Activation of Leishmania (Leishmania) amazonensis arginase at low temperature by binuclear Mn(2+) center formation of the immobilized enzyme on a Ni(2+) resin

In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from L. (L.) amazonensis were studied by varying the concentration of Mn(2+) applied to the nickel column at 23 degrees C. The intensity of the binding of the enzyme to the Ni(2+) resin was directly proportional to the concentration of Mn(2+). Conformational changes of the enzyme may occur when the enzyme interacts with immobilized Ni(2+), allowing the following to occur: (1) entrance of Mn(2+) and formation of the metal bridge; (2) stabilization and activation of the enzyme at 23 degrees C; and (3) an increase in the affinity of the enzyme to Ni(2+) after the Mn(2+) activation step. The conformational alterations can be summarized as follows: the interaction with the Ni(2+) simulates thermal heating in the artificial activation by opening a channel for Mn(2+) to enter. (C) 2010 Elsevier Inc. All rights reserved.

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.

Circulating renin-angiotensin system and catecholamines in childhood: is there a role for birthweight?

There have been only a few reports on the sympathoadrenal and renin-angiotensin systems in children of small gestational age. The purpose of the present study was to investigate plasma levels of ACE (angiotensin-converting enzyme) activity, angiotensin and catecholamines in 8- to 13-year-old children and to determine whether there are correlations between the components of these systems with both birthweight and BP (blood pressure) levels. This clinical study included 66 children (35 boys and 31 girls) in two groups: those born at term with an appropriate birthweight [AGA (appropriate-for-gestational age) group, n = 31] and those born at term but with a small birthweight for gestational age [SGA (small-for-gestational age) group, n = 35]. Concentrations of angiotensin, catecholamines and ACE activity were determined in plasma. Circulating noradrenaline levels were significantly elevated in SGA girls compared with AGA girls (P = 0.036). In addition, angiotensin 11 and ACE activity were higher in SGA boys (P = 0.024 and P = 0.050 respectively). There was a significant association of the circulating levels of both angiotensin 11 and ACE activity with BP levels in our study population. Although the underlying mechanisms that link restricted fetal growth with later cardiovascular events are not fully understood, the findings in the present study support the link between low birthweight and overactivity of both sympathoadrenal and renin-angiotensin systems into later childhood.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B(2) and M1 muscarinic acetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (< EKWAP), a member of this structurally related peptide family, was essential for the development of captopril, the first site-directed ACE inhibitor used for the treatment of human hypertension. Nowadays, more Bj-PROs have been identified with higher ACE inhibition potency compared to Bj-PRO-5a. However, despite its modest inhibitory effect of ACE inhibition, Bj-PRO-5a reveals strong bradykinin-potentiating activity, suggesting the participation of other mechanisms for this peptide. In the present study, we have shown that Bj-PRO-5a induced nitric oxide (NO) production depended on muscarinic acetylcholine receptor M1 subtype (mAchR-M1) and bradykinin B(2) receptor activation, as measured by a chemiluminescence assay using a NO analyzer. Intravital microscopy based on transillumination of mice cremaster muscle also showed that both bradykinin B(2) receptor and mAchR-M1 contributed to the vasodilatation induced by Bj-PRO-5a. Moreover, Bj-PRO-5a-mediated vasodilatation was completely blocked in the presence of a NO synthase inhibitor. The importance of this work lies in the definition of novel targets for Bj-PRO-5a in addition to ACE, the structural model for captopril development. (C) 2011 Elsevier Inc. All rights reserved.

