Ultrasensitive Simultaneous Quantification of 1,N(2)-Etheno-2 `-deoxyguanosine and 1,N(2-)Propano-2 `-deoxyguanosine in DNA by an Online Liquid Chromatography-Electrospray Tandem Mass Spectrometry Assay

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo) and 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2`-deoxyguanosine and ca. 500 amol of adducts in 100 mu g of hydrolyzed DNA M the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2`-deoxyguanosine and 1,N2-propano-2`-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilon dGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilon dGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/108 dGuo; brain, 2.96 +/- 1.43,N(2)-epsilon dGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanoclGuo/10(8) dGuo; and lung, 0,87 +/- 0.34 1,N(2)-edGuo/ 10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental an with pathological conditions and after air pollution exposure.

Association of soluble tumor necrosis factor receptors 1 and 2 with nephropathy, cardiovascular events, and total mortality in type 2 diabetes

AIMS/HYPOTHESIS: Soluble tumor necrosis factor receptors 1 and 2 (sTNFR1 and sTNFR2) contribute to experimental diabetic kidney disease, a condition with substantially increased cardiovascular risk when present in patients. Therefore, we aimed to explore the levels of sTNFRs, and their association with prevalent kidney disease, incident cardiovascular disease, and risk of mortality independently of baseline kidney function and microalbuminuria in a cohort of patients with type 2 diabetes. In pre-defined secondary analyses we also investigated whether the sTNFRs predict adverse outcome in the absence of diabetic kidney disease. METHODS: The CARDIPP study, a cohort study of 607 diabetes patients [mean age 61 years, 44 % women, 45 cardiovascular events (fatal/non-fatal myocardial infarction or stroke) and 44 deaths during follow-up (mean 7.6 years)] was used. RESULTS: Higher sTNFR1 and sTNFR2 were associated with higher odds of prevalent kidney disease [odd ratio (OR) per standard deviation (SD) increase 1.60, 95 % confidence interval (CI) 1.32-1.93, p < 0.001 and OR 1.54, 95 % CI 1.21-1.97, p = 0.001, respectively]. In Cox regression models adjusting for age, sex, glomerular filtration rate and urinary albumin/creatinine ratio, higher sTNFR1 and sTNFR2 predicted incident cardiovascular events [hazard ratio (HR) per SD increase, 1.66, 95 % CI 1.29-2.174, p < 0.001 and HR 1.47, 95 % CI 1.13-1.91, p = 0.004, respectively]. Results were similar in separate models with adjustments for inflammatory markers, HbA1c, or established cardiovascular risk factors, or when participants with diabetic kidney disease at baseline were excluded (p < 0.01 for all). Both sTNFRs were associated with mortality. CONCLUSIONS/INTERPRETATIONS: Higher circulating sTNFR1 and sTNFR2 are associated with diabetic kidney disease, and predicts incident cardiovascular disease and mortality independently of microalbuminuria and kidney function, even in those without kidney disease. Our findings support the clinical utility of sTNFRs as prognostic markers in type 2 diabetes.

Disfunção autonômica e doença vascular periférica em pacientes com diabete melito tipo 2

