A 4.2 kDa synthetic peptide as a potential probe to evaluate the antibacterial activity of coumarin drugs

The coumarin antibiotics are potent inhibitors of DNA replication whose target is the enzyme DNA gyrase, an ATP-dependent bacterial type II topoisomerase. The coumarin drugs inhibit gyrase action by competitive binding to the ATP-binding site of DNA gyrase B protein. The production of new biologically active products has stimulated additional studies on coumarin-gyrase interactions. In this regard, a 4.2 kDa peptide mimic of DNA gyrase B protein from Escherichia coli has been designed and synthesized. The peptide sequence includes the natural fragment 131-146 (coumarin resistance-determining region) and a segment containing the gyrase-DNA interaction region (positions 753-770). The peptide mimic binds to novobiocin (K-a = 1.4 +/- 0.3 x 10(5) m(-1)), plasmid (K-a = 1.6 +/- 0.5 x 10(6) m(-1)) and ATP (K-a = 1.9 f 0.4 x 10(3) m(-1)), results previously found with the intact B protein. on the other hand, the binding to novobiocin was reduced when a mutation of Arg-136 to Leu-136 was introduced, a change previously found in the DNA gyrase B protein from several coumarin-resistant clinical isolates of Escherichia coLi. In contrast, the binding to plasmid and to ATP was not altered. These results suggest that synthetic peptides designed in a similar way to that described here could be used as mimics of DNA gyrase in studies which seek a better understanding of the ATP, as well as coumarin, binding to the gyrase and also the mechanism of action of this class of antibacterial drugs. Copyright (C) 2004 European Peptide Society and John Wiley Sons, Ltd.

Thermal diffusivity analysis of doped poly(methyl-methacrylate) with electron donor and acceptor molecules

A computer-assisted method for analysing photoacoustic spectra has been developed in the Windows(TM) environment with the use of an easy graphical interface, the computer simulation was carried out with the aim of using the entire expression of the Rosencwaig-Gersho theory, thus permitting multiple applications. The simulation was applied to a system that mimics the electron transfer process in which the concentration of octaethylporphin donor molecules was constant whereas the concentration of duroquinone and 2,3-dichloro-5,6-dicyano-1, l-benzoquinone acceptor molecules varied. The increment of the acceptor concentration influenced the photoacoustic amplitude and phase signals. In the phase signal a significant shift to smaller values was observed, denoting a faster heat generation. The analysis of the photoacoustic signal enabled the determination of the thermal diffusivity, the result obtained through the simulation was about (7 +/- 1) x 10(-7) m(2) s(-1) indicating that changes in the photoacoustic phase signals were due to the electron transfer process rather than changes in the thermal properties of the sample.

Prostaglandin E-2 inhibits Paracoccidioides brasiliensis killing by human monocytes

Human monocytes lacked fungicidal activity against high virulence strain of Paracoccidioides brasiliensis, even after IFN-gamma activation. However, monocytes treated with indomethacin exhibited an effective killing against this fungus, suggesting a role of prostaglandin E-2 (PGE(2)) in the inhibition process. Thus, the purpose of this work was to determine whether the effect of PGE2 in fungicidal activity was related with decrease on H2O2 release, the metabolite involved in P. brasiliensis killing, and changes in the levels of TNF-alpha, IL-6 and IL-10. Human monocytes challenged with the fungus produced high PGE(2) levels, which in turn inhibited the fungicidal activity of these cells by reducing H2O2 and TNF-alpha production. (C) 2007 Elsevier Masson SAS. All rights reserved.

Decolorization of textile indigo dye by ligninolytic fungi

The indigo dye is extensively used by textile industries and is considered a recalcitrant substance, which causes environmental concern. Chemical products used on textile processing, which affect the environment through effluents, can be voluminous, colored and varied. Vat textile dyes, like indigo, are often used and dye mainly cellulosic fibers of cotton. Decolorization of this dye in liquid medium was tested with ligninolytic basidiomycete fungi from Brazil. Decolorization started in a few hours and after 4 days the removal of dye by Phellinus gilvus culture was in 100%, by Pleurotus sajor-caju 94%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No color decrease was observed in a sterile control. Thin layer chromatography of fungi culture extracts revealed only one unknown metabolite of Rf = 0.60, as a result of dye degradation. (C) 2001 Published by Elsevier B.V. B.V.

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger-fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane em,, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behaviour. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P 1-30 was estimated by measuring the permeability PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the PEGs of different pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St 11 conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation. (c) 2005 Wiley Periodicals, Inc.