Comparison of signal-to-cutoff values in first, second, and third generation enzyme immunoassays for the diagnosis of HTLV-1/2 infection in ""at-risk"" individuals from Sao Paulo, Brazil

Data obtained during routine diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) and 2 (HTLV-2) in ""at-risk"" individuals from Sao Paulo, Brazil using signal-to-cutoff (S/C) values obtained by first, second, and third generation enzyme immunoassay (EIA) kits, were compared. The highest S/C values were obtained with third generation EIA kits, but no correlation was detected between these values and specific antibody reactivity to HTLV-1, HTLV-2, or untyped HTLV (p = 0.302). In addition, use of these third generation kits resulted in HTLV-1/2 false-positive samples. In contrast, first and second generation EIA kits showed high specificity, and the second generation EIA kits showed the highest efficiency, despite lower S/C values. Using first and second generation EIA kits, significant differences in specific antibody detection of HTLV-1, relative to HTLV-2 (p = 0.019 for first generation and p < 0.001 for second generation EIA kits) and relative to untyped HTLV (p = 0.025 for first generation EIA kits), were observed. These results were explained by the composition and format of the assays. In addition, using receiver operating characteristics (ROC) analysis, a slight adjustment in cutoff values for third generation EIA kits improved their specificities and should be used when HTLV ""at-risk"" populations from this geographic area are to be evaluated. (C) 2009 Elsevier B.V. All rights reserved.

Increased aerobic metabolism is essential for the beneficial effects of caloric restriction on yeast life span

Calorie restriction is a dietary regimen capable of extending life span in a variety of multicellular organisms. A yeast model of calorie restriction has been developed in which limiting the concentration of glucose in the growth media of Saccharomyces cerevisiae leads to enhanced replicative and chronological longevity. Since S. cerevisiae are Crabtree-positive cells that present repression of aerobic catabolism when grown in high glucose concentrations, we investigated if this phenomenon participates in life span regulation in yeast. S. cerevisiae only exhibited an increase in chronological life span when incubated in limited concentrations of glucose. Limitation of galactose, raffinose or glycerol plus ethanol as substrates did not enhance life span. Furthermore, in Kluyveromyces lactis, a Crabtree-negative yeast, glucose limitation did not promote an enhancement of respiratory capacity nor a decrease in reactive oxygen species formation, as is characteristic of conditions of caloric restriction in S. cerevisiae. In addition, K. lactis did not present an increase in longevity when incubated in lower glucose concentrations. Altogether, our results indicate that release from repression of aerobic catabolism is essential for the beneficial effects of glucose limitation in the yeast calorie restriction model. Potential parallels between these changes in yeast and hormonal regulation of respiratory rates in animals are discussed.

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

OBJECTIVES To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil. METHODS Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani. Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus. RESULTS Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi. All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed. CONCLUSIONS Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi.

Depot-specific modulation of adipokine levels in rat adipose tissue by diet-induced obesity: The effect of aerobic training and energy restriction

The present study examined the effects of aerobic training and energy restriction on adipokines levels in mesenteric (MEAT) and retroperitoneal (RPAT) white adipose tissue from obese rats. Male Wistar rats were fed with standard laboratory diet (Control group) or high fat diet (HFD). After 15 weeks, HFD rats were randomly assigned to the following groups: rats submitted to HFD, which were sedentary (sedentary HFD, n = 8) or trained (trained HFD, n = 8); or submitted to energy-restriction (ER), which were sedentary (sedentary ER, n = 8) or trained (trained ER, n = 8). Trained rats ran on a treadmill at 55% VO(2max) for 60 min/day, 5 days/week, for 10 weeks. ER rats were submitted to a reduction of 20% daily caloric ingestion compared to the Control group. ER and aerobic training decreased body weight, MEAT and RPAT absolute weight, and fat mass. IL-6, IL-10 and TNF-alpha levels were decreased and adiponectin did not change in RPAT in response to ER protocol. On the other hand, ER and the aerobic training protocol decreased IL-6, TNF-alpha and adiponectin levels in MEAT. Absolute MEAT weight showed a positive correlation with IL-6 (r = 0.464), INF-alpha (r = 0.508); and adiponectin (r = 0.342). These results suggest a tissue-specific heterogeneous response in adipokines level. The combination of the protocols (aerobic training and energy restriction) did not induce an enhanced effect. Published by Elsevier Ltd.

