Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures.

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure.

Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags.

BACKGROUND: Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites. STUDY DESIGN AND METHODS: A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry. RESULTS: Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%). CONCLUSIONS: The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.

Thermal Degradation of Small Molecules: A Global Metabolomic Investigation.

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolite extracts. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250 ��C) at different exposure times (30 s, 60 s, and 300 s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online ( xcmsonline.scripps.edu ). The results showed that heating at an elevated temperature of 100 ��C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250 ��C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250 ��C, 300s) with a significant formation of degradation and transformation products. The analysis of 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30 s). For example, tri- and diorganophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was identified in both the underivatized and derivatized forms of the plasma extracts and small molecule standard mixture, and was likely generated from oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions that mimic sample preparation and analysis in GC/MS experiments.

PEC1/AtABCG32 transport function in cuticle biosynthesis

Most aerial parts of the plants are covered by a hydrophobic coating called cuticle. The cuticle is formed of cutin, a complex mixture of esterified fatty acids that are embedded and associated with waxes. The cuticle often appears as a superposition of layers of different composition: The cuticle proper formed of cutin and a mixture of waxes and underneath, the cuticle layer containing cutin, intracuticular waxes and polysaccharides of the cell wall. In addition to its involvement in plant development by preventing organ fusions, the cuticle acts as a barrier to prevent water loss and protect plants against environmental aggressions such as excessive radiation or pathogens attacks. PEC1/AtABCG32 is an ABC transporter from the PDR family involved in cutin biosynthesis. Characterization of the peci mutant in Arabidopsis thaliana showed that PEC1 plays a significant role in the diffusion barrier formation in leaves and petals. The cuticles of leaves and flowers of peci are permeable and the cuticular layer rather than the cuticular proper was altered in the petals, underlining the importance of this particular layer in the maintenance of the diffusion barrier. Chemical analysis on the flower cutin monomer composition of ped mutant revealed a decrease in hydroxylated cutin monomers, suggesting a function of PEC1 in the incorporation of these monomers in the polymer cutin. However, the exact nature of the substrates of PEC1 remained elusive. PEC1 homologues in barley and rice, respectively HvABCG31/EIBI1 and OsABCG31, are also implicated in cuticle biosynthesis. Interestingly, the rice mutant displays more severe phenotypes such as dwarfism and spreading necrosis conducting to the seedling death. In this work, we further characterized osabcg31 mutant and hairpin-RNAi downregulated OsABCG31 plant lines showing reduced growth and cuticle permeability. Our analysis showed a decrease in hydroxylated cutin monomers and severe disruptions in the cuticle, which explain the permeability. Further insights into the function of the cuticle in rice resistance/susceptibility to Pathogens were obtained after inoculation with Magnaporthe oryzae, the fungus responsible for the rice blast disease. Osabcg31 as well as the transgenic lines downregulating OsABCG31 showed increased resistance to the fungus. However, only later steps of infection are reduced . and no impact is obseived on the germination or penetration stages, suggesting that the cuticle disruption per se is not responsible for the resistance. We further investigated the cause of the resistance by analyzing the expression of defense related gene in osabcg31 prior to infection. We found that osabcg31 constitutively express defense related genes, which may explain the resistance, the dwarfism and the cell death. osabcg31 is thus a tool to study the connection between cuticle, plant development and defense signaling networks in rice. The transport function of PEC1 family members is still unknown. In order to link cutin biosynthesis and transport activity, we combined ped mutation with mutations in cutin synthesis related genes. Here, we show that PEC1 acts independently from GPAT4 and GPAT8 pathway and partially overlaps with GPAT6 biosynthesis pathway that leads to the production of hydroxylated C16 cutin precursor 2-Mono(10,16-dihydroxyhexadecanoylJglycerol (2-MHG). In addition, we noticed that despite a comparable cutin monomer composition, ped mutant leaves cuticle are permeable while that of gpat6 mutant are not. This finding raises the possibility of PEC1 being required for the incorporation of C16 hydroxylated monomers and their structural arrangement rather than their direct transport towards the