Glucose uptake in the mammalian stages of Trypanosoma cruzi

Trypanosoma cruzi, the agent of Chagas` disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T cruzi. The infective trypomastigotes showed the highest transport activity (V(max) = 5.34 +/- 0.54 nmol/min per mg of protein: K(m) = 0.38 +/- 0.01 mM) when compared to intracellular epimastigotes (V(max) = 2.18 +/- 0.20 nmol/min per mg of protein; K(m) = 0.39 +/- 0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T cruzi. (C) 2009 Elsevier B.V. All rights reserved.

Glucose-induced heat production, insulin secretion and lactate production in isolated Wistar rat pancreatic islets

Transplantation of pancreatic islets is efficient in improving the metabolic control and quality of life and in preventing severe hypoglycemia in patients with brittle type I diabetes mellitus. More accurate methods to assess islet viability would be extremely useful in designing target interventions for islet cytoprorection and in reducing the number of islets required to achieve insulin independence. Here we report on an application of calorimetry to evaluate the metabolic response of pancreatic islets to glucose stimulation. A significant increase in metabolic heat was produced by islet samples when consecutively subjected to 2.8 and 16.3 mmol L-1 glucose. Under these glucose concentrations, 1000 islets released average heat values of 9.16 +/- 0.71 mJ and 14.90 +/- 1.21 mJ over 50 min, respectively. Additionally, the glucose stimulation indexes were 1.67 +/- 0.30 for insulin. 1.72 +/- 0.13 for heat and 2.91 +/- 0.50 for lactate, raising the important possibility of substituting the secreted insulin index/ratio by the index/ratio of the heat released in the evaluation of Langerhans islets viability for transplantation. Altogether, Our results demonstrate the applicability of calorimetry to assess the quality of isolated pancreatic islets and to study vital islet functions. (c) 2008 Published by Elsevier B.V.

The Arabidopsis bZIP Gene AtbZIP63 Is a Sensitive Integrator of Transient Abscisic Acid and Glucose Signals

Glucose modulates plant metabolism, growth, and development. In Arabidopsis (Arabidopsis thaliana), Hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. Here, we show that the intensity of short-term responses to glucose can vary with ABA activity. We report that the transient (2 h/4 h) repression by 2% glucose of AtbZIP63, a gene encoding a basic-leucine zipper (bZIP) transcription factor partially involved in the Snf1-related kinase KIN10-induced responses to energy limitation, is independent of HXK1 and is not mediated by changes in ABA levels. However, high-concentration (6%) glucose-mediated repression appears to be modulated by ABA, since full repression of AtbZIP63 requires a functional ABA biosynthetic pathway. Furthermore, the combination of glucose and ABA was able to trigger a synergistic repression of AtbZIP63 and its homologue AtbZIP3, revealing a shared regulatory feature consisting of the modulation of glucose sensitivity by ABA. The synergistic regulation of AtbZIP63 was not reproduced by an AtbZIP63 promoter-5`-untranslated region:beta-glucuronidase fusion, thus suggesting possible posttranscriptional control. A transcriptional inhibition assay with cordycepin provided further evidence for the regulation of mRNA decay in response to glucose plus ABA. Overall, these results indicate that AtbZIP63 is an important node of the glucose-ABA interaction network. The mechanisms by which AtbZIP63 may participate in the fine-tuning of ABA-mediated abiotic stress responses according to sugar availability (i.e., energy status) are discussed.

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance. (C) 2011 Elsevier Inc. All rights reserved.

PREDICTION OF METAL CATION TOXICITY TO THE BIOLUMINESCENT FUNGUS GERRONEMA VIRIDILUCENS

A correlation between the physicochemical properties of mono- [Li(I), K(I), Na(I)] and divalent [Cd(II), Cu(II), Mn(II), Ni(II), Co(II), Zn(II), Mg(II), Ca(II)] metal cations and their toxicity (evaluated by the free ion median effective concentration. EC50(F)) to the naturally bioluminescent fungus Gerronema viridilucens has been studied using the quantitative ion character activity relationship (QICAR) approach. Among the 11 ionic parameters used in the current study, a univariate model based on the covalent index (X(m)(2)r) proved to be the most adequate for prediction of fungal metal toxicity evaluated by the logarithm of free ion median effective concentration (log EC50(F)): log EC50(F) = 4.243 (+/-0.243) -1.268 (+/-0.125).X(m)(2)r (adj-R(2) = 0.9113, Alkaike information criterion [AIC] = 60.42). Additional two- and three-variable models were also tested and proved less suitable to fit the experimental data. These results indicate that covalent bonding is a good indicator of metal inherent toxicity to bioluminescent fungi. Furthermore, the toxicity of additional metal ions [Ag(I), Cs(I), Sr(II), Ba(II), Fe(II), Hg(II), and Pb(II)] to G. viridilucens was predicted, and Pb was found to be the most toxic metal to this bioluminescent fungus (EC50(F)): Pb(II) > Ag(I) > Hg(I) > Cd(II) > Cu(II) > Co(II) Ni(II) > Mn(II) > Fe(II) approximate to Zn(II) > Mg(II) approximate to Ba(II) approximate to Cs(I) > Li(I) > K(I) approximate to Na(I) approximate to Sr(II)> Ca(II). Environ. Toxicol. Chem. 2010;29:2177-2181. (C) 2010 SETAC