Leucine supplementation favors liver protein status but does not reduce body fat in rats during 1 week of food restriction

An important role in protein-energy metabolism has been attributed to leucine because of its long-term effects on body fat reduction and on the improvement of some indicators of protein status in rodents. The present study investigated the influence of leucine supplementation on the body composition and protein status of rats during the early phase of weight loss, which is characterized by a rapid loss of body weight. Thirty adult male Wistar rats were divided into 2 groups, a control and a leucine group (diet supplemented with 0.59% L-leucine), and were submitted to 1 week of 50% food restriction. The following parameters were evaluated: chemical carcass composition, protein and RNA content in liver and gastrocnemius muscle, and serum concentrations of insulin-like growth factor-1 and corticosterone. A higher liver weight and liver protein content were observed in the supplemented group (p < 0.05). However, no difference in body fat was found between groups (p > 0.05). The results indicate that low-dose leucine supplementation favors liver protein status but does not reduce body fat in rats during the early phase of rapid weight loss.

Immobilization of glucose oxidase enzyme (GOD) in large pore ordered mesoporous cage-like FDU-1 silica

Large pore ordered mesoporous silica FDU-1 with three-dimensional (3D) face-centered cubic, Fm3m arrangement of rnesopores, was synthesized under strong acid media using B-50-6600 poly(ethylene oxide)-poly(butylene oxide)-poly(ethylene oxide) triblock copolymer (EO(39)BO(47)EO(39)), tetraethyl orthosilicate (TEOS) and trimethyl-benzene (TMB). Large pore FDU-1 silica was obtained by using the following gel composition 1TEOS:0.00735B50-6600:0.00735TMB:6HCl:155H(2)O. The pristine material exhibited a BET specific surface area of 684 m(2) g(-1), total pore volume of 0.89 cm(3) g(-1), external surface area of 49 m(2) g(-1) and microporous volume of 0.09 cm(3) g(-1). The enzyme activity was determined by the Flow Injection Analysis-Chemiluminescence (FIA-CL) method. For GOD immobilized on the FDU-1 silica, GOD supernatant and GOD solution, the FIA-CL results were 9.0, 18.6 and 34.0 U, respectively. The value obtained for the activity of the GOD solution with FIA-CL method is in agreement with the 35 U, obtained by spectrophotometry. (C) 2011 Elsevier B.V. All rights reserved.

An asymmetric sandpile model with height restriction

We analyze by numerical simulations and mean-field approximations an asymmetric version of the stochastic sandpile model with height restriction in one dimension. Each site can have at most two particles. Single particles are inactive and do not move. Two particles occupying the same site are active and may hop to neighboring sites following an asymmetric rule. Jumps to the right or to the left occur with distinct probabilities. In the active state, there will be a net current of particles to the right or to the left. We have found that the critical behavior related to the transition from the active to the absorbing state is distinct from the symmetrical case, making the asymmetry a relevant field.

Capacitive electrolyte-insulator-semiconductor structures functionalised with a polyelectrolyte/enzyme multilayer: New strategy for enhanced field-effect biosensing

A novel strategy for enhanced field-effect biosensing using capacitive electrolyte-insulator-semiconductor (EIS) structures functionalised with pH-responsive weak polyelectrolyte/enzyme or dendrimer/enzyme multilayers is presented. The feasibility of the proposed approach is exemplarily demonstrated by realising a penicillin biosensor based on a capacitive p-Si-SiO(2) EIS structure functionalised with a poly(allylamine hydrochloride) (PAH)/penicillinase and a poly(amidoamine) dendrimer/penicillinase multilayer. The developed sensors response to changes in both the local pH value near the gate surface and the charge of macromolecules induced via enzymatic reaction, resulting in a higher sensitivity. For comparison, an EIS penicillin biosensor with adsorptively immobilised penicillinase has been also studied. The highest penicillin sensitivity of 100 mV/dec has been observed for the EIS sensor functionalised with the PAH/penicillinase multilayer. The lower and upper detection limit was around 20 mu M and 10 mM, respectively. In addition, an incorporation of enzymes in a multilayer prepared by layer-by-layer technique provides a larger amount of immobilised enzymes per sensor area, reduces enzyme leaching effects and thus, enhances the biosensor lifetime (the loss of penicillin sensitivity after 2 months was 10-12%). (C) 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Enzyme activity of horseradish peroxidase immobilized in chitosan matrices in alternated layers

