Transdermal penetration of vasoconstrictors - Present understanding and assessment of the human epidermal flux and retention of free bases and ion-pairs

Purpose. As reductions in dermal clearance increase the residence time of solutes in the skin and underlying tissues we compared the topical penetration of potentially useful vasoconstrictors (VCs) through human epidermis as both free bases and ion-pairs with salicylic acid (SA). Methods. We determined the in vitro epidermal flux of ephedrine, naphazoline, oxymetazoline, phenylephrine, and xylometazoline applied as saturated solutions in propylene glycol: water (1: 1) and of ephedrine, naphazoline and tetrahydrozoline as 10% solutions of 1: 1 molar ratio ion-pairs with SA in liquid paraffin. Results. As free bases, ephedrine had the highest maximal flux, Jmax = 77.4 +/- 11.7 mug/cm(2)/h, being 4-fold higher than tetrahydrozoline and xylometazoline, 6-fold higher than phenylephrine, 10-fold higher than naphazoline and 100-fold higher than oxymetazoline. Stepwise regression of solute physicochemical properties identified melting point as the most significant predictor of flux. As ion-pairs with SA, ephedrine and naphazoline had similar fluxes (11.5 +/- 2.3 and 12.0 +/- 1.6 mug/cm(2)/h respectively), whereas tetrahydrozoline was approximately 3-fold slower. Corresponding fluxes of SA from the ion-pairs were 18.6 +/- 0.6, 7.8 +/- 0.8 and 1.1 +/- 0.1 respectively. Transdermal transport of VC's is discussed. Conclusions. Epidermal retention of VCs and SA did not correspond to their molar ratio on application and confirmed that following partitioning into the stratum corneum, ion-pairs separate and further penetration is governed by individual solute characteristics.

χ-conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter

The interactions of chi-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [H-3] norepinephrine (K-i 1.89 muM) than [H-3] dopamine (K-i 4.33 muM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA ( to 7 muM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [H-3] norepinephrine uptake for three mutations ( in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine.

Expression of hNP22 is altered in the frontal cortex and hippocampus of the alcoholic human brain

Background: Human neuronal protein (hNP22) is a gene with elevated messenger RNA expression in the prefrontal cortex of the human alcoholic brain. hNP22 has high homology with a rat protein (rNP22). These proteins also share homology with a number of cytoskeleton-interacting proteins. Methods: A rabbit polyclonal antibody to an 18-amino acid epitope was produced for use in Western and immunohistochemical analysis. Samples from the human frontal and motor cortices were used for Western blots (n = 10), whereas a different group of frontal cortex and hippocampal samples were obtained for immunohistochemistry (n = 12). Results: The hNP22 antibody detected a single protein in both rat and human brain. Western blots revealed a significant increase in hNP22 protein levels in the frontal cortex but not the motor cortex of alcoholic cases. Immunohistochemical studies confirmed the increased hNP22 protein expression in all cortical layers. This is consistent with results previously obtained using Northern analysis. Immunohistochemical analysis also revealed a significant increase of hNP22 immunoreactivity in the CA3 and CA4 but not other regions of the hippocampus. Conclusions: It is possible that this protein may play a role in the morphological or plastic changes observed after chronic alcohol exposure and withdrawal, either as a cytoskeleton-interacting protein or as a signaling molecule.

Fighting climate change in the air: lessons from the EU directive on global aviation

The European Union's (EU) decision to include aviation into the Emissions Trade Scheme was heatedly contested. Countries around the world, but mainly the Brazil, Russia, India, China and South Africa group (BRICS) and the US, denounced the EU's initiate as illegal and unilateral. Following a decade of frustrated negotiations at the International Civil Aviation Organization (ICAO), this paper interrogates why such measure, in principle climate-friendly, inspired so much global resentment. I argue that concerns with competitiveness and risks of legal inconsistency are important, but insufficient elements to explain the core of the conflict. The paper suggests that the EU was strongly criticized because third countries perceived this action as an imposed solution, which fostered an environment of distrust. Therefore, I claim that the problem has more to do with a normative divide than with a substantive divergence on what should be done regarding aviation emissions. My analysis is informed by the present literature on the links between trade and climate change, but gives particular weight to first-hand information through interviews with key stakeholders. The paper is divided in three parts. First, it presents the scope of the EU directive in historical perspective. Second, it explores the EU's measure through three different angles: legal, economical and political. The final part explores some possible solutions to overcome these divergences.

