Molecular basis for reduced estrone sulfate transport and altered modulator sensitivity of transmembrane helix (TM) 6 and TM17 mutants of multidrug resistance protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys(332) in transmembrane helix (TM) 6 and Trp(1246) in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp(1246) is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non-GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4'-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.

Perfis de resistência a agentes antimicrobianos de 5 estirpes do género Mycobacterium isoladas de ambiente Hospitalar

As infecções nosocomiais têm aumentado ao longo dos anos, resultando num aumento do tempo de permanência do doente no hospital, e permanecem como elevada causa de elevada morbilidade e mortalidade. As micobactérias são organismos que se encontram amplamente distribuídos no meio ambiente (M. mucogenicum, M. obuense e M. gordonae), incluindo, habitats marinhos (Mycobacterium marinum), sendo muitos deles patogénicos de mamíferos, e causadores de diferentes patologias, como a Lepra e a Tuberculose. M. marinum causa uma doença sistémica tal como tuberculose em peixes e pode causar infecções da pele em seres humanos (Granuloma de Aquário) que se podem propagar para estruturas mais profundas como ossos (osteomielite). Enquanto que M. obuense é causador de infecções do tracto respiratório, M. mucogenicum e M. gordonae promovem bacteremias. Este estudo teve como principal objectivo a identificação das populações bacterianas e o seu isolamento, em particular micobactérias ambientais em dois hospitais, que sabe serem responsáveis, cada vez mais por infecções atípicas como bacteremias (M. mucogenicum e M. gordonae), infecções pulmonares (M. obuense) e infecções cutâneas (M. marinum). Pretendeu-se também avaliar a resistência aos antibióticos e desinfectantes comummente utilizados no tratamento de infecções causadas por micobactérias não tuberculosas (MNT) através do cálculo da Concentração Mínima Inibitória (CMI) para aferir os perfis de resistência. Os resultados deste estudo demonstram a identificação de 186 espécies de bactérias em dois hospitais amostrados das quais se identificaram 5 estirpes de micobactérias – “M. gardonae” (10AIII, 29AIII e 35AIII), “M. obuense” (22DIII) e “M. mucogenicum” (24AIII). Das 5 estirpes de micobactérias identificadas “M. gardonae” 10AIII apresenta perfil de resistência ao imipenemo (CMI = 16 mg/L); “M. gardonae” 29AIII apresenta perfil de resistência à claritromicina (CMI = 8 mg/L) e “M. gardonae” 35AIII apresenta, por sua vez, apenas perfil de susceptibilidade intermédia ao imipenem (CMI = 8 mg/L). M. obuense 22DIII apresenta perfil de resistência ao imipenem (CMI = 32 mg/L), à tobramicina (CMI=32 mg/L) e à ciprofloxacina (CMI = 8 mg/L). “M. mucogenicum” apresenta perfil de resistência ao sulfametoxazol (CMI > 128 mg/L), à doxiciclina (CMI>64 mg/L), à tobramicina (CMI=16 mg/L) e à ciprofloxacina (CMI=4 mg/L).Em conclusão pôde-se verificar que além da presença de um grande leque de bactérias capazes de causar infecções nosocomiais nos hospitais, MNT também existem na forma multirresistente, o que revela uma problemática a ter em atenção. Esta requer mais estudo dos mecanismos de resistência e da sua disseminação, e obtenção de novos medicamentos com novos alvos, mais eficazes para combater as estirpes multirresistentes que ao longo dos anos tem aumentado.

Flat-top supercontinuum and tunable femtosecond fiber laser sources at 1.9-2.5 μm

