Probing the hormonal activity of fractionated molecular humic components in tomato auxin mutants

We progressively reduced the complexity of humic matter by a mild sequential removal of unbound or free components, weakly, and strongly bound molecules. The auxin-like response of residues from each step was tested using tomato (cv. Micro-Tom) seedlings expressing DR5 auxin synthetic promoter fused to the beta-glucuronidase (GUS) reporter gene and the low auxin-sensitivity diageotropica (dgt) mutant. Both exogenous auxin and humic matter promoted lateral root emergence in the control, but failed to induce lateral roots in the dgt mutant. When strongly bound components were removed from humic matter by breaking the ester and ether bonds, the humic residues lost their ability to induce the DR5

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M(r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always < 2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

Peripheral Oxidative Stress Biomarkers in Mild Cognitive Impairment and Alzheimer`s Disease

Oxidative stress has been associated with normal aging and Alzheimer`s disease (AD). However, little is known about oxidative stress in mild cognitive impairment (MCI) patients who present a high risk for developing AD. The aim of this study was to investigate plasma production of the lipid peroxidation marker, malonaldehyde (MDA) and to determine, in erythrocytes, the enzymatic antioxidant activity of catalase, glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) in 33 individuals with MCI, 29 with mild probable AD and 26 healthy aged subjects. GR/GPx activity ratio was calculated to better assess antioxidant defenses. The relationship between oxidative stress and cognitive performance was also evaluated by the Mini Mental State Examination (MMSE). AD patients showed higher MDA levels than both MCI and healthy elderly subjects. MCI subjects also exhibited higher MDA levels compared to controls. Catalase and GPx activity were similar in MCI and healthy individuals but higher in AD. GR activity was lower in MCI and AD patients than in healthy aged subjects. Additionally, GR/GPx ratio was higher in healthy aged subjects, intermediate in MCI and lower in AD patients. No differences in GST activity were detected among the groups. MMSE was negatively associated with MDA levels (r = -0.31, p = 0.028) and positively correlated with GR/GPx ratio in AD patients (r = 0.68, p < 0.001). MDA levels were also negatively correlated to GR/GPx ratio (r = -0.31, p = 0.029) in the AD group. These results suggest that high lipid peroxidation and decreased antioxidant defenses may be present early in cognitive disorders.

Structural and chemical basis for anticancer activity of a series of 'beta'-tubulin ligands: molecular modeling and 3D QSAR studies

An important approach to cancer therapy is the design of small molecule modulators that interfere with microtubule dynamics through their specific binding to the ²-subunit of tubulin. In the present work, comparative molecular field analysis (CoMFA) studies were conducted on a series of discodermolide analogs with antimitotic properties. Significant correlation coefficients were obtained (CoMFA(i), q² =0.68, r²=0.94; CoMFA(ii), q² = 0.63, r²= 0.91), indicating the good internal and external consistency of the models generated using two independent structural alignment strategies. The models were externally validated employing a test set, and the predicted values were in good agreement with the experimental results. The final QSAR models and the 3D contour maps provided important insights into the chemical and structural basis involved in the molecular recognition process of this family of discodermolide analogs, and should be useful for the design of new specific ²-tubulin modulators with potent anticancer activity.

Lercanidipine reduces matrix metalloproteinase-2 activity and reverses vascular dysfunction in renovascular hypertensive rats

Increased expression/activity of matrix metalloproteinases (MMPs), especially MMP-2, plays a role in the vascular alterations induced by hypertension, and increased oxidative stress is a major factor activating MMPs. Here, we hypothesized that lercanidipine, a calcium channel blocker, could attenuate the increases in oxidative stress and MMP-2 expression/activity in the two-kidney, one-clip (2K-1C) hypertensive rats. Sham-operated or 2K-1C hypertension rats were treated with lercanidipine 2.5 mg/kg/day (or vehicle) starting three weeks after hypertension was induced. Systolic blood pressure was monitored weekly. After five weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall were studied in hematoxylin/eosin sections. Aortic MMP-2 levels were determined by gelatin zymography. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real-time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined using a fluorometric method. Lercanidipine attenuated 2K-1C hypertension (224 12 versus 183 11 mm Hg in 2K-1C rats and 2K-1C + Lercandipine rats, respectively; P < 0.01) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of 2K-1C rats (all P < 0.05). Lercandipine attenuated 2K-1C-induced increases in MMP-2 by more than 60% and blunted 2K-1C-induced increases in oxidative stress (both P < 0.001). While hypertension-induced significant aortic wall hypertrophy and approximately 9-fold increases in the ratio of MMP-2MMP-2 mRNA expression (both P < 0.05), lercandipine did not affect these changes. These results suggest that lercanidipine produces antihypertensive effects and reverses the endothelial dysfunction associated with 2K-1C hypertension, probably through mechanisms involving antioxidant effects leading to lower MMP-2 activation. (C) 2008 Elsevier B.V. All rights reserved.