A disfunção autonômica está associada com aumento da mortalidade em pacientes diabéticos, especialmente naqueles com doença cardiovascular. Neuropatia periférica, mau controle glicêmico, dislipidemia e hipertensão são alguns dos fatores de risco para o desenvolvimento de doença vascular periférica (DVP) nestes pacientes. O objetivo deste estudo foi avaliar os fatores de risco associados com a presença de DVP em pacientes com DM tipo 2. Um estudo transversal foi realizado em 84 pacientes com DM tipo 2 ( 39 homens, idade média de 64,9 ± 7,5 anos). Os pacientes foram submetidos a uma avaliação clínica e laboratorial. A presença de DVP foi definida, utilizando-se um um aparelho manual de ultrasom com doppler (índice perna-braço < 0,9). A atividade autonômica foi avaliada através da análise da variabilidade da freqüência cardíaca (HRV) por métodos no domínio do tempo e da freqüência (análise espectral), e pelo mapa de retorno tridimensional durante o período do dia e da noite. Para a análise da HRV, um eletrocardiograma de 24 horas foi gravado e as fitas analisadas em um analisador de Holter Mars 8000 (Marquete). A potência espectral foi quantificada pela área em duas bandas de freqüência: 0,04-0,15 Hz – baixa freqüência (BF), 0,015-0,5 Hz – alta freqüência (AF). A razão BF/AF foi calculada em cada paciente. O mapa de retorno tridimensional foi construído através de um modelo matemático onde foram analisados os intervalos RR versus a diferença entre os intervalos RR adjacentes versus o número de contagens verificadas, e quantificado por três índices refletindo a modulação simpática (P1) e vagal (P2 e P3). DVP estava presente em 30 (36%) pacientes. Na análise univariada, pacientes com DVP apresentaram índices que refletem a modulação autonômica (análise espectral) diminuídos quando comparados aos pacientes sem DVP, respectivamente: BF = 0,19 ± 0,07 m/s2 vs. 0,29 ± 0,11 m/s2 P = 0,0001; BF/AF = 1,98 ± 0,9 m/s2 vs. 3,35 ± 1,83 m/s2 p = 0,001. Além disso, o índice que reflete a atividade simpática no mapa de retorno tridimensional (P1), foi mais baixo em pacientes com DVP (61,7 ± 9,4 vs. 66,8 ± 9,7 unidades arbitrárias, P = 0,04) durante a noite, refletindo maior ativação simpática neste período. Estes pacientes também apresentavam uma maior duração do diabetes (20 ± 8,1 vs. 15,3 ± 6,7 anos, P = 0,006), níveis de pressão arterial sistólica (154 ± 20 vs. 145 ± 20 mmHg, P = 0,04), razão cintura-quadril ( 0,98 ± 0,09 vs.0,92 ± 0,08, P = 0,01), e níveis de HbA1c mais elevados (7,7 ± 1,6 vs. 6,9 ± 1,7 %, P = 0,04), bem como valores de triglicerídeos ( 259 ± 94 vs. 230 ± 196 mg/dl, P= 0,03) e de excreção urinária de albumina ( 685,5 ± 1359,9 vs. 188,2 ± 591,1 μ/min, P = 0,02) superiores aos dos pacientes sem DVP.. Nos pacientes com DVP observou-se uma presença aumentada de nefropatia diabética (73,3% vs. 29,6% P = 0,0001), de retinopatia (73,3% vs. 44,4% P = 0,02) e neuropatia periférica (705 vs. 35,1% P = 0,006). Os grupos não diferiram quanto à idade, índice de massa corporal, tabagismo e presença de doença arterial coronariana. Na análise logística multivariada, a DVP permaneceu associada com a disfunção autonômica, mesmo após ter sido controlada pela pressão arterial sistólica, duração do DM, HbA1c, triglicerídeos e excreção urinária de albumina. Concluindo, pacientes com DVP e DM tipo 2 apresentam índices que refletem a modulação autonômica diminuídos, o que pode representar um fator de risco adicional para o aumento da mortalidade nestes pacientes.

Causas da indisciplina escolar no 2º e 3º Ciclo do Ensino Básico

É consensual na literatura e nos meios de comunicação social a crescente divulgação dos casos de indisciplina escolar, tendo estes assumido uma posição sociomediática a nível nacional e internacional. A frequência e complexidade desta temática fazem com que sejam cada vez mais crescentes os estudos científicos onde se pretende encontrar os factores/causas, as estratégias e o modo de prevenção da indisciplina escolar. Esta investigação teve como principal intuito compreender quais as causas da indisciplina escolar, tendo em conta a análise de quatro variáveis explícitas: contexto familiar, rendimento socioeconómico, motivação escolar e rendimento escolar. A amostra é composta 66 sujeitos entre os 10 e os 17 anos de idade (10 raparigas e 56 rapazes), divididos em dois grupos distintos (grupo de indisciplina e grupo comparação), que se encontram a frequentar uma escola do 2.º e 3.º Ciclo do ensino básico, situada no Funchal, Região Autónoma da Madeira, Portugal. Para a recolha parcial de dados foi utilizado o Questionário de Motivação Escolar - QME. Os resultados indicam uma clara associação entre baixa motivação escolar e baixo rendimento escolar com a ocorrência de indisciplina. Quanto ao contexto familiar e o rendimento socioeconómico, a associação já não é tão evidente, dado que somente o estado civil dos pais dos alunos (divorciados) e o tipo de habitação (social) se encontra maioritariamente associado aos alunos com comportamentos indisciplinados.

Estudo clínico das manifestações orais e fatores relacionados em pacientes diabéticos tipo 2

Conselho Nacional de Desenvolvimento Científico e Tecnológico

3-(1-Hydroxy-2-phenylprop-2-en-1-yl)phenol

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ethyl (E)-2-methoxyimino-2-(4-nitrobenzoyl)acetate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

(Z)-Ethyl 2-hydroxyimino-2-(4-nitrobenzyl)ethanoate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of day 2 embryo quality after conventional ICSI versus intracytoplasmic morphologically selected sperm injection (IMSI) using sibling oocytes