We report the high-energy flat-top supercontinuum covering the mid-infrared wavelength range of 1.9-2.5 μm as well as electronically tunable femtosecond pulses between 1.98-2.22 μm directly from the thulium-doped fiber laser amplifier. Comparison of experimental results with numerical simulations confirms that both sources employ the same nonlinear optical mechanism - Raman soliton frequency shift occurring inside the Tm-fiber amplifier. To illustrate that, we investigate two versions of the compact diode-pumped SESAM mode-locked femtosecond thulium-doped all-silica-fiber-based laser system providing either broadband supercontinuum or tunable Raman soliton output, depending on the parameters of the system. The first system operates in the Raman soliton regime providing femtosecond pulses tunable between 1.98-2.22 μm. Wide and continuous spectral tunability over 240 nm was realized by changing only the amplifier pump diode current. The second system generates high-energy supercontinuum with the superior spectral flatness of better than 1 dB covering the wavelength range of 1.9-2.5 μm, with the total output energy as high as 0.284 μJ, the average power of 2.1 W at 7.5 MHz repetition rate. We simulate the amplifier operation in the Raman soliton self-frequency shift regime and discuss the role of induced Raman scattering in supercontinuum formation inside the fiber amplifier. We compare this system with a more traditional 1.85-2.53 μm supercontinuum source in the external highly-nonlinear commercial chalcogenide fiber using the Raman soliton MOPA as an excitation source. The reported systems1 can be readily applied to a number of industrial applications in the mid-IR, including sensing, stand-off detection, medical surgery and fine material processing.

Teoria da mente e linguagem metacognitiva em crianças com idades compreendidas entre os 3 e os 5 anos de idade

Dissertação de Mestrado apresentada ao Instituto Superior de Psicologia Aplicada para obtenção de grau de Mestre na especialidade de Psicologia Educacional.

Introdução ao conceito de número racional: análise de Manuais do 5º ano de escolaridade

Dissertação de Mestrado apresentada no ISPA – Instituto Universitário para obtenção do grau de Mestre em Psicologia especialidade de Psicologia Educacional.

Levantamento epidemiológico da cárie dentária de escolares de 5 a 12 anos, residentes na zona rural e urbana do município de Natércia, Minas Gerais, Brasil, 2010.

A maior exposição da população brasileira ao flúor das águas e dos dentifrícios fluoretados tem sido apontada como principal responsável pela redução da prevalência e incidência de cárie. Dessa forma, o objetivo do trabalho foi avaliar a prevalência de cárie através dos índices CPO e ceo dos alunos, com idade entre 5 e 12 anos, estudantes das Escolas Municipal e Estadual da cidade de Natércia - MG no ano de 2010. Para tanto foram realizadas comparações dos alunos residentes na zona urbana, que possuem água fluoretada, com os da zona rural, que não possuem água fluoretada. Foram avaliadas clinicamente todas as crianças em relação aos índices de dentes cariados, perdidos e obturados para dentes permanentes (CPOD) e decíduos (ceo). A análise desta frequência acumulada da doença em relação aos alunos da zona urbana e rural mostrou uma prevalência mais elevada da doença nos alunos da zona rural, que apresentam índices mais altos do que os residentes na zona urbana. Os serviços odontológicos de Natércia têm conseguido cuidar da demanda acumulada, restaurado as lesões de cárie dentária, pois o componente obturado é maior em muitas faixas etárias. Estes serviços, entretanto, não conseguem diminuir a velocidade da incidência da instalação da cárie dentária, nem tão pouco compensar a falta dos efeitos benéficos do flúor, inexistente na água de abastecimento na zona rural . Assim sendo, conclui-se que a água de abastecimento público fluoretada tem influência na redução dos índices da cárie e que os serviços odontológicos públicos do Município de Natércia precisam criar novas estratégias coletivas de educação e promoção de saúde em paralelo aos atendimentos clínicos individuais, principalmente na zona rural do município. Deve-se procurar intensificar as mudanças de hábitos nocivos alimentares e o uso de dentifrícios fluoretados com uma boa técnica de escovação e uma dieta equilibrada.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Pyrimidine-5'-nucleotidase Campinas, A New Mutation (p.r56g) In The Nt5c3 Gene Associated With Pyrimidine-5'-nucleotidase Type I Deficiency And Influence Of Gilbert's Syndrome On Clinical Expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Chemo-, Regio- And Stereoselective Heck Arylation Of Allylated Malonates: Mechanistic Insights By Esi-ms And Synthetic Application Toward 5-arylmethyl-γ-lactones.

We describe herein a general method for the controlled Heck arylation of allylated malonates. Both electron-rich and electron-poor aryldiazonium salts were readily employed as the aryl-transfer agents in good yields and in high chemo-, regio-, and stereoselectivity without formation of decarboxylated byproducts. Reaction monitoring via ESI-MS was used to support the formation of chelated Pd species through the catalytic cycle. Additionally, some Heck adducts were successfully used in the total synthesis of pharmacologically active γ-lactones.