Effect of oxidative stress upon absorption of glucose by the human placenta: in vitro studies with BeWo cells

Pregnancy is a dynamic state and the placenta is a temporary organ that, among other important functions, plays a crucial role in the transport of nutrients and metabolites between the mother and the fetus, which is essential for a successful pregnancy. Among these nutrients, glucose is considered a primary source of energy and, therefore, fundamental to insure proper fetus development. Several studies have shown that glucose uptake is dependent on several morphological and biochemical placental conditions. Oxidative stress results from the unbalance between reactive oxygen species (ROS) and antioxidants, in favor of the first. During pregnancy, ROS, and therefore oxidative stress, increase, due to increased tissue oxygenation. Moreover, the relation between ROS and some pathological conditions during pregnancy has been well established. For these reasons, it becomes essential to understand if oxidative stress can compromise the uptake of glucose by the placenta. To make this study possible, a trophoblastic cell line, the BeWo cell line, was used. Experiments regarding glucose uptake, either under normal or oxidative stress conditions, were conducted using tert-butylhydroperoxide (tBOOH) as an oxidative stress inducer, and 3H-2-deoxy-D-glucose (3H-DG) as a glucose analogue. Afterwards, studies regarding the involvement of glucose facilitative transporters (GLUT) and the phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC) pathways were conducted, also under normal and oxidative stress conditions. A few antioxidants, endogenous and from diet, were also tested in order to study their possible reversible effect of the oxidative effect of tBOOH upon apical 3H-DG uptake. Finally, transepithelial studies gave interesting insights regarding the apical-to-basolateral transport of 3H-DG. Results showed that 3H-DG uptake, in BeWo cells, is roughly 50% GLUT-mediated and that tBOOH (100 μM; 24h) decreases apical 3H-DG uptake in BeWo cells by about 33%, by reducing both GLUT- (by 28%) and non-GLUT-mediated (by 40%) 3H-DG uptake. Uptake of 3H-DG and the effect of tBOOH upon 3H-DG uptake are not dependent on PKC and PI3K. Moreover, the effect of tBOOH is not associated with a reduction in GLUT1 mRNA levels. Resveratrol, quercetin and epigallocatechin-3-gallate, at 50 μM, reversed, by at least 45%, the effect of tBOOH upon 3H-DG uptake. Transwell studies show that the apical-to-basolateral transepithelial transport of 3H-DG is increased by tBOOH.In conclusion, our results show that tBOOH caused a marked decrease in both GLUT and non-GLUT-mediated apical uptake of 3H-DG by BeWo cells. Given the association of increased oxidative stress levels with several important pregnancy pathologies, and the important role of glucose for fetal development, the results of this study appear very interesting.

Sequencing of a 17.6 kb segment on the right arm of yeast chromosome VII reveals 12 ORFs, including CCT, ADE3 and TR-I genes, homologues of the yeast PMT and EF1G genes, of the human and bacterial electron-transferring flavoproteins (beta-chain) and of the Escherichia coli phosphoserine phosphohydrolase, and five new ORFs.

A 17.6 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains twelve open reading frames (ORFs) longer than 100 amino acids. Three genes had already been cloned and sequenced: CCT, ADE3 and TR-I. Two ORFs are similar to other yeast genes: G7722 with the YAL023 (PMT2) and PMT1 genes, encoding two integral membrane proteins, and G7727 with the first half of the genes encoding elongation factors 1gamma, TEF3 and TEF4. Two other ORFs, G7742 and G7744, are most probably yeast orthologues of the human and Paracoccus denitrificans electron-transferring flavoproteins (beta chain) and of the Escherichia coli phosphoserine phosphohydrolase. The five remaining identified ORFs do not show detectable homology with other protein sequences deposited in data banks. The sequence has been deposited in the EMBL data library under Accession Number Z49133.

Exploring bioactive properties of marine cyanobacteria Isolated from the Portuguese coast: high potential as a source of anticancer compounds

The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase release (LDH) assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria.

Transitional justice in the judiciary: lessons from the portuguese democratisation

The nature of the Portuguese transition to democracy and the following state crises (1974-1975) created a ‘window of opportunity’ in which the ‘reaction to the past’ was much stronger than in the other Southern or even of Central and Eastern European transitions. In Portugal, initiatives of symbolic rupture with the past began soon after the April 25, 1974, coup d’état and transitional justice policies assumed mainly three formulas. First, the institutional reforms directed primarily to abusive state institutions such as the political police (PIDE-DGS) and political courts (Plenary courts) in order to dismantle the repressive apparatus and prevent further human rights abuses and impunity. Secondly, the criminal prosecutions addressed to perpetrators considered as being the most responsible for repression and abuses. Finally, lustration or political purges (saneamentos, the term used in Portugal to designate political purges) which were, in fact, the most common form of political justice in Portuguese transition to democracy. This paper deals with the peculiarities of transitional justice in Portugal devoting a particular attention to the judicial, a key sector to understand the way the Portuguese dealt with their authoritarian past.

Managing Informal Learning in professional contexts: the learner’s perspective

Informal Learning is present in everyone's life but its awareness only recently has been reported. The need to keep track of the knowledge acquired this way is increasing as its sources diversity also increases. This work presents the pilots trials on the use of a tool developed to help keeping track of the learners’ informal learning, within a number of companies spread out in three countries. This tool developed through the European Commission funded project TRAILER, is still under development, which will allow integrating the set of improving suggestions obtained from users during the piloting phase. The overall idea of managing one’s informal learning was well accepted and welcomed, which validated the emerging need for a tool with this purpose.