The successful immobilization of enzymes such as horseradish peroxidase (HRP) in solid films is essential for applications in sensors and for fundamental studies aimed at identifying possible biotechnological devices. In this study we show that HRP can be immobilized in alternated layers with chitosan as the template material. The activity of HRP in HRP/chitosan films was preserved for several weeks, and could be detected optically upon monitoring the reaction with pyrogallol. The morphology of the film displayed stripes that disappeared after reaction with pyrogallol. Though the activity in the HRP/chitosan film was lower than in a homogeneous solution or in an LB film investigated earlier, the response was linear for a considerable period of time, which may be advantageous for sensing hydrogen peroxide. (C) 2009 Elsevier B.V. All rights reserved.

Enzyme immobilization on Ag nanoparticles/polyaniline nanocomposites

We show a simple strategy to obtain all efficient enzymatic broelectrochemical device, in which urease was immobilized oil electroactive nanostructured membranes (ENMs) made with polyaniline and silver nanoparticles (AgNP) stabilized in polyvinyl alcohol (PAni/PVA-AgNP). Fabrication of the modified electrodes comprised the chemical deposition of polyaniline followed by drop-coating of PVA-AgNP and urease, resulting in a final ITO/PAni/PVA-AgNP/urease electrode Configuration. For comparison. the electrochemical performance of ITO/PAni/urease electrodes (without Ag nanoparticles) was also studied. The performance of the modified electrodes toward Urea hydrolysis was investigated via amperometric measurements, revealing a fast increase in cathodic current with a well-defined peak upon addition of urea to the electrolytic solution. The cathodic currents for the ITO/PAni/PVA-AgNP urease electrodes were significantly higher than for the ITO/PAni/urease electrodes. The friendly environment provided by the ITO/PAni/PVA-AgNP electrode to the immobilized enzyme promoted efficient catalytic conversion of urea into ammonium and bicarbonate tons. Using the Michaelis-Menten kinetics equation, a K(M)(aPP) of 2.7 mmol L(-1) was obtained. indicating that the electrode architecture employed may be advantageous for fabrication of enzymatic devices with improved biocatalytic properties. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Enzyme immobilisation on electroactive nanostructured membranes (ENM): Optimised architectures for biosensing

Electroactive nanostructured membranes have been produced by the layer-by-layer (LbL) technique, and used to make electrochemical enzyme biosensors for glucose by modification with cobalt hexacyanoferrate redox mediator and immobilisation of glucose oxidase enzyme. Indium tin oxide (ITO) glass electrodes were modified with up to three bilayers of polyamidoamine (PAMAM) dendrimers containing gold nanoparticles and poly(vinylsulfonate) (PVS). The gold nanoparticles were covered with cobalt hexacyanoferrate that functioned as a redox mediator, allowing the modified electrode to be used to detect H(2)O(2), the product of the oxidase enzymatic reaction, at 0.0 V vs. SCE. Enzyme was then immobilised by cross-linking with glutaraldehyde. Several parameters for optimisation of the glucose biosensor were investigated, including the number of deposited bilayers, the enzyme immobilisation protocol and the concentrations of immobilised enzyme and of the protein that was crosslinked with PAMAM. The latter was used to provide glucose oxidase with a friendly environment, in order to preserve its bioactivity. The optimised biosensor, with three bilayers, has high sensitivity and operational stability, with a detection limit of 6.1 mu M and an apparent Michaelis-Menten constant of 0.20 mM. It showed good selectivity against interferents and is suitable for glucose measurements in natural samples. (C) 2008 Elsevier B.V. All rights reserved.