cuticle. A careful investigation of the cuticle permeability, cutin composition and ultrastructure during leave development in Wt plants and ped mutants revealed a possible different regulation of several pathways of cutin biosynthesis and showed the importance of PEC1 function early during leave cuticle maturation. In order to elucidate the transport activity of PEC1, we successfully expressed PEC1 in Nicotiana benthamiana plant system for direct transport experiments. This system will be used to test the PEC 1-dependent transport of potential substrates such as sn-2-monoacylglycerol loaded with a hydroxylated C16 fatty acid. -- Toutes les parties a��riennes des plantes sont recouvertes d'une couche hydrophobe appel��e ��cuticule��. Cette cuticule est compos��e de cutine, un polym��re d'acides gras est��rifi��s, et de cires. La cuticule appara��t souvent sous forme de couches superpos��es: une premi��re couche ext��rieure appel��e ��cuticle proper�� form��e de cutine et d'un m��lange de cires, et une deuxi��me couche, la ��cuticle layer��, form��e de cutine associ��e �� des cires intracuticulaires et des polysaccharides pari��taux. La cuticule joue le r��le de barri��re pr��venant contre la perte d'eau et les agressions environnementales. AtABCG32/PEC1 est un transporteur ABC de la famille des PDR impliqu�� dans la synth��se de la cutine. L'��tude du mutant peci d'Arabidopsis thaliana a r��v��l�� une fonction de PEC1 dans la formation de la barri��re de diffusion. La cuticule des feuilles et fleurs de peci est perm��able. Des alt��rations de la ��cuticle layer�� ont ��t�� d��montr��es, soulignant son importance dans le maintien de la barri��re. L'analyse de la composition de la cutine de peci a montr�� une r��duction sp��cifique en monom��res hydroxyl��s, sugg��rant un r��le de PEC1 dans leur incorporation dans la cuticule. Cependant, la nature exacte des substrats de PEC1 n'a pas ��t�� identifi��e. PEC1 poss��de deux homologues chez l'orge et le riz, respectivement HvABCG31 et OsABCG31, et qui sont impliqu��s dans la biosynth��se de la cuticule. Chez le riz, des ph��notypes plus s��v��res ont ��t�� observ��s tels que nanisme et n��croses conduisant �� la mort des jeunes plants. Dans cette ��tude, nous avons continu�� la caract��risation de osabcg31 ainsi que des lign��es de riz sous exprimant le g��ne OsABCG31 et pr��sentant une cuticule perm��able tout en ayant une meilleure croissance. Notre ��tude a d��montr�� une r��duction des monom��res hydroxyl��s de cutine et une d��sorganisation de la structure de la cuticule, aggrav��e dans le mutant osabcg31. Ce r��sultat explique la perm��abilit�� observ��e. Des mformations P|us approfondies sur l'implication de la cuticule dans la r��sistance aux pathog��nes ont ��t�� obtenues apr��s inoculation du mutant osabcg31 et les lign��es sous- exprimant OsABCG31 avec une souche virulente de Magnaporthe Oryzae, le champignon responsable de la pyriculariose du riz. Les diff��rentes lign��es test��es ont d��montr�� une r��sistance au pathog��ne. Cependant, seules les ��tapes tardives de l'infection sont r��duites et aucun impact n'est observ�� sur la germination des spores ou la p��n��tration du champignon, sugg��rant que les modifications de la cuticule ne sont pas directement �� l'origine de la r��sistance. L'analyse de l'expression de g��nes impliqu��s dans la r��sistance �� Magnaporthe.oryzae a mis en ��vidence l'expression constitutive de ces g��nes en l'absence de tout contact avec le pathog��ne. Ceci explique la r��sistance, le nanisme et la mort cellulaire observ��s. Ainsi, osabcg31 repr��sente un outil efficace pour l'��tude int��gr��e des syst��mes de r��gulation de la d��fense, de d��veloppement des plantes et la cuticule. La nature des substrats transport��s par PEC1/AtABCG32 reste inconnue. Dans le but d'��tablir une liaison entre biosynth��se de cutine et transport des pr��curseurs par PEC1, la mutation peci a ��t�� combin��e avec des mutants impliqu��s dans diff��rentes voies de biosynth��se. Cette ��tude a d��montr�� une fonction ind��pendante de PEC1 de la voie de biosynth��se impliquant les enzymes GPAT4 et GPAT8, et une fonction partiellement ind��pendante de la voie impliquant GPAT6 qui m��ne �� la production de pr��curseurs sn-2- monoacylglycerol charg��s en acides gras en C16 (2-MHG). De plus, malgr�� un profil similaire en monom��res de cutine, gpat6 conserve une cuticule imperm��able alors que celle de PEC1 est perm��able. Ceci sugg��re que PEC1 est n��cessaire �� l'incorporation des monom��res en C16 et leur arrangement structurel plut��t que simplement �� leur transport direct. L'��tude approfondie de la perm��abilit�� cuticulaire, de la structure ainsi que de la composition en cutine pendant le d��veloppement des feuilles de peci et la plante sauvage a r��v��l�� l'existence de diff��rentes r��gulations des voies de biosynth��ses des monom��res et a d��montr�� l'importance de PEC1 dans les premi��res ��tapes de la mise en place de la cuticule. Pour identifier les substrats transport��s, l'expression de PEC1 chez le syst��me h��t��rologue Nicotiana benthamiana a ��t�� conduite avec succ��s. Ce syst��me sera utilis�� pour tester le transport de substrats potentiels tels que le sn-2-monoacylglycerol charg�� en acide gras en C16.