Institutional factors governing the deployment of remote experiments: lessons from the rexnet project

Remote labs offer many unique advantages to students as they provide opportunities to access experiments and learning scenarios that would be otherwise unavailable. At the same time, however, these opportunities introduce real challenges to the institutions hosting the remote labs. This paper draws on the experiences of the REXNET project consortium to expose a number of these issues as a means of furthering the debate on the value of remote labs and the best practices in deploying them. The paper presents a brief outline of the various types of remote lab scenarios that might be deployed. It then describes the key human and technological actors that have an interest in or are intrinsic to a remote lab instance, with a description of the role of each actor and their interest. Some relationships between these various actors are then discussed with some factors that might influence those relationships. Finally some general issues are briefly described.

Preliminar characterization of a new virus isolated from the spinal fluid of patients with meningitis in São Paulo, Brazil

A total of 138 patients with the age of 4 months to 57 years were attended in different hospitals of São Paulo State with aseptic meningitis. A probable new agent was isolated from the cerebrospinal fluid of 35 of 53 specimens examined. Replication of the agent with similar characteristics was detected by CPE produced in the MDCK cell line. Virus-like particles measuring about 40 nm in diameter were observed by negative staining electron microscopy. No hemaglutinating activity was detected at pH 7.2 by using either human, guinea pig, chicken and at pH ranged 6.0 - 7.2 with goose red blood cells. The agent was not pathogenic to newborn or adult mice. Virus infectivity as measured by CPE was sensitive to chloroform and not inhibited by BuDR, suggesting that agent is an enveloped virus with RNA genome.

Seasonal patterns of polycyclic aromatic hydrocarbons in digestive gland and arm of octopus (Octopus vulgaris) from the Northwest Atlantic

Among organic pollutants existing in coastal areas, polycyclic aromatic hydrocarbons (PAHs) are of great concern due to their ubiquity and carcinogenic potential. The aim of this study was to evaluate the seasonal patterns of PAHs in the digestive gland and arm of the common octopus (Octopus vulgaris) from the Northwest Atlantic Portuguese coast. In the different seasons, 18 PAHs were determined and the detoxification capacity of the species was evaluated. Ethoxyresorufin O-deethylase (EROD) and ethoxycoumarin O-deethylase (ECOD) activities were measured to assess phase I biotransformation capacity. Individual PAH ratios were used for major source (pyrolytic/petrogenic) analysis. Risks for human consumption were determined by the total toxicity equivalence approach. Generally, low levels of PAHs were detected in the digestive gland and in the arm of octopus, with a predominance of low molecular over high molecular weight compounds. PAHs exhibited seasonality in the concentrations detected and in their main emission sources. In the digestive gland, the highest total PAH levels were observed in autumn possibly related to fat availability in the ecosystem and food intake. The lack of PAH elimination observed in the digestive gland after captivity could be possibly associated to a low biotransformation capacity, consistent with the negligible/undetected levels of EROD and ECOD activity in the different seasons. The emission sources of PAHs found in the digestive gland varied from a petrogenic profile observed in winter to a pyrolytic pattern in spring. In the arm, the highest PAH contents were observed in June; nevertheless, levels were always below the regulatory limits established for food consumption. The carcinogenic potential calculated for all the sampling periods in the arm were markedly lower than the ones found in various aquatic species from different marine environments. The results presented in this study give relevant baseline data for environmental monitoring of organic pollution in coastal areas.

St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

Characterization and Biological Activities of Ocellatin Peptides from the Skin Secretion of the Frog Leptodactylus pustulatus

Eight new peptides were isolated from the skin secretion of the frog Leptodactylus pustulatus and their amino acid sequences determined by de novo sequencing and by cDNA cloning. Structural similarities between them and other antimicrobial peptides from the skin secretion of Leptodactylus genus frogs were found. Ocellatins-PT1 to -PT5 (25 amino acid residues) are amidated at the C-terminus, while ocellatins-PT6 to -PT8 (32 amino acid residues) have free carboxylates. Antimicrobial activity, hemolytic tests, and cytotoxicity against a murine fibroblast cell line were investigated. All peptides, except for ocellatin-PT2, have antimicrobial activity against at least one Gram negative strain. Ocellatin-PT8 inhibited the growth of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella choleraesuis strains with MICs in the 60−240 μM range. No significant effect was observed in human erythrocytes and in a murine fibroblast cell line after exposure to the peptides at MICs. A comparison between sequences obtained by both direct HPLC-MS de novo sequencing and cDNA cloning demonstrates the secretion of mature peptides derived from a pre-pro-peptide structure.