Role of early GnRH administration in sexual behavior disorders of rat pups perinatally exposed to lead

The effects of maternal exposure to lead (Pb) during the perinatal ( 1% and 0.1% Pb) periods of sexual brain differentiation were studied in adult male offspring. Maternal Pb levels were measured after treatment. Behavioral (open field and sexual behavior), physical (sexual maturation, body and organ weights), and biochemical (testosterone levels and hypothalamic monoamine and respective metabolite levels) data were assessed in perinatally exposed offspring. The effects of gonadrotopin-releasing hormone (GnRH) administration to pups at birth on puberty and sexual behavior were also investigated in offspring postnatally exposed to the metal. Results showed that perinatal administration of the two Pb concentrations did not modify maternal weight gain; 1% Pb exposure reduced offspring body weight during the 7 days of treatment while no changes were observed after 0.1% Pb exposure; neither ph concentration altered offspring sexual maturation; the higher Pb concentration improved sexual behavior while the 0.1% concentration reduced it; exposure to 0.1% Pb caused decrease in testis weight, an increase in seminal vesicle weight and no changes in plasma testosterone levels; hypothalamic VMA levels were increased compared to the control group; GnRH administration reversed the effects of 0.1% Ph administration on male sexual behavior. These results show that perinatal exposure to ph had a dose-dependent effect on the sexual behavior of rats and that a decrease in GnRH source in the offspring was probably involved in the reduction of their sexual performance. (C) 2001 Elsevier B.V. All rights reserved.

Chemical evolution in the Asteraceae. The Oxidation-Reduction mechanism and production of secondary metabolites

This work describes the application of partial least squares (PLS) regression to variables that represent the oxidation data of several types of secondary metabolite isolated from the family Asteraceae. The oxidation states were calculated for each carbon atom of the involved compounds after these had been matched with their biogenetic precursor. The states of oxidation variations were named oxidation steps. This methodology represents a new approach to inspect the oxidative changes in taxa. Partial least square (PLS) regression was used to inspect the relationships among terpenoids, cournarins, polyacetylenes, and flavonoids from a data base containing approximately 27,000 botanical entries. The results show an interdependence between the average oxidation states of each class of secondary metabolite at tribe and sub tribe levels.

Dehydromonocrotaline inhibits mitochondrial complex 1. A potential mechanism accounting for hepatotoxicity of monocrotaline

Monocrotaline is a pyrrolizidine alkaloid present in plants of the Crotalaria species, which causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline. We evaluated the effects of monocrotaline and its metabolite on respiration, membrane potential and ATP levels in isolated rat liver mitochondria, and on respiratory chain complex I NADH oxidase activity in submitochondrial particles. Dehydromonocrotaline, but not the parent compound, showed a concentration-dependent inhibition of glutamate/malate-supported state 3 respiration (respiratory chain complex 1), but did not affect succinate-supported respiration (complex II). Only dehydromonocrotaline dissipated mitochondrial membrane potential, depleted ATP, and inhibited complex I NADH oxidase activity (IC50 = 62.06 mu M) through a non-competitive type of inhibition (K-I = 8.1 mu M). Therefore, dehydromonocrotaline is an inhibitor of the activity of respiratory chain complex I NADH oxidase, an action potentially accounting for the well-documented monocrotaline's hepatotoxicity to animals and humans. The mechanism probably involves change of the complex I conformation resulting from modification of cysteine thiol groups by the metabolite. (c) 2007 Elsevier Ltd. All rights reserved.

CHIRAL SCHWINGER MODEL WITH A PODOLSKY TERM AT FINITE-TEMPERATURE

We study an exactly solvable two-dimensional model which mimics the basic features of the standard model. This model combines chiral coupling with an infrared behavior which resembles low energy QCD. This is done by adding a Podolsky higher-order derivative term in the gauge field to the Lagrangian of the usual chiral Schwinger model. We adopt a finite temperature regularization procedure in order to calculate the non-trivial fermionic Jacobian and obtain the photon and fermion propagators, first at zero temperature and then at finite temperature in the imaginary and real time formalisms. Both singular and non-singular cases, corresponding to the choice of the regularization parameter, are treated. In the nonsingular case there is a tachyonic mode as usual in a higher order derivative theory, however in the singular case there is no tachyonic excitation in the spectrum.

