Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6

Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K-l of 0.9 muM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.

Epizootic activity of Murray Valley encephalitis and Kunjin viruses in an aboriginal community in the Southeast Kimberley region of Western Australia: Results of mosquito fauna and virus isolation studies

We undertook annual surveys of flavivirus virus activity in the community of Billiluna of Western Australia in the southeast Kimberley region between 1989 and 2001. Culex annulirostris was the dominant mosquito species, particularly in years of above average rains and flooding. Murray Valley encephalitis (MVE) virus was isolated in 8 of the 13 years of the study from seven mosquito species, but more than 90% of the isolates were from Cx. annulirostris. The results suggest that MVE virus is epizootic in the region, with activity only apparent in years with average or above average rainfall and increased numbers of Cx. annulirostris. High levels of MVE virus activity and associated human cases were detected only once (in 1993) during the survey period. Activity of MVE virus could only be partially correlated with wet season rainfall and flooding, suggesting that a number of other factors must also be considered to accurately predict MVE virus activity at such communities.

Nitrification of high strength ammonia wastewtaer treatment - process selection is the major factor.

Biological nitrogen removal via the nitrite pathway in wastewater treatment is very important in Saving the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulation were carried out in a biofilm airlift reactor with autotrophic nitrifying biofilm. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.5 to 3.5 kg N/m3.d. Nitrite accumulation was stably achieved by the selective inhibition of nitrite oxidizers with free ammonia and dissolved oxygen limitation. Stable 100% conversion to nitrite could also be achieved even under the absence of free ammonia inhibition on nitrite oxidizers. Batch ammonium oxidation and nitrite oxidation with nitrite accumulating nitrifying biofilm showed that nitrite Oxidation was completely inhibited when free ammonia is higher than 0.2 mg N/L. However, nitrite oxidation activity was recovered as soon as the free ammonia concentration was below the threshold level when dissolved oxygen concentration was not the limiting factor. Fluorescence in situ hybridization analysis of cryosectioned nitrite accumulating nitrifying biofilm showed that the β-subclass of Proteobacteria, where ammonia oxidizers belong, was distributed outside the biofilm whereas the α-subclass of Proteobacteria, where nitrite oxidizers belong, was found mainly in the inner part of the biofilm. It is likely that dissolved oxygen deficiency or limitation in the inner part of the nitrifying biofilm, where nitrite oxidizers exist, is responsible for the complete shut down of the nitrite oxidizers activity under the absence of free ammonia inhibition.

Physical activity and sedentary behavior: A population-based study of barriers, enjoyment, and preference

The associations of physical activity and sedentary behavior with barriers, enjoyment, and preferences were examined in a population-based mail survey of 1,332 adults. Respondents reporting high enjoyment and preference for physical activity were more likely to report high levels of activity. Those reporting cost, the weather, and personal barriers to physical activity were less likely to be physically active. Preference for sedentary behavior was associated with the decreased likelihood of being physically active, and the weather as a barrier to physical activity was associated with the increased likelihood of sedentary behavior. These constructs can be used to examine individual and environmental influences on physical activity and sedentary behavior in specific populations and could inform the development of targeted interventions.

Cytotoxic iron chelators: Characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto-enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe-II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.

Dehydration converts DsbG crystal diffraction from low to high resolution

Diffraction quality crystals are essential for crystallographic studies of protein structure, and the production of poorly diffracting crystals is often regarded as a dead end in the process. Here we show a dramatic improvement of poorly diffracting DsbG crystals allowing high-resolution diffraction data measurement. Before dehydration, the crystals are fragile and the diffraction pattern is streaky, extending to 10 Angstrom resolution. After dehydration, there is a spectacular improvement, with the diffraction pattern extending to 2 Angstrom resolution. This and other recent results show that dehydration is a simple, rapid, and inexpensive approach to convert poor quality crystals into diffraction quality crystals.

Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group) underwent left coronary artery ligation (infarcted, Inf) or sham-operation (Sham). Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e) and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 ± 6; Inf 3 = 130 ± 9; Inf 30 = 92 ± 7 mmHg; P < 0.05). The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 ± 3; Inf 3 = 45 ± 2; Inf 30 = 29 ± 3 mmHg; P < 0.05), while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

QSAR modeling of antitubercular activity of diverse organic compounds

Tuberculosis (TB) is a worldwide infectious disease that has shown over time extremely high mortality levels. The urgent need to develop new antitubercular drugs is due to the increasing rate of appearance of multi-drug resistant strains to the commonly used drugs, and the longer durations of therapy and recovery, particularly in immuno-compromised patients. The major goal of the present study is the exploration of data from different families of compounds through the use of a variety of machine learning techniques so that robust QSAR-based models can be developed to further guide in the quest for new potent anti-TB compounds. Eight QSAR models were built using various types of descriptors (from ADRIANA.Code and Dragon software) with two publicly available structurally diverse data sets, including recent data deposited in PubChem. QSAR methodologies used Random Forests and Associative Neural Networks. Predictions for the external evaluation sets obtained accuracies in the range of 0.76-0.88 (for active/inactive classifications) and Q(2)=0.66-0.89 for regressions. Models developed in this study can be used to estimate the anti-TB activity of drug candidates at early stages of drug development (C) 2011 Elsevier B.V. All rights reserved.