Objective: To evaluate whether intracytoplasmic morphologically selected sperm injection (IMSI) could influence early paternal effects by observing embryo quality at day 2.Study design: The study included 30 couples with at least one of the following criteria: male factor infertility, at least 2 previous failures of implantation or previous miscarriages after IVF/ICSI. Sibling oocytes of each patient were randomly assigned to either the ICSI group or the IMSI group. For IMSI, spermatozoa were selected at 8400x magnification through an inverted microscope equipped with Nomarski differential interference contrast optics, Uplan Apo 100x oil/1.35 objective lens and variable zoom lens. For conventional ICSI, spermatozoa were selected at 400x magnification. An embryo was defined as top quality if there were four identical blastomeres on day 2 with no fragments or multinucleation of blastomeres. Data were analysed using the Wilcoxon and chi-squared tests. The significance level was set at P < 0.05. The variables were analysed in relation to the general population and the subpopulations with or without male factor.Results: A total of 331 MII oocytes (30 oocyte retrievals) were selected and injected by the ICSI (n: 172) or IMSI (n: 159) procedure. For IMSI, only spermatozoa classified as morphologically normal at high magnification were used. No differences (P > 0.05) in fertilisation rate (ICSI: 70.9%; IMSI: 70.4%), early embryo cleavage rate (ICSI: 66.9%; IMSI: 60.4%) or cleavage rate (ICSI: 99.2%; IMSI: 99.1%) were observed. on day 2, as compared to ICSI, IMSI provided a similar proportion of top quality embryos (ICSI: 57.8%; IMSI: 52.2%; P > 0.05). These results were not influenced by the presence or absence of male factor.Conclusion: In terms of embryo quality at day 2, IMSI had the same performance as conventional ICSI. However, we cannot exclude the possibility that IMSI effects occur only as a positive later paternal effect. (C) 2010 Elsevier B.V. All rights reserved.

Plasma concentrations of 13,14-dihydro-15-keto prostaglandin F-2-alpha (PGFM), progesterone and estradiol in pregnant and nonpregnant diestrus cross-bred bitches

The canine corpus luteum (CL) typically sustains elevated plasma progesterone concentrations for 2 months or more, with a peak approximately 15-25 days after ovulation, followed by a slow decline. The processes involved in the slow, protracted regression of the CL over the remaining 1.5-2-month period in nonpregnant bitches and until shortly prepartum in pregnant bitches are not well characterized. The rapid luteolysis that occurs immediately prepartum appears to be a result of a prepartum rise in peripheral PGF. The potential role of PGF in the slow regression process in the several weeks preceding parturition and in nonpregnant bitches after 15-25 days after ovulation is not known. Therefore, plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F-2-alpha (PGFM), progesterone (P-4) and estradiol (E-2) Were determined and compared in bitches during nonpregnant diestrus (n = 9) or pregnancy (n = 8). During the gradual decrease in plasma concentrations of progesterone in both groups, the P-4 pattern appeared unrelated to changes in either E-2 or PGFM concentrations. The PGFM pattern was different between diestrus and pregnant bitches (P > 0.01); there was an apparent progressive but slow increase in PGFM in pregnant bitches from Days 30 to 60, followed by a large increase prior to parturition; concentrations declined immediately postpartum. However, there were no increases in PGFM during the same interval in nonpregnant bitches. Mean estradiol concentrations were sporadically elevated during the last third of pregnancy and less so in nonpregnant diestrus; there was no acute prepartum increase in estradiol associated with the PGFM increase. In summary, although there were no apparent changes in peripheral PGF(2)alpha concentration involved in regulating the slow protracted phase of luteal regression in nonpregnant bitches, modest increases in PGFM may play a role in ovarian function after mid-gestation in pregnant bitches. Furthermore, the acute prepartum rise in PGFM was not dependent on any concomitant increase in estradiol concentrations. (c) 2006 Elsevier B.V. All rights reserved.

Biocompatibility of an adhesive system and 2-hydroxyethylmethacrylate

The aim of the study was to evaluate the biocompatibility of an adhesive system and a resin component when implanted into connective tissue of rats. Forty sponges embedded in both materials: Scotchbond MP (SBMP/3M - Group A) and 2 - hydroxyethyl-methacrylate (HEMA - Group B), were implanted into dorsal connective tissue of 20 animals. After 7, 15, 30, or 60 days of the implantation, the animals were sacrificed; implant sites were excised and immersed for 24 hours in Kamovisky's fixative. The samples were processed under routine histologic technique, being stained with H & E. Histological evaluation showed that both materials promoted at 7 days intense inflammatory response with predominance of neutrophils and macrophages. The intense connective reaction was replaced for fibroblastic proliferation associated with macrophages and foreign body giant cells over time. The persistent moderate inflammatory reaction adjacent to scattered fragments of materials was greater to HEMA than to the SBMP. Both experimental materials did not show acceptable biocompatibility with connective tissue of rats in spite of allowing an evident connective tissue healing.

Effect of incorporation of 2-tert-butylaminoethyl methacrylate on flexural strength of a denture base acrylic resin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A(2) homologue from Bothrops pirajai venom complexed with rosmarinic acid

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization of the Lys49 PLA(2) homologue, myotoxin II, from the venom of Atropoides nummifer

Myotoxin H, a Lys49 catalytically inactive phospholipase A(2) homologue from Atropoides nummifer venom, was purified, characterized and crystallized. The crystals belongs to the tetragonal system, space group P4(3)2(1)2, with unit cell parameters (a=b=68.66 and c=63.87 Angstrom). Diffraction data were collected to a resolution of 2.32 Angstrom. The crystal structure is currently being determined using molecular replacement techniques. (C) 2004 Elsevier B.V. All rights reserved.