Chemical modification of a nanocrystalline TiO(2) film for efficient electric connection of glucose oxidase

A novel biosensor for glucose was prepared by adsorption of 1,1`-bis(4-carboxybenzyl)-4,4`-bipyridinium di-bromide compound (H(2)BpybcBr(2)) onto the surface of a nanocrystalline TiO(2) film deposited onto FTO glasses, which was used as a platform to assemble the enzyme glucose oxidase to the electrode surface. The H(2)BpybcBr(2)/TiO(2)/FTO modified electrode was characterized by scanning electron microscopy, X-ray fluorescence image, cyclic voltammograms and spectroelectrochemical measurements. The immobilization of GOD on functionalized TiO(2) film led to stable amperometric biosensing for glucose with a linear range from 153 mu mol L(-1) to 1.30 mmol L(-1) and a detection limit of 51 mu mol L(-1). The apparent Michaelis-Menten constant (K(m)) was estimated to be 3.76 mmol L(-1), which suggested a high enzyme-substrate affinity. The maximum electrode sensitivity was 1.25 mu A mmol L(-1). The study proved that the combination of viologen mediators with TiO(2) film retains the electrocatalytic activity of the enzyme, and also enhances the electron transfer process, and hence regenerating the enzyme in the reaction with glucose. (C) 2010 Elsevier Inc. All rights reserved.

EVALUATION OF METAL TOXICITY BY A MODIFIED METHOD BASED ON THE FUNGUS GERRONEMA VIRIDILUCENS BIOLUMINESCENCE IN AGAR MEDIUM

Metal cation toxicity to basidiomycete fungi is poorly understood, despite its well-known importance in terrestrial ecosystems. Moreover, there is no reported methodology for the routine evaluation of metal toxicity to basidiomycetes. In the present study, we describe the development of a procedure to assess the acute toxicity of metal cations (Na(+), K(+), Li(+), Ca(2+), Mg(2+), Co(2+), Zn(2+), Ni(2+), Mn(2+), Cd(2+), and Cu(2+)) to the bioluminescent basidiomycete fungus Gerronema viridilucens. The method is based on the decrease in the intensity of bioluminescence resulting from injuries sustained by the fungus mycelium exposed to either essential or nonessential metal toxicants. The assay described herein enables LIS to propose a metal toxicity series to Gerronenia viridilucens based on data obtained from the bioluminescence intensity (median effective concentration [EC50] values) versus metal concentration: Cd(2+) > Cu(2+) > Mn(2+) approximate to Ni(2+) approximate to Co(2+) > Zn(2+) > Mg(2+) > Li(+) > K(+) approximate to Na(+) > Ca(2+), and to shed some li-ht on the mechanism of toxic action of metal cations to basidiomycete fungi. Environ. Toxicol. Chem. 2010;29:320-326. (C) 2009 SETAC

Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)(2)] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38(MAPK)/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N`]copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

Micro/nanostructured carbon composite modified with a hybrid redox mediator and enzymes as a glucose biosensor

A carbon micro/nanostructured composite based on cup-stacked carbon nanotubes (CSCNTs) grown onto a carbon felt has been found to be an efficient matrix for enzyme immobilization and chemical signal transduction. The obtained CSCNT/felt was modified with a copper hexacyanoferrate/polypyrrole (CuHCNFe/Ppy) hybrid mediator, and the resulting composite electrode was applied to H(2)O(2) detection, achieving a sensitivity of 194 +/- 15 mu A mmol(-1) L. The results showed that the CSCNT/felt matrix significantly increased the sensitivity of CuHCNFe/Ppy-based sensors compared to those prepared on a felt unrecovered by CSCNTs. Our data revealed that the improved sensitivity of the as-prepared CuHCNFe/Ppy-CSCNT/felt composite electrode can be attributed to the electronic interactions taking place among the CuHCNFe nanocrystals, Ppy layer and CSCNTs. In addition, the presence of CSCNTs also seemed to favor the dispersion of CuHCNFe nanocrystals over the Ppy matrix, even though the CSCNTs were buried under the conducting polymer layer. The CSCNT/felt matrix also enabled the preparation of a glucose biosensor whose sensitivity could be tuned as a function of the number of glucose oxidase (GOx) layers deposited through a Layer-by-Layer technique with an sensitivity of 11 +/- 2 mu A mmol(-1) L achieved at 15 poly(diallyldimethylammoniumchloride)/GOx bilayers. (C) 2011 Elsevier Ltd. All rights reserved.