Coronary anomalies: what the radiologist should know

AbstractCoronary anomalies comprise a diverse group of malformations, some of them asymptomatic with a benign course, and the others related to symptoms as chest pain and sudden death. Such anomalies may be classified as follows: 1) anomalies of origination and course; 2) anomalies of intrinsic coronary arterial anatomy; 3) anomalies of coronary termination. The origin and the proximal course of anomalous coronary arteries are the main prognostic factors, and interarterial course or a coronary artery is considered to be malignant due its association with increased risk of sudden death. Coronary computed tomography angiography has become the reference method for such an assessment as it detects not only anomalies in origination of these arteries, but also its course in relation to other mediastinal structures, which plays a relevant role in the definition of the therapeutic management. Finally, it is essential for radiologists to recognize and characterize such anomalies.

Determina����o de ��cidos Carbox��licos na Atmosfera Urbana de S��o Paulo: uma abordagem anal��tica e ambiental

In this work, analytical strategies are evaluated in order to measure accurately the ambient levels of atmospheric organic acids. Environmental considerations about the determination of low molecular weight mono- and di-carboxylic acids from urban areas of S��o Paulo are described.

Prepara����o de nanocomp��sitos atrav��s do encapsulamento de pol��meros condutores em 2H-MoS2 e 1T-TiS2

Nanocomposites obtained by the encapsulation of conducting polymers such as polyaniline and polydiphenylamine in 2H-MoS2 and 1T-TiS2 are synthesized and characterized by X-ray diffraction and infrared spectrophotometry. The synthesis consists in intercalating the layered compound with n-butyllithium and subsequent exfoliation in water and organic solvents. The nanocomposites are obtained by the adsorption of the polymers into the single-layers sulfides and posterior restacking. The X-ray diffraction measurements showed that the organic conducting polymers are encapsulated in mono and bilayers arrangement in a well-ordered fashion to produce single phase compounds.

S��ntese e caracteriza����o dos nanocomp��sitos K0,1(PEO)xMoS2 (X= 0,5; 1,2)

The reaction of an aqueous solution of poly(ethylene oxide) (peo - mw 100.000) with a neutral aqueous suspension of single layers of MoS2 was studied. The single layers aqueous suspension was prepared by first intercalating lithium (using n-Butyllithium in n-hexane) and reaction of these ternary compound with water under ultrasound stirring. The suspension was washed several times with water until neutral pH. The suspension was mixed with the PEO aqueous solution in the presence of KCl. Two single phase compounds were obtained with the expansion of 4,8 and 9,0��, attributed to the solvation of the intercalated potassium cations with mono and double layers, respectively.

Lamellarin D bioconjugates I: synthesis and cellular internalization of PEG-derivatives

Herein is reported the design and synthesis of poly(ethylene glycol) derivatives of Lamellarin D with the aim of modulating their physicochemical properties, and improving the biological activity. Mono-, di- and tri-PEG conjugates with improved solubility were obtained in 18-57% overall yields from the corresponding partially protected phenolic derivatives of Lamellarin D. Conjugates 1-9 were tested in a panel of three human tumor cell lines (MDA-MB-231 breast, A-549 lung and HT-29 colon) to evaluate their cytotoxicity. Several compounds exhibited enhanced cellular internalization, and more than 85% of the derivatives showed a lower GI50 than Lam-D. Furthermore, cell cycle arrest at G2 phase, and apoptotic cell-death pathways were determined for Lamellarin D and these derivatives.

