Differential regulation of the renin-angiotensin system by nicotine in WKY and SHR glia

Given that (1) the renin-angiotensin system (RAS) is compartmentalized within the central nervous system in neurons and glia (2) the major source of brain angiotensinogen is the glial cells, (3) the importance of RAS in the central control of blood pressure, and (4) nicotine increases the probability of development of hypertension associated to genetic predisposition; the objective of the present study was to evaluate the effects of nicotine on the RAS in cultured glial cells from the brainstem and hypothalamus of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Ligand binding, real-time PCR and western blotting assays were used to compare the expression of angiotensinogen, angiotensin converting enzyme, angiotensin converting enzyme 2 and angiotensin II type1 receptors. We demonstrate, for the first time, that there are significant differences in the basal levels of RAS components between WKY and SHR rats in glia from 1-day-old rats. We also observed that nicotine is able to modulate the renin-angiotensin system in glial cells from the brainstem and hypothalamus and that the SHR responses were more pronounced than WKY ones. The present data suggest that nicotine effects on the RAS might collaborate to the development of neurogenic hypertension in SHR through modulation of glial cells.

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.

The Central Nervous System as Target for Antihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (system. In agreement with its supposed neuronal action, this peptide dose-dependently evoked elevations of intracellular calcium concentration ([Ca(2+)](i)) in primary culture from postnatal rat brain. The N-terminal sequence of the peptide was not essential for induction of calcium fluxes, while any changes of C-terminal Pro or Ile residues affected Bj-PRO-10c`s activity. Using calcium imaging by confocal microscopy and fluorescence imaging plate reader analysis, we have characterized Bj-PRO-10c-induced [Ca(2+)](i) transients in rat brain cells as being independent from bradykinin-mediated effects and ACE inhibition. Bj-PRO-10c induced pertussis toxin-sensitive G(i/o)-protein activity mediated through a yet unknown receptor, influx and liberation of calcium from intracellular stores, as well as reduction of intracellular cAMP levels. Bj-PRO-10c promoted glutamate and GABA release that may be responsible for its antihypertensive activity and its effect on HR. (C) 2010 International Society for Advancement of Cytometry

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.

16-Bromoepiandrosterone, an activator of the mammalian immune system, inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the pentose-phosphate pathway which supplies cells with ribose 5-phosphate (R5P) and NADPH. R5P is the precursor for the biosynthesis of nucleotides while NADPH is the cofactor of several dehydrogenases acting in a broad range of biosynthetic processes and in the maintenance of the cellular redox state. RNA interference-mediated reduction of G6PDH levels in bloodstream-form Trypanosoma brucei validated this enzyme as a drug target against Human African Trypanosomiasis. Dehydroepiandrosterone (DHEA), a human steroidal pro-hormone and its derivative 16 alpha-bromoepiandrosterone (16BrEA) are uncompetitive inhibitors of mammalian G6PDH. Such steroids are also known to enhance the immune response in a broad range of animal infection models. It is noteworthy that the administration of DHEA to rats infected by Trypanosoma cruzi, the causative agent of Human American Trypanosomiasis (also known as Chagas` disease), reduces blood parasite levels at both acute and chronic infection stages. In the present work, we investigated the in vitro effect of DHEA derivatives on the proliferation of T. cruzi epimastigotes and their inhibitory effect on a recombinant form of the parasite`s G6PDH (TcG6PDH). Our results show that DHEA and its derivative epiandrosterone (EA) are uncompetitive inhibitors of TcG6PDH, with K(i) values of 21.5 +/- 0.5 and 4.8 +/- 0.3 mu M, respectively. Results from quantitative inhibition assays indicate 16BrEA as a potent inhibitor of TcG6PDH with an IC(50) of 86 +/- 8 nM and those from in vitro cell viability assays confirm its toxicity for T. cruzi epimastigotes, with a LD(50) of 12 +/- 8 mu M. In summary, we demonstrated that, in addition to host immune response enhancement, 16BrEA has a direct effect on parasite viability, most likely as a consequence of TcG6PDH inhibition. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

Superoxide radical protects liposome-contained cytochrome c against oxidative damage promoted by peroxynitrite and free radicals