Fat intake interacts with polymorphisms of Caspase9, FasLigand and PPARgamma apoptotic genes in modulating Crohn's disease activity

Background & aims: Crohn’s disease (CD) is a multifactorial disease where resistance to apoptosis is one major defect. Also, dietary fat intake has been shown to modulate disease activity. We aimed to explore the interaction between four single nucleotide polymorphisms (SNPs) in apoptotic genes and dietary fat intake in modulating disease activity in CD patients. Methods: Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) techniques were used to analyze Caspase9þ93C/T, FasLigand-843C/T, Peroxisome Proliferator-Activated Receptor gammaþ161C/T and Peroxisome Proliferator-Activated Receptor gamma Pro12Ala SNPs in 99 patients with CD and 116 healthy controls. Interactions between SNPs and fat intake in modulating disease activity were analyzed using regression analysis. Results: None of the polymorphisms analyzed influenced disease susceptibility and/or activity, but a high intake of total, saturated and monounsaturated fats and a higher ratio of n-6/n-3 polyunsaturated fatty acids (PUFA), was associated with a more active phenotype (p < 0.05). We observed that the detrimental effect of a high intake of total and trans fat was more marked in wild type carriers of the Caspase9þ93C/T polymorphism [O.R (95%CI) 4.64 (1.27e16.89) and O.R (95%CI) 4.84 (1.34e17.50)]. In the Peroxisome Proliferator-Activated Receptor gamma Pro12Ala SNP, we also observed that a high intake of saturated and monounsaturated fat was associated to a more active disease in wild type carriers [OR (95%CI) 4.21 (1.33e13.26) and 4.37 (1.52e12.51)]. Finally, a high intake of n-6 PUFA was associated with a more active disease in wild type carriers for the FasLigand-843C/T polymorphism [O.R (95%CI) 5.15 (1.07e24.74)]. Conclusions: To our knowledge, this is the first study to disclose a synergism between fat intake and SNPs in apoptotic genes in modulating disease activity in CD patients.

Synthesis, structural characterization and leishmanicidal activity evaluation of ferrocenyl N-heterocyclic compounds

Agência Financiadora: FCT - PTDC/QUI/72656/2006 ; SFRH/BPD/27454/2006; SFRH/BPD/44082/2008; SFRH/BPD/41138/2007

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.

Food consumption, physical activity and socio-economic status related to BMI, waist circumference and waist-to-height ratio in adolescents

Objective: To examine the association between obesity and food group intakes, physical activity and socio-economic status in adolescents. Design: A cross-sectional study was carried out in 2008. Cole’s cut-off points were used to categorize BMI. Abdominal obesity was defined by a waist circumference at or above the 90th percentile, as well as a waist-to-height ratio at or above 0?500. Diet was evaluated using an FFQ, and the food group consumption was categorized using sex-specific tertiles of each food group amount. Physical activity was assessed via a self-report questionnaire. Socio-economic status was assessed referring to parental education and employment status. Data were analysed separately for girls and boys and the associations among food consumption, physical activity, socio-economic status and BMI, waist circumference and waist-to-height ratio were evaluated using logistic regression analysis, adjusting the results for potential confounders. Setting: Public schools in the Azorean Archipelago, Portugal. Subjects: Adolescents (n 1209) aged 15–18 years. Results: After adjustment, in boys, higher intake of ready-to-eat cereals was a negative predictor while vegetables were a positive predictor of overweight/ obesity and abdominal obesity. Active boys had lower odds of abdominal obesity compared with inactive boys. Boys whose mother showed a low education level had higher odds of abdominal obesity compared with boys whose mother presented a high education level. Concerning girls, higher intake of sweets and pastries was a negative predictor of overweight/obesity and abdominal obesity. Girls in tertile 2 of milk intake had lower odds of abdominal obesity than those in tertile 1. Girls whose father had no relationship with employment displayed higher odds of abdominal obesity compared with girls whose father had high employment status. Conclusions: We have found that different measures of obesity have distinct associations with food group intakes, physical activity and socio-economic status.

Relationship of milk intake and physical activity to abdominal obesity among adolescents

Background: Diet and physical activity (PA) are recognized as important factors to prevent abdominal obesity (AO), which is strongly associated with chronic diseases. Some studies have reported an inverse association between milk consumption and AO. Objective: This study examined the association between milk intake, PA and AO in adolescents. Methods: A cross-sectional study was conducted with 1209 adolescents, aged 15–18 from the Azorean Archipelago, Portugal in 2008. AO was defined by a waist circumference at or above the 90th percentile. Adolescent food intake was measured using a semi-quantitative food frequency questionnaire, and milk intake was categorized as ‘low milk intake’ (<2 servings per day) or ‘high milk intake’ ( 2 servings per day). PA was assessed via a self-report questionnaire, and participants were divided into active (>10 points) and low-active groups ( 10 points) on the basis of their reported PA. They were then divided into four smaller groups, according to milk intake and PA: (i) low milk intake/low active; (ii) low milk intake/active; (iii) high milk intake/low active and (iv) high milk intake/active. The association between milk intake, PA and AO was evaluated using logistic regression analysis, and the results were adjusted for demographic, body mass index, pubertal stage and dietary confounders. Results: In this study, the majority of adolescents consumed semi-skimmed or skimmed milk (92.3%). The group of adolescents with high level of milk intake and active had a lower proportion of AO than did other groups (low milk intake/low active: 34.2%; low milk intake/active: 26.9%; high milk intake/low active: 25.7%; high milk intake/active: 21.9%, P = 0.008). After adjusting for confounders, low-active and active adolescents with high levels of milk intake were less likely to have AO, compared with low-active adolescents with low milk intake (high milk intake/low active, odds ratio [OR] = 0.412, 95% confidence intervals [CI]: 0.201– 0.845; high milk intake/active adolescents, OR = 0.445, 95% CI: 0.235–0.845).Conclusion: High milk intake seems to have a protective effect on AO, regardless of PA level

Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata

The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmolL−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The shortterm assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μgL−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.