Lamellarin D bioconjugates I: synthesis and cellular internalization of PEG-derivatives

Herein is reported the design and synthesis of poly(ethylene glycol) derivatives of Lamellarin D with the aim of modulating their physicochemical properties, and improving the biological activity. Mono-, di- and tri-PEG conjugates with improved solubility were obtained in 18-57% overall yields from the corresponding partially protected phenolic derivatives of Lamellarin D. Conjugates 1-9 were tested in a panel of three human tumor cell lines (MDA-MB-231 breast, A-549 lung and HT-29 colon) to evaluate their cytotoxicity. Several compounds exhibited enhanced cellular internalization, and more than 85% of the derivatives showed a lower GI50 than Lam-D. Furthermore, cell cycle arrest at G2 phase, and apoptotic cell-death pathways were determined for Lamellarin D and these derivatives.

S��ntese e atividade citot��xica de alguns azido-ciclopaladados estabilizados com ligantes bifosf��nicos

Some cyclopalladated compounds containing the azido group ligand and the (C-N) ring of N,N-dimethylbenzylamine have been prepared by bridge opening reactions of dimmer azide complex precursor with some diphosphines in different stoichiometric quantities. The neutral or ionic, mono or binuclear complexes synthesized were characterized by elemental analyses, I. R. spectroscopy and NMR techniques. The series of complexes was screened for cytotoxicity against a panel three human tumour cells lines(C6,Hep-2,HeLa). All complexes were found to be cytotoxic (IC50) at ��M concentrations while one complex having the coordination bond N-Pd ruptured also displayed some differential cytotoxicity.

Terapia fotodin��mica: aspectos farmacol��gicos, aplica����es e avan��os recentes no desenvolvimento de medicamentos

Photodynamic Therapy (PDT) is a clinical procedure, which utilize a photosensitive compound and light. This is a new modality of treatment for cancer, aged related macular degenerescence (AMD), psoriasis, arthritis, arterial restenosis, etc which exhibits efficiency, less traumatic effects, low recovery time and few co-lateral effects. The first officially approved drug for PDT by the Food and Drug Administration (EUA) is Photofrin<FONT FACE=Symbol>��</FONT>, which is applied for cancer. A new generation drug for PDT, Visudyne<FONT FACE=Symbol>��</FONT> was recently approved to treat AMD; its photoactive compound is BPDMA, a benzoporphyrin mono-acid derivative (chlorin-type molecule). A concise history, technical information and some drugs for PDT are reported.

Uma nova s��ntese de alfa-bromo-acetofenonas e sua aplica����o na obten����o de 2-benzoil-benzofuranas

alpha-Bromoacetophenones are important in organic synthesis. They have been widely used as precursors of several natural products. Several methods of bromination aiming at their synthesis have been described, however they furnish a mixture of starting material 10, mono 8 and dibromide 11 products. We developed a novel, simple and efficient synthesis of these compounds with applications in the synthesis of benzoylbenzofurans 9, compounds with important pharmacological properties, such as the ability of dilating the coronary artery and analgesic action. Such compounds have also been used as key intermediates to obtain quinone systems.

Biossensor enzim��tico para detec����o de fungicidas ditiocarbamatos: estudo cin��tico da enzima alde��do desidrogenase e otimiza����o do biossensor

Initially, all major factors that affect the rate of the AldH-catalyzed reaction (enzyme concentration, substrate concentration, temperature and pH) were investigated. Optimal activity was observed between pH values of 7.5 and 9.5 in the temperature range of 25 to 50 ��C. Kinetic parameters, such as Km (2.92 ��mol L-1) and Vmax (1.33 10-2 ��mol min-1) demonstrate a strong enzyme-substrate affinity. The sensors were based on screen-printed electrodes modified with the Meldola Blue-Reinecke salt (MBRS) combination. Operational conditions (NAD+ and substrate contents, enzyme loading and response time) were optimized. Also, two enzyme immobilization procedures were tested: entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) and crosslinking with glutaraldehyde. Chronoamperometry was employed to observe the biosensor responses during enzymatic hydrolysis of propionaldehyde and also to construct inhibition curves with maneb and zineb fungicides. Best results were found with the following conditions: [NAD+] = 0.25 mmol L-1; [propionaldehyde] = 80 ��mol L-1; enzyme loading = 0.8 U per electrode; response time = 10 min, and inhibition time = 10 min. Current intensities around 103 &plusmn; 13 nA with the sensors and good stability was obtained for both immobilization procedures. Detection limits, calculated using 10% inhibition were 31.5 ��g L-1 and 35 ��g L-1 for maneb and zineb, respectively. Results obtained with other MBRS-modified electrodes consisting of mono and bi-enzymic sensors were compared. The ability to catalyze NADH oxidation by MB was also highlighted.