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress. we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201 +/- 5 vs. 163 +/- 7 mm Hg in diabetic rats untreated and treated with lercaniclipine, respectively; P < 0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P < 0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P < 0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P < 0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2MMP-2 expression. (C) 2008 Published by Elsevier B.V.

Electronic Structure and Spectro-Structural Correlations of Fe(III)Zn(II) Biomimetics for Purple Acid Phosphatases: Relevance to DNA Cleavage and Cytotoxic Activity

Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe(II)M(II) center (M(II) = Fe, Mn, Zn) in the active site and are able to catalyze the hydrolysis of a variety of phosphoric acid esters. The dinuclear complex [(H(2)O)Fe(III)(mu-OH)Zn(II)(L-H)](CIO(4))(2) (2) with the ligand 2-[N-bis(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H(2)L-H) has recently been prepared and is found to closely mimic the coordination environment of the Fe(III)Zn(II) active site found in red kidney bean PAP (Neves et al. J. Am. Chem. Soc. 2007, 129, 7486). The biomimetic shows significant catalytic activity in hydrolytic reactions. By using a variety of structural, spectroscopic, and computational techniques the electronic structure of the Fe(III) center of this biomimetic complex was determined. In the solid state the electronic ground state reflects the rhombically distorted Fe(III)N(2)O(4) octahedron with a dominant tetragonal compression align ad along the mu-OH-Fe-O(phenolate) direction. To probe the role of the Fe-O(phenolate) bond, the phenolate moiety was modified to contain electron-donating or -withdrawing groups (-CH(3), -H, -Br, -NO(2)) in the 5-position. Tie effects of the substituents on the electronic properties of the biomimetic complexes were studied with a range of experimental and computational techniques. This study establishes benchmarks against accurate crystallographic struck ral information using spectroscopic techniques that are not restricted to single crystals. Kinetic studies on the hydrolysis reaction revealed that the phosphodiesterase activity increases in the order -NO(2)<- Br <- H <- CH(3) when 2,4-bis(dinitrophenyl)phosphate (2,4-bdnpp) was used as substrate, and a linear free energy relationship is found when log(k(cat)/k(0)) is plotted against the Hammett parameter a. However, nuclease activity measurements in the cleavage of double stranded DNA showed that the complexes containing the electron-withdrawing -NO(2) and electron-donating CH3 groups are the most active while the cytotoxic activity of the biomimetics on leukemia and lung tumoral cells is highest for complexes with electron-donating groups.

A modified signed likelihood ratio test in elliptical structural models

In this paper we deal with the issue of performing accurate testing inference on a scalar parameter of interest in structural errors-in-variables models. The error terms are allowed to follow a multivariate distribution in the class of the elliptical distributions, which has the multivariate normal distribution as special case. We derive a modified signed likelihood ratio statistic that follows a standard normal distribution with a high degree of accuracy. Our Monte Carlo results show that the modified test is much less size distorted than its unmodified counterpart. An application is presented.

Enzyme kinetics, structural analysis and molecular modeling studies on a series of Schistosoma mansoni PNP inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the development of novel drugs against schistosomiasis, a neglected tropical disease that affects about 200 million people worldwide. In the present work, enzyme kinetic studies were carried out in order to determine the potency and mechanism of inhibition of a series of SmPNP inhibitors. In addition to the biochemical investigations, crystallographic and molecular modeling studies revealed important molecular features for binding affinity towards the target enzyme, leading to the development of structure-activity relationships (SAR).

Synthesis, screening for antiacetylcholinesterase activity and binding mode prediction of a new series of [3-(disubstituted-phosphate)-4,4,4-trifluoro-butyl]-carbamic acid ethyl esters

A series of nine new [3-(disubstituted-phosphate)-4,4,4-trifluoro-butyl]-carbamic acid ethyl esters (phosphate-carbamate compounds) was obtained through the reaction of (4,4,4-trifluoro-3-hydroxybut-1-yl)-carbamic acid ethyl esters with phosphorus oxychloride followed by the addition of alcohols. The products were characterized by ¹H, 13C, 31P, and 19F NMR spectroscopy, GC-MS, and elemental analysis. All the synthesized compounds were screened for acetylcholinesterase (AChE) inhibitory activity using the Ellman method. All compounds containing phosphate and carbamate pharmacophores in their structures showed enzyme inhibition, being the compound bearing the diethoxy phosphate group (2b) the most active compound. Molecular modeling studies were performed to investigate the detailed interactions between AChE active site and small-molecule inhibitor candidates, providing valuable structural insights into AChE inhibition.