The effects of nitrosative species on cyt c structure and peroxidase activity were investigated here in the presence of O(2)(center dot-) and anionic and zwitterionic vesicles. Nitrosative species were generated by 3-morpholinesydnonymine (SIN1) decomposition, using cyt c heme iron and/or molecular oxygen as electron acceptor. Far-and near-UV CD spectra of SIN1-treated cyt c revealed respectively a slight decrease of a-helix content (from 39 to 34%) and changes in the tryptophan structure accompanied by increased fluorescence. The Soret CD spectra displayed a significant decrease of the positive signal at 403 nm. EPR spectra revealed the presence of a low-spin cyt c form (S = 1/2) with g(1) = 2.736, g(2) = 2.465, and g(3) = 2.058 after incubation with SIN1. These data suggest that the concomitant presence of NO(center dot) and O(2)(center dot-) generated from dissolved oxygen, in a system containing cyt c and liposomes, promotes chemical and conformational modi. cations in cyt c, resulting in a hypothetical bis-histidine hexacoordinated heme iron. We also show that, paradoxically, O(2)(center dot-) prevents not only membrane lipoperoxidation by peroxide-derived radicals but also oxidation of cyt c itself due to the ability of O(2)(center dot-) to reduce heme iron. Finally, lipoperoxidation measurements showed that, although it is a more efficient peroxidase, SIN1-treated cyt c is not more effective than native cyt c in promoting damage to anionic liposomes in the presence of tert-ButylOOH, probably due to loss of affinity with negatively charged lipids. (C) 2009 Elsevier Inc. All rights reserved.

Effects of Micelles and Vesicles on the Oximolysis of p-Nitrophenyl Diphenyl Phosphate: A Model System for Surfactant-Based Skin-defensive Formulations against Organophosphates

The rates of oximolysis of p-nitrophenyl diphenyl phosphate (PNPDPP) by Acetophenoxime; 10-phenyl-10-hydi-oxyiminodecanoic acid; 4-(9-carboxynonanyl)-1-(9-carboxy-1-hydroyiminononanyl) benzene; 1-dodecyl-2-[(hydroxyimino)methyl]-pyridinium chloride (IV) and N-methylpyridinium-2-aldoxime chloride were determined in micelles of N-hexadecyl-N,N,N-trimethylammonium chloride (CTAC), N-hexadecyl-N,N-dimethylammonium propanesulfonate and dioctadecyldimethylammonium chloride (DODAC) vesicles. The effects of CTAC micelles and DODAC vesicles on the rates of oxymolysis of O,O-Diethyl O-(4-nitrophenyl) phosphate (paraoxon) by oxime IV were also determined. Analysis of micellar and vesicular effects on oximolysis of PNPDPP, using pseudophase or pseudophase with explicit consideration of ion exchange models, required the determination of the aggregate`s effects on the pK(a), of oximes and on the rates of PNPDPP hydrolysis. All aggregates increased the rate of oximolysis of PNPDPP and the results were analyzed quantitatively. In particular, DODAC vesicles catalyzed the reaction and increased the rate of oximolysis of PNPDPP by IV several million fold at pH`s compatible with pharmaceutical formulations. The rate increase produced by DODAC vesicles on the rate of oximolysis paraoxon by IV demonstrates the pharmaceutical potential of this system, since the substrate is used as an agricultural defensive agent and the surfactant is extensively employed in cosmetic formulations. (C) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:1040-1052, 2009

Aminoacetone, a putative endogenous source of methylglyoxal, causes oxidative stress and death to insulin-producing RINm5f cells

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4+ ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O-2(center dot-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 mu M) and Fe3+ ion (100 mu M) addition to the cell cultures had no effect, whereas Cu2+ (5.0-100 mu M) significantly increased cell death. Supplementation of the AA- and Cu2+-containing culture medium with antioxidants, such as catalase (5.0 mu M), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu2+ ions (10-50 mu M) relative to control cultures. This may imply higher activity of antioxidant enzymes C, in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) Plus Cu2+ (100 mu M) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

Development of a heavy metal sorption system through the P=S functionalization of coconut (Cocos nucifera) fibers

Lignocellulosic residues are interesting materials for the production of heavy metal adsorbents for aquatic systems. Whole fibers taken from coconut (Cocos nucifera) husks were functionalized with the thiophosphoryl (P=S) group by means of the direct reaction with CI(3)P=S, (CH(3)O)(2)CIP=S or (CH(3)CH(2)O)(2)CIP=S in order to obtain an adsorptive system for `soft` metal ions, particularly Cd(2+). These functionalized fibers (FFs) were characterized by means of elemental analysis, infrared spectroscopy, thermal analysis and acid-base titration. Adsorption isotherms for Cd(2+) fitted the Langmuir model, with binding capacities of 0.2-5 mmol g(-1) of FF at 25 degrees C. (C) 2009 Elsevier Ltd. All rights reserved.