Serotonergic mechanisms on breathing modulation in the rat locus coeruleus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kinetic properties of glycerophosphate oxidase isolated from dry baker's yeast

The glycerophosphate oxidase is a flavoprotein responsible for the catalysis of the oxidation of the glycerophosphate to dihydroxyacetone phosphate, through the reduction of the oxygen to hydrogen peroxide. The glycerophosphate oxidase from baker's yeast was specific for L-alpha-glycerol phosphate. It was estimated by monitoring the consumption of oxygen with an oxygraph. An increase of 32% in consumption of oxygen was obtained when the enzyme was concentrated 16-fold. The assay of enzyme was determined by the peroxidase chromogen method followed at 500 nm. The procedure for the standardization of the activity of the glycerophosphate oxidase from baker's yeast was accomplished, and the pH and temperature stability showed that the enzyme presented a high stability at pH 8.0, and the thermal stability was maintained up to 60 degrees C during I h. Such method allowed quantifying in the range 92-230 mM of glycerol phosphate, an important intermediate metabolite from lipid biosynthesis and glycolytic routes. (C) 2007 Elsevier B.V. All rights reserved.

Inhibitory effect of PGE(2) on the killing of Paracoccidioides brasiliensis by human monocytes can be reversed by cellular activation with cytokines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Acupuntura e implante de fragmentos de ouro em pontos de acupuntura e pontos Acupuntura e implante de fragmentos de ouro em pontos de acupuntura e pontos gatilho para o tratamento de displasia coxo-femoral em Pastor Alemão

Background: Canine hip dysplasia (HD) is characterized by hip joint laxity and subluxation. It is the most common cause of osteoarthritis in dogs, especially in larger breeds. Its management includes nutritional supplements, nonsteroidal anti-inflammatory drugs, physical therapy, acupuncture or surgical procedures. Implantation of gold beads in acupuncture points and trigger points around a joint has been used in the treatment of osteoarthritis in dogs for at least 30 years. Gold bead implants(GBI) acts as continuous acupuncture stimulation and trigger point treatment in canine HD with long lasting results. Electrophysiological investigations of trigger points reveal dysfunctional muscle spindles which indicate that the electrical activity of active loci arises from extrafusal motor endplates.Case: This is a report on the use of acupuncture and GBI for bilateral HD in a nine year old female German Shepherd. The patient has a HD non-responsive to anti-inflammatory drugs and was unable to stand up or walk by its own. Radiographs showed marked dysplasia, significant subluxation with the femoral head partly out of a shallow acetabulum and massive secondary arthritic bone changes, mainly on the right side. The animal was submitted to eight acupuncture sessions with seven days interval. After the first acupuncture session the use of NSAID was interrupted. After eight weeks the dog was considered rehabilitated and underwent GBI in acupoints and trigger points as maintenance treatment. During the one-year follow-up period the improvement remained unchanged with no need of analgesics.Discussion: It has been suggested that acupuncture or GBI can treat the chronic pain resulting from osteoarthritis induced by HD. According to AP theory, GBI is permanent and long-lasting acupoint stimulation. Moreover, the method is inexpensive, quick and easy to perform, with no postoperative pain or need of exercise restriction. Although gold is extremely corrosion-resistant, the surface of the gold implants stimulates a reaction from the immune system causing an oxidative liberation of gold ions with anti-inflammatory actions. It is well known that gold ions are effective inhibitors of the respiratory burst of neutrophils and monocytes and the proliferation of lymphocytes. These findings suggest that gold implantation, on a local scale, mimics the anti-inflammatory and pain-relieving effect of drugs with chemically bound gold ions. The relatively slow speed of the process results in a limited liberation of gold ions securing that they are taken up almost exclusively by cells close to the implant. The nine year old female German shepherd had a positive response to acupuncture with pain relieve and locomotor rehabilitation. For the nine year old female German shepherd previous acupuncture sessions to GBI resulted in no post-implant worsening period. Indeed, the association acupuncture/GBI does not have the anti-inflammatory drugs undesirable effects and brings long lasting results. In conclusion, GBI therefore should be considered for canine HD when conservative or medical treatments fail to give the desired effect.

Cytotoxicity of monocrotaline in isolated rat hepatocytes: Effects of dithiothreitol and fructose

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

EFEITO DE SURFACTANTES NA ESTABILIDADE DE COMPOSTOS DE USO TERAPEUTICO

A critical revision of literature as regards to the drug stability in the presence of surfactants were realized. The functional groups envolved in the drug decomposition were used to the development of the discussion. The analysis indicated that the detergent effect can be used to control the rates and mechanisms of drug decomposition and to obtain specific information about the drug reactivity in the environment of pharmacological action.