Association Between Physical Activity in Parks and Perceived Environment: A Study With Adolescents

Background: The purpose of this study was to analyze the relationship between adolescents' physical activity practice and their perception about the environment of urban parks. Methods: A school-based representative sample (n = 1,718; boys = 40.4%) of teenagers of Curitiba, Southern region of Brazil. A questionnaire was employed to identify perceived parks environmental features as well as physical activity practice in the parks (PAP), habitual physical activity (HPA) and demographics. The relationship between PAP and parks environments was analyzed through multivariate logistic regression controlling for age and socioeconomic status, HPA and parks distance. Results: After controlling for confounders PAP was associated with lack of space to be physically active, activities to choose from and equipments for both boys and girls, (odds ratio (OR)-ranging from 1.5 to 1.8). Among boys, having people of same age (OR = 1.5) and accessibility (OR = 2.0) showed association with PAP only in crude analysis. However, among girls, to be bulled or teased (OR = 1.4) and accessibility (OR = 1.7) were associated with PAP after confounding control. Conclusions: The results showed that specific attributes in parks may be considered and offered to increase the likelihood of physical activity practice among adolescents in such locations.

Effectiveness of a School-Based Intervention on Physical Activity for High School Students in Brazil: The Saude na Boa Project

Background: We evaluated the effectiveness of a school-based intervention on the promotion of physical activity among high school students in Brazil: the Saude no Boa project. Methods: A school-based, randomized trial was carried out in 2 Brazilian cities: Recife (northeast) and Florianopolis (south). Ten schools in each city were matched by size and location, and randomized into intervention or control groups. The intervention included environmental/organizational changes, physical activity education, and personnel training and engagement. Students age 15 to 24 years were evaluated at baseline and 9 months later (end of school year). Results: Although similar at baseline, after the intervention, the control group reported significantly fewer d/wk accumulating 60 minutes+ moderate-to-vigorous physical activity (MVPA) in comparison with the intervention group (2.6 versus 3.3, P < .001). The prevalence of inactivity (0 days per week) rose in the control and decreased in the intervention group. The odds ratio for engaging at least once per week in physical activity associated with the intervention was 1.83 (95% CI = 1.24-2.71) in the unadjusted analysis and 1.88 (95% CI = 1.27-2.79) after controlling for gender. Conclusion: The Saude no Boa intervention was effective at reducing the prevalence of physical inactivity. The possibility of expanding the intervention to other locations should be considered.

The burden of physical activity on type 2 diabetes public healthcare expenditures among adults: a retrospective study

Background: Determinants of public healthcare expenditures in type 2 diabetics are not well investigated in developing nations and, therefore, it is not clear if higher physical activity decreases healthcare costs. The purpose of this study was to analyze the relationship between physical activity and the expenditures in public healthcare on type 2 diabetes mellitus treatment. Methods: Cross-sectional study carried out in Brazil. A total of 121 type 2 diabetics attended to in two Basic Healthcare Units were evaluated. Public healthcare expenditures in the last year were estimated using a specific standard table. Also evaluated were: socio-demographic variables; chronological age; exogenous insulin use; smoking habits; fasting glucose test; diabetic neuropathy and anthropometric measures. Habitual physical activity was assessed by questionnaire. Results: Age (r = 0.20; p = 0.023), body mass index (r = 0.33; p = 0.001) and waist-to-hip ratio (r = 0.20; p = 0.025) were positively related to expenditures on medication for the treatment of diseases other than diabetes. Insulin use was associated with increased expenditures. Higher physical activity was associated with lower expenditure, provided medication for treatment of diseases other than diabetes (OR = 0.19; p = 0.007) and medical consultations (OR = 0.26; p = 0.029). Conclusions: Type 2 diabetics with higher enrollment in physical activity presented consistently lower healthcare expenditures for the public healthcare system.

Pimarane-type Diterpenes: Antimicrobial Activity against Oral Pathogens

Seven pimarane type-diterpenes re-isolated from Viguiera arenaria Baker and two semi-synthetic pimarane derivatives were evaluated in vitro against the following main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. The compounds ent-pimara-8(14), 15-dien-19-oic acid (PA); ent-8(14), 15-pimaradien-3 beta-ol; ent-15-pimarene-8 beta, 19-diol; ent-8(14), 15-pimaradien-3 beta-acetoxy and the sodium salt derivative of PA were the most active compounds, displaying MIC values ranging from 2 to 8 mu g.mL(-1). Thus, this class of compounds seems promising as a class of new effective anticariogenic agents. Furthermore, our results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new natural compounds that could be employed in the development of oral care products.