Root system of Panicum maximum Jacq. cv. IPR-86 Mil(e)over-capnio fertilized with nitrogen and submitted to rotational stocking

The effect of nitrogen on the root system of the species Panicum maximum Jacq. cv. IPR-86 Mil (e) over cap nio, under grazing, was evaluated. The N rates were 0; 150; 300 and 450 kg/ha. year. The root density was evaluated during pregrazing at five years of successive N application, in three depths (0-10; 10-20 and 20-40 cm) and the root growth at 7, 14, 21, and 35 days after grazing. The grazing method adopted was rotational stocking. Root length and root mass densities in pre-and post-grazing presented maximum values at rates 204, 206, 192, and 197 kg/ha of N, respectively. The root growth (in root length density) increased, on average, until 29 days after grazing at rates 0, 150, and 300 kg/ha; at 450 kg/ha N rate, the increase was linear. Independently of N rates, around 60 and 25% of IPR-86 Mil (e) over cap nio cultivar root system was concentrated in 0-10 and 10-20 cm depth, respectively.

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper. we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4 h of incubation at 25 degreesC. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395 nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degreesC.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degreesC. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degreesC.Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme.An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Recombinant expression of glycerol-3-phosphate dehydrogenase using the Pichia pastoris system

In the present study, the GPD2 gene from Saccharomyces cerevisiae, which codifies for the enzyme glycerol-3-phosphate dehydrogenase (GPDH), was cloned from the pPICZ-alpha expression vector and used with the purpose of inducing the extracellular expression of the glycerol-3-phosphate dehydrogenase under the control of the methanol-regulated AOX promoter. The presence of the GPD2 insert was confirmed by PCR analysis. Pichia pastoris X-33 (Mut(+)) was transformed with linearized plasmids by electroporation and transformants were selected on YPDS plates containing 100 mu g/mL of zeocin. Several clones were selected and the functionality of this enzyme obtained in a culture medium was assayed. Among the mutants tested, one exhibited 3.1 x 10(-2) U/mg of maximal activity. Maximal enzyme activity was achieved at 6 days of growth. Medium composition and pre-induction osmotic stress influenced protein production. Pre-induction osmotic stress (culturing cells in medium with either 0.35 M sodium chloride or 1.0 M sorbitol for 4h prior to induction) led to an increase in cell growth with sorbitol and resulted in a significant increase in GPDH productivity with sodium chloride in 24h of induction approximately fivefold greater than under standard conditions (without pre-induction). (C) 2010 Elsevier B.V. All rights reserved.

Exploring S-type orbits in the Pluto-Charon binary system

This work generates, through a sample of numerical simulations of the restricted three-body problem, diagrams of semimajor axis and eccentricity which defines stable and unstable zones for particles in S-type orbits around Pluto and Charon. Since we consider initial conditions with 0 <= e <= 0.99, we found several new stable regions. We also identified the nature of each one of these newly found stable regions. They are all associated to families of periodic orbits derived from the planar circular restricted three-body problem. We have shown that a possible eccentricity of the Pluto-Charon system slightly reduces, but does not destroy, any of the stable regions.

Serological diagnosis of visceral leishmaniasis by an enzyme immunoassay using protein A in naturally infected dogs

Um ensaio de imunoadsorção enzimática para detecção de anticorpos contra Leishmania chagasi, utilizando antígeno total de formas promastigotas lisados foi desenvolvido. Cinqüenta cães com sintomas clínicos de leishmaniose visceral foram examinados. Esta técnica utilizou anti-IgG de cão conjugado a peroxidase ou proteína A conjugado a peroxidase. Foi verificado que nos animais positivos diagnosticados por exame parasitológico direto o ensaio ELISA utilizando proteína A conjugada a peroxidase (média da densidade óptica ± desvio padrão 2,078 ± 0,631) detecta mais anticorpos do que o sistema utilizando anti-IgG de cão conjugado a peroxidase (média da densidade óptica ± desvio padrão 1,008 ± 0,437), enquanto para os animais negativos o resultado obtido nos dois sistemas de detecção são similares. Esse resultado sugere que o sistema de ELISA utilizando proteína A conjugado a peroxidase pode ser útil na detecção de animais na fase aguda da infecção e desta forma auxiliar na identificação dos animais positivos e no controle desta importante zoonose.