Direct Adhesive Pin-retained Restorations For Severely Worn Dentition Treatment: A 1.5-year Follow-up Report.

Excessive occlusal surface wear can result in occlusal disharmony, functional and esthetic impairment. As a therapeutic approach, conventional single crowns have been proposed, but this kind of treatment is complex, highly invasive and expensive. This case report describes the clinical outcomes of an alternative minimally invasive treatment based on direct adhesive-pin retained restorations. A 64-year-old woman with severely worn dentition, eating problems related to missing teeth and generalized tooth hypersensitivity was referred for treatment. Proper treatment planning based on the diagnostic wax-up simulation was used to guide the reconstruction of maxillary anterior teeth with direct composite resin over self-threading dentin pins. As the mandibular remaining teeth were extremely worn, a tooth-supported overdenture was installed. A stabilization splint was also used to protect the restorations. This treatment was a less expensive alternative to full-mouth rehabilitation with positive esthetic and functional outcomes after 1.5 years of follow-up.

Synthesis And Antitumor Activity Of Novel 1-substituted Phenyl 3-(2-oxo-1,3,4-oxadiazol-5-yl) β-carbolines And Their Mannich Bases.

A series of novel 1-(substituted phenyl)-3-(2-oxo-1,3,4-oxadiazol-5-yl) β-carbolines (4a-e) and the corresponding Mannich bases 5-9(a-c) were synthesized and evaluated for their in vitro antitumor activity against seven human cancer cell lines. Compounds of 4a-e series showed a broad spectrum of antitumor activity, with GI50 values lower than 15μM for five cell lines. The derivative 4b, having the N,N-dimethylaminophenyl group at C-1, displayed the highest activity with GI50 in the range of 0.67-3.20μM. A high selectivity and potent activity were observed for some Mannich bases, particularly towards resistant ovarian (NCI-ADR/RES) cell lines (5a, 5b, 6a, 6c and 9b), and ovarian (OVCAR-03) cell lines (5b, 6a, 6c, 9a, 9b and 9c). In addition, the interaction of compound 4b with DNA was investigated by using UV and fluorescence spectroscopic analysis. These studies indicated that 4b interact with ctDNA by intercalation binding.

S-nitrosoglutathione Accelerates Recovery From 5-fluorouracil-induced Oral Mucositis.

Mucositis induced by anti-neoplastic drugs is an important, dose-limiting and costly side-effect of cancer therapy. To evaluate the effect of the topical application of S-nitrosoglutathione (GSNO), a nitric oxide donor, on 5-fluorouracil (5-FU)-induced oral mucositis in hamsters. Oral mucositis was induced in male hamsters by two intraperitoneal administrations of 5-FU on the first and second days of the experiment (60 and 40 mg/kg, respectively) followed by mechanical trauma on the fourth day. Animals received saline, HPMC or HPMC/GSNO (0.1, 0.5 or 2.0 mM) 1 h prior to the 5-FU injection and twice a day for 10 or 14 days. Samples of cheek pouches were harvested for: histopathological analysis, TNF-α and IL-1β levels, immunohistochemical staining for iNOS, TNF-α, IL-1β, Ki67 and TGF-β RII and a TUNEL assay. The presence and levels of 39 bacterial taxa were analyzed using the Checkerboard DNA-DNA hybridization method. The profiles of NO released from the HPMC/GSNO formulations were characterized using chemiluminescence. The HPMC/GSNO formulations were found to provide sustained release of NO for more than 4 h at concentration-dependent rates of 14 to 80 nmol/mL/h. Treatment with HPMC/GSNO (0.5 mM) significantly reduced mucosal damage, inflammatory alterations and cell death associated with 5-FU-induced oral mucositis on day 14 but not on day 10. HPMC/GSNO administration also reversed the inhibitory effect of 5-FU on cell proliferation on day 14. In addition, we observed that the chemotherapy significantly increased the levels and/or prevalence of several bacterial species. Topical HPMC/GSNO accelerates mucosal recovery, reduces inflammatory parameters, speeds up re-epithelization and decreases levels of periodontopathic species in mucosal ulcers.

Recolonization Of Mutans Streptococci After Application Of Chlorhexidine Gel.

