold was reduced by two to three times. These results show that the obstacles to the evolution of the extended peptide to the folded structure can be overcome when the temperature of the peptide is not directly controlled.

Dynamics of free and H-3-labelled glutamine concentrations during zygotic and somatic embryogenesis of Feijoa [Acca sellowiana (O. Berg.) Burret]

Amino acids play fundamental roles in plant morphogenesis. Among sources of organic nitrogen (N), glutamine has frequently been used during the establishment and maintenance of cell and tissue cultures. The aim of this study was analyse endogenous levels of glutamine during somatic and zygotic embryogenesis of Acca sellowiana (Feijoa or pineapple guava). The in vitro absorption of H-3-labelled glutamine was investigated. Zygotic embryos and embryogenic cultures (EC) were evaluated at 30 d and 70 d after explant inoculation onto the medium. Endogenous levels of glutamine were similar during zygotic and somatic embryogenesis, and showed a gradual decline until day-24 in culture. The highest rates of H-3-labelled glutamine uptake were observed during the first 2 h of incubation, resulting in values of 6.29 mu mol g(-1) fresh weight (FW) for zygotic embryos, 14.43 mu mol g(-1) FW for EC after 30 d, and 13.85 mu mol g(-1) FW for EC after 70 d. These results showed that the decreased levels of glutamine observed during the initial phase of development may be related to de novo protein synthesis and mobilisation during embryo maturation. The absorption of glutamine in the first 2 h of incubation also emphasises its involvement as an important source of N during morphogenesis of somatic and zygotic embryos.

Extensive structural change of the envelope protein of dengue virus induced by a tuned ionic strength: conformational and energetic analyses

The Dengue has become a global public health threat, with over 100 million infections annually; to date there is no specific vaccine or any antiviral drug. The structures of the envelope (E) proteins of the four known serotype of the dengue virus (DENV) are already known, but there are insufficient molecular details of their structural behavior in solution in the distinct environmental conditions in which the DENVs are submitted, from the digestive tract of the mosquito up to its replication inside the host cell. Such detailed knowledge becomes important because of the multifunctional character of the E protein: it mediates the early events in cell entry, via receptor endocytosis and, as a class II protein, participates determinately in the process of membrane fusion. The proposed infection mechanism asserts that once in the endosome, at low pH, the E homodimers dissociate and insert into the endosomal lipid membrane, after an extensive conformational change, mainly on the relative arrangement of its three domains. In this work we employ all-atom explicit solvent Molecular Dynamics simulations to specify the thermodynamic conditions in that the E proteins are induced to experience extensive structural changes, such as during the process of reducing pH. We study the structural behavior of the E protein monomer at acid pH solution of distinct ionic strength. Extensive simulations are carried out with all the histidine residues in its full protonated form at four distinct ionic strengths. The results are analyzed in detail from structural and energetic perspectives, and the virtual protein movements are described by means of the principal component analyses. As the main result, we found that at acid pH and physiological ionic strength, the E protein suffers a major structural change; for lower or higher ionic strengths, the crystal structure is essentially maintained along of all extensive simulations. On the other hand, at basic pH, when all histidine residues are in the unprotonated form, the protein structure is very stable for ionic strengths ranging from 0 to 225 mM. Therefore, our findings support the hypothesis that the histidines constitute the hot points that induce configurational changes of E protein in acid pH, and give extra motivation to the development of new ideas for antivirus compound design.

Fhos encodes a Drosophila Formin-Like Protein participating in autophagic programmed cell death

Larval tissues undergo programmed cell death (PCD) during Drosophila metamorphosis. PCD is triggered in a stage and tissue-specific fashion in response to ecdysone pulses. The understanding of how ecdysone induces the stage and tissue-specificity of cell death remains obscure. Several steroid-regulated primary response genes have been shown to act as key regulators of cellular responses to ecdysone by inducing a cascade of transcriptional regulation of late responsive genes. In this article, the authors identify Fhos as a gene that is required for Drosophila larval salivary gland destruction. Animals with a P-element mutation in Fhos possess persistent larval salivary glands, and precise excisions of this P-element insertion resulted in reversion of this salivary gland mutant phenotype. Fhos encodes the Drosophila homolog of mammalian Formin Fhos. Fhos is differentially transcribed during development and responds to ecdysone in a method that is similar to other cell death genes. Similarly to what has been shown for its mammalian counterpart, FHOS protein is translocated to the nucleus at later stages of cell death. Fhos mutants posses disrupted actin cytoskeleton dynamics in persistent salivary glands. Together, our data indicate that Fhos is a new ecdysone-regulated gene that is crucial for changes in the actin cytoskeleton during salivary gland elimination in Drosophila. genesis 50:672684, 2012. (c) 2012 Wiley Periodicals, Inc.

Phylogenetic relationship of dengue virus type 3 isolated in Brazil and Paraguay and global evolutionary divergence dynamics

Background: Dengue is the most important mosquito-borne viral disease worldwide. Dengue virus comprises four antigenically related viruses named dengue virus type 1 to 4 (DENV1-4). DENV-3 was re-introduced into the Americas in 1994 causing outbreaks in Nicaragua and Panama. DENV-3 was introduced in Brazil in 2000 and then spread to most of the Brazilian States, reaching the neighboring country, Paraguay in 2002. In this study, we have analyzed the phylogenetic relationship of DENV-3 isolated in Brazil and Paraguay with viruses isolated worldwide. We have also analyzed the evolutionary divergence dynamics of DENV-3 viruses. Results: The entire open reading frame (ORF) of thirteen DENV-3 isolated in Brazil (n = 9) and Paraguay (n = 4) were sequenced for phylogenetic analysis. DENV-3 grouped into three main genotypes (I, II and III). Several internal clades were found within each genotype that we called lineage and sub-lineage. Viruses included in this study belong to genotype III and grouped together with viruses isolated in the Americas within the lineage III. The Brazilian viruses were further segregated into two different sub-lineage, A and B, and the Paraguayan into the sub-lineage B. All three genotypes showed internal grouping. The nucleotide divergence was in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages. Phylogenetic trees constructed with any of the protein gene sequences showed the same segregation of the DENV-3 in three genotypes. Conclusion: Our results showed that two groups of DENV-3 genotypes III circulated in Brazil during 2002-2009, suggesting different events of introduction of the virus through different regions of the country. In Paraguay, only one group DENV-3 genotype III is circulating that is very closely related to the Brazilian viruses of sub-lineage B. Different degree of grouping can be observed for DENV-3 and each group showed a characteristic evolutionary divergence. Finally, we have observed that any protein gene sequence can be used to identify the virus genotype.

Characterization of suramin binding sites on the human group IIA secreted phospholipase A(2) by site-directed mutagenesis and molecular dynamics simulation

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.