In vitro basal cytotoxicity assay applied to estimate acute oral systemic toxicity of grandisin and its major metabolite

Preclinical investigations can start with preliminary in vitro studies before using animal models. Following this approach, the number of animals used in preclinical acute toxicity testing can be reduced. In this study, we employed an in-house validated in vitro cytotoxicity test based on the Spielmann approach for toxicity evaluation of the lignan grandisin, a candidate anticancer agent, and its major metabolite. the 4-O-demethylgrandisin, by neutral red uptake (NRU) assay, on mouse fibroblasts Balb/c 3T3 cell line. Using different concentrations of grandisin and its major metabolite (2.31; 1.16; 0.58; 0.29; 0.14; 0.07; 0.04; 0.002 mu M) in Balb/c 3T3-A31 NRU cytotoxicity assay, after incubation for 48 h, we obtained IC(50) values for grandisin and its metabolite of 0.078 and 0.043 mu M, respectively. The computed LD(50) of grandisin and 4-O-demethylgrandisin were 617.72 and 429.95 mg/kg, respectively. Both were classified under the Globally Harmonized System as category 4. Since pharmacological and toxicological data are crucial in the developmental stages of drug discovery, using an in vitro assay we demonstrated that grandisin and its metabolite exhibit distinct toxicity profiles. Furthermore, results presented in this work can contribute to reduce the number of animals required in subsequent pharmacological/toxicological studies. (C) 2010 Elsevier GmbH. All rights reserved.

Triphenylmethane dyes, an alternative for mediated electronic transfer systems in glucose oxidase biofuel cells

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bio-electrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 mu W cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 mu W cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxiclase bioanodes and/or biofuel cells. (C) 2011 Elsevier Inc. All rights reserved.

Heterogeneous photocatalytic degradation of acid dyes in aqueous TiO2 suspensions: Kinetics and toxicity

This work assesses the photocatalytic (TiO2/UV) degradation of a simulated acid dye bath (Yellow 3, Red 51, Blue 74, and auxiliary chemicals). Color and phytotoxicity removal were monitored by spectrophotometry and lettuce (Lactuca sativa) seeds as the test organism, respectively. Mineralization was determined by DOC analyses. Photocatalytic, photolytic, and adsorption experiments were performed, showing that adsorption was negligible. After 240 minutes of irradiation, it was achieved 96% and 78% of color removal with photocatalysis and photolysis, respectively. 37% of mineralization occurred with photocatalysis only. The dye bath was rendered completely non-toxic after 60 minutes of photocatalytic treatment; the same result was only achieved with photolysis after 90 minutes. A kinetic model composed of two first-order in series reactions was used. The first photocatalytic decolorization rate constant was k(1) = 0.062 min(-1) and the second k(2) = 0.0043 min(-1), approximately two times greater than the photolytic ones.

Synthesis, characterization and biological analysis of the complex [VO(Hdhp)(2)] (H(2)dhp=2,3-dihydroxypyridine)

A new vanadium (IV) complex with the monoanion of 2,3-dihydroxypyridine (H(2)dhp), or 3-hydroxy-2(1H)-pyridone, was synthesized, characterized by physicochemical techniques and tested biologically. The EPR data for the [VO(Hdhp)(2)] complex in DMF are: g(x) = 1.9768, g(y) = 1.9768 and g(z) = 1.9390; A values (10(-4) cm(-1)): A(x), 59.4; A(y//), 59.4; A(z), 171.0. The vV=O band in the IR spectrum of the complex is at 986 cm(-1). The complex is paramagnetic, with mu(eff) = 1.65 BM (d(1), spin-only) at 25 degrees C. The irreversible oxidation process [V(V)/V(IV)] of the [VO(Hdhp)(2)] complex, as revealed in a cyclic voltammogram, occurs at 876 mV. The calculated molecular structure of [VO(Hdhp)(2)] shows the vanadium(IV) center in a distorted square pyramidal environment, with the oxo ligand in the apical position and the oxygen donor atoms of the Hdhp ligands in the basal positions. The ability of [VO(Hdhp)(2)] to mimic insulin, and its toxicity to hepato-biliary functions, were investigated in streptozotocin-induced diabetic rats and it was concluded that the length of treatment and the amount of [VO(Hdhp)(2)] administered were effective in reducing experimental diabetes.

TiO(2)-PHOTOCATALYZED DEGRADATION OF PHENOL IN SALINE MEDIA IN AN ANNULAR REACTOR: HYDRODYNAMICS, LUMPED KINETICS, INTERMEDIATES, AND ACUTE TOXICITY

The photocatalytic degradation of phenol in aqueous suspensions of TiO(2) under different salt concentrations in an annular reactor has been investigated. In all cases, complete removal of phenol and mineralization degrees above 90% were achieved. The reactor operational parameters were optimized and its hydrodynamics characterized in order to couple mass balance equations with kinetic ones. The photodegradation of the organics followed a Langmuir-Hinshelwood-Hougen- Watson lumped kinetics. From GC/MS analyses, several intermediates formed during oxidation have been identified. The main ones were catechol, hydroquinone, and 3-phenyl-2-propenal, in this order. The formation of negligible concentrations of 4-chlorophenol was observed only in high salinity medium. Acute toxicity was determined by using Artemia sp. as the test organism, which indicated that intermediate products were all less toxic than phenol and a significant abatement of the overall toxicity was accomplished, regardless of the salt concentration.