Streptococcus mutans is specifically suppressed by intensive treatment with chlorhexidine gel, but the time for recolonization and the effect on other oral bacteria are not totally clear. In this study, recolonization of mutans streptococci was evaluated in nine healthy adult volunteers, who were highly colonized with this microorganism. Stimulated saliva was collected before (baseline) and at 1, 7, 14, 21 and 28 days after application of 1% chlorhexidine gel on volunteers' teeth for two consecutive days. On each day, the gel was applied using disposable trays for 3 x 5 min with intervals of 5 min between each application. Saliva was plated on blood agar to determine total microorganisms (TM); on mitis salivarius agar to determine total streptococci (TS) and on mitis salivarius agar plus bacitracin to determine mutans streptococci (MS). Chlorhexidine was capable of reducing the counts of MS and the proportion of MS with regard to total microorganisms (%MS/TM) (p<0.05), but these values did not differ statistically from baseline (p>0.05) after 14 days for MS and 21 days for %MS/TM. The counts of TM and TS and the proportion of MS to total streptococci did not differ statistically from baseline (p>0.05) after chlorhexidine treatment. The results suggest that the effect of chlorhexidine gel treatment on suppression of mutans streptococci is limited to less than a month in highly colonized individuals.

Effects Of Pde Type 5 Inhibitors On Left Ventricular Diastolic Dysfunction In Resistant Hypertension.

Resistant hypertension (RHTN) is a multifactorial disease characterized by blood pressure (BP) levels above goal (140/90 mmHg) in spite of the concurrent use of three or more antihypertensive drugs of different classes. Moreover, it is well known that RHTN subjects have high prevalence of left ventricular diastolic dysfunction (LVDD), which leads to increased risk of heart failure progression. This review gathers data from studies evaluating the effects of phosphodiesterase-5 (PDE-5) inhibitors (administration of acute sildenafil and short-term tadalafil) on diastolic function, biochemical and hemodynamic parameters in patients with RHTN. Acute study with sildenafil treatment found that inhibition of PDE-5 improved hemodynamic parameters and diastolic relaxation. In addition, short-term study with the use of tadalafil demonstrated improvement of LVDD, cGMP and BNP-32 levels, regardless of BP reduction. No endothelial function changes were observed in the studies. The findings of acute and short-term studies revealed potential therapeutic effects of IPDE-5 drugs on LVDD in RHTN patients.A Hipertensão arterial resistente (HAR) é uma doença multifatorial caracterizada por níveis pressóricos acima das metas (140/90 mmHg), a despeito de tratamento farmacológico otimizado de 3 ou mais fármacos anti-hipertensivos de diferentes classes. Pacientes diagnosticados como hipertensos resistentes apresentam alta prevalência de disfunção diastólica do ventrículo esquerdo (DDVE) que proporciona risco aumentado para insuficiência cardíaca. Esta revisão reúne dados de estudos prévios avaliando os efeitos dos inibidores de fosfodiesterase-5 (PDE-5) (administração aguda de sildenafil e de curto prazo de tadalafil) na função diastólica e nos parâmetros bioquímicos e hemodinâmicos em pacientes com HAR. O estudo agudo com sildenafil demonstrou que a inibição da PDE-5 melhorou os parâmetros hemodinâmicos e de relaxamento diastólico. Além disso, o estudo curto prazo com o uso de tadalafil revelou melhora da DDVE e dos níveis de GMPc e BNP-32, independente de redução de pressão arterial. A função endotelial não apresentou alteração com ambos os tratamentos. Os resultados dos estudos agudo e de curto prazo sugerem efeitos terapêuticos potenciais dos fármacos inibidores da PDE-5 na disfunção diastólica em pacientes com HAR.

Indução assimétrica 1,5-Anti na adição de enolatos de boro de metilcetonas beta-oxigenadas a aldeídos

High levels of substrate-based 1,5-stereoinduction are obtained in the boron-mediated aldol reactions of beta-oxygenated methyl ketones with achiral and chiral aldehydes. Remote induction from the boron enolates gives the 1,5-anti adducts, with the enolate pi-facial selectivity critically dependent upon the nature of the beta-alkoxy protecting group. This 1,5-anti aldol methodology has been strategically employed in the total synthesis of several natural products. At present, the origin of the high level of 1,5-anti induction obtained with the boron enolates is unclear, although a model based on a hydrogen bonding between the alkoxy oxygen and the formyl hydrogen has been recently proposed.