In vitro and in vivo experiments with iron oxide nanoparticles functionalized with DEXTRAN or polyethylene glycol for medical applications: magnetic targeting.

In this research work, DEXTRAN- and polyethylene glycol (PEG)-coated iron-oxide superparamagnetic nanoparticles were synthetized and their cytotoxicity and biodistribution assessed. Well-crystalline hydrophobic Fe3 O4 SPIONs were formed by a thermal decomposition process with d = 18 nm and σ = 2 nm; finally, the character of SPIONs was changed to hydrophilic by a post-synthesis procedure with the functionalization of the SPIONs with PEG or DEXTRAN. The nanoparticles present high saturation magnetization and superparamagnetic behavior at room temperature, and the hydrodynamic diameters of DEXTRAN- and PEG-coated SPIONs were measured as 170 and 120 nm, respectively. PEG- and DEXTRAN-coated SPIONs have a Specific Power Absorption SPA of 320 and 400 W/g, respectively, in an ac magnetic field with amplitude of 13 kA/m and frequency of 256 kHz. In vitro studies using VERO and MDCK cell lineages were performed to study the cytotoxicity and cell uptake of the SPIONs. For both cell lineages, PEG- and DEXTRAN-coated nanoparticles presented high cell viability for concentrations as high as 200 µg/mL. In vivo studies were conducted using BALB/c mice inoculating the SPIONs intravenously and exposing them to the presence of an external magnet located over the tumour. It was observed that the amount of PEG-coated SPIONs in the tumor increased by up to 160% when using the external permanent magnetic as opposed to those animals that were not exposed to the external magnetic field.

Protective properties of melatonin-loaded nanoparticles against lipid peroxidation.

The aim of this study was to prepare melatonin-loaded nanoparticles (nanocapsules and nanospheres) by nanoprecipitation, using Eudragit S100 as polymer. The potential of these systems to protect lipids against peroxidation was evaluated in comparison to melatonin in aqueous solution and nanoemulsion. Liposomes and microsomes were used as model of a lipid membrane and lipid peroxidation was induced by free radical ascorbyl. Nanocapsule and nanosphere suspensions presented total recoveries of melatonin near 100% and associated drug around 55%. The zeta potential values were negative and the hydrodynamic diameter of particles were lower than 255 nm. The results demonstrate that the lipids were protected against peroxidation from 8 to 51% due to the presence of the melatonin and that this effect depended on the drug dose, the type of the lipid substrate and the type of colloid, in which melatonin was incorporated. Nanocapsules and nanospheres provided an important increase in the antioxidant effect of melatonin against lipid peroxidation.

Energetic metabolism of Chromobacterium violaceum

Chromobacterium violaceum is a free-living microorganism, normally exposed to diverse environmental conditions; it has a versatile energy-generating metabolism. This bacterium is capable of exploiting a wide range of energy resources by using appropriate oxidases and reductases. This allows C. violaceum to live in both aerobic and anaerobic conditions. In aerobic conditions, C. violaceum is able to grow in a minimal medium with simple sugars, such as glucose, fructose, galactose, and ribose; both Embden-Meyerhoff, tricarboxylic acid and glyoxylate cycles are used. The respiratory chain supplies energy, as well as substrates for other metabolic pathways. Under anaerobic conditions, C. violaceum metabolizes glucose, producing acetic and formic acid, but not lactic acid or ethanol. C. violaceum is also able to use amino acids and lipids as an energy supply

Genetic analysis of violacein biosynthesis by Chromobacterium violaceum

Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule

Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum.

Natural products are an inexhaustible source of compounds with promising pharmacological activities including antiviral action. Violacein, the major pigment produced by Chromobacterium violaceum, has been shown to have antibiotic, antitumoral and anti-Trypanosoma cruzi activities. The goal of the present work was to evaluate the cytotoxicity of violacein and also its potential antiviral properties. The cytotoxicity of violacein was investigated by three methods: cell morphology evaluation by inverted light microscopy and cell viability tests using the Trypan blue dye exclusion method and the MTT assay. The cytotoxic concentration values which cause destruction in 50% of the monolayer cells (CC50) were different depending on the sensitivity of the method. CC50 values were > or =2.07 +/- 0.08 microM for FRhK-4 cells: > or =2.23 +/- 0.11 microM for Vero cells; > or =2.54 +/- 0.18 microM for MA104 cells; and > or =2.70 +/- 0.20 microM for HEp-2 cells. Violacein showed no cytopathic inhibition of the following viruses: herpes simplex virus type 1 (HSV-1) strain 29-R/acyclovir resistant, hepatitis A virus (strains HM175 and HAF-203) and adenovirus type 5 nor did it show any antiviral activity in the MTT assay. However violacein did show a weak inhibition of viral replication: 1.42 +/- 0.68%, 14.48 +/- 5.06% and 21.47 +/- 3.74% for HSV-1 (strain KOS); 5.96 +/- 2.51%, 8.75 +/- 3.08% and 17.75 +/- 5.19% for HSV-1 (strain ATCC/VR-733); 5.13 +/- 2.38 %, 8.18 +/- 1.11% and 8.51 +/- 1.94% for poliovirus type 2; 8.30 +/- 4.24%; 13.33 +/- 4.66% and 24.27 +/- 2.18% for simian rotavirus SA11, at 0.312, 0.625 and 1.250 mM, respectively, when measured by the MTT assay.

Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum

Natural products are an inexhaustible source of compounds with promising pharmacological activities including antiviral action. Violacein, the major pigment produced by Chromobacterium violaceum, has been shown to have antibiotic, antitumoral and anti-Trypanosoma cruzi activities. The goal of the present work was to evaluate the cytotoxicity of violacein and also its potential antiviral properties.The cytotoxicity of violacein was investigated by three methods: cell morphology evaluation by inverted light microscopy and cell viability tests using the Trypan blue dye exclusion method and the MTT assay. The cytotoxic concentration values which cause destruction in 50 percent of the monolayer cells (CC50) were different depending on the sensitivity of the method. CC50 values were > 2.07 ± 0.08 æM for FRhK-4 cells: > 2.23 ± 0.11 æM for Vero cells; > 2.54 ± 0.18 æM for MA104 cells; and > 2.70 ± 0.20 æM for HEp-2 cells. Violacein showed no cytopathic inhibition of the following viruses: herpes simplex virus type 1 (HSV-1) strain 29-R/acyclovir resistant, hepatitis A virus (strains HM175 and HAF-203) and adenovirus type 5 nor did it show any antiviral activity in the MTT assay. However violacein did show a weak inhibition of viral replication: 1.42 ± 0.68 percent, 14.48 ± 5.06 percent and 21.47 ± 3.74 percent for HSV-1 (strain KOS); 5.96 ± 2.51 percent, 8.75 ± 3.08 percent and 17.75 ± 5.19 percent for HSV-1 (strain ATCC/VR-733); 5.13 ± 2.38 percent, 8.18 ± 1.11 percent and 8.51 ± 1.94 percent for poliovirus type 2; 8.30 ± 4.24 percent; 13.33 ± 4.66 percent and 24.27 ± 2.18 percent for simian rotavirus SA11, at 0.312, 0.625 and 1.250 mM, respectively, when measured by the MTT assay

Complexes trans-[RuCl(2)(nic)(4)] and trans-[RuCl(2)(i-nic)(4)] as free radical scavengers.

This study evaluates the action of the new ruthenium complexes trans-RuCl(2)(nic)(4)] (I) and trans-[RuCl(2)(i-nic)(4)] (II) as free radical scavengers. In our experiments, both compounds acted as scavengers of superoxide anion (O(2)*(-)), hydroxyl radicals (HO*) and nitrogen monoxide (formally known as 'nitric oxide'; NO*). In addition, complexes I and II potentiated the release of NO* from S-nitroso-N-acetyl-DL-penicilamine (SNAP), a NO* donor. Complex II, but not I, also decreased the nitrite levels in culture media of activated macrophages. A hypsochromic shift of lambda(max) and a significant change in half-wave potential (E(1/2)) was observed when NO* was added to the Complex II. Thiobarbituric reactive substance (TBARS) levels were significantly reduced in rats treated for 1 week with Complex II plus tert-butylhydroperoxide, when compared to rats treated only with tert-butylhydroperoxide. None of the complexes showed cytotoxicity. These findings support the suggestion that the new ruthenium complexes, especially trans-[RuCl(2)(i-nic)(4)] or its derivatives, might provide potential therapeutic benefits in disorders where reactive nitrogen (RNS) or oxygen (ROS) species are involved.

Characterization of nucleotide binding sites of the isolated H(+)-ATPase from spinach chloroplasts, CF(0)F(1).

Soluble purified CF(0)F(1) from chloroplasts was either oxidized or reduced and then incubated with [alpha-(32)P]ATP in the presence or in the absence of Mg(2+). Depending on the conditions of incubation, the enzyme showed different tight-nucleotide binding sites. In the presence of EDTA, two sites bind [alpha-(32)P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [alpha-(32)P]ATP and [alpha-(32)P]ADP are bound to the enzyme. In the presence of Mg(2+), only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg(2+). However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[alpha-(32)P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg(2+), there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic.

Synthesis, potentiometric titration, electrochemical investigation and biological properties of trans-[RuCl2(dinic)4] (dinic = 3,5-pyridinecarboxylic acid).

This work discusses both the synthesis of trans-[RuCl2(dinic)4], dinic = 3,5-pyridinecarboxylic acid, and its main characteristics including potentiometric titration, spectroscopic and electrochemical properties, and some biological properties. The complex was synthesized using ruthenium blue solution as the precursor in a synthetic route. The complex was characterized using electronic spectroscopy, vibrational FT-IR spectroscopy, and Raman spectroscopy, as well as 1H and 13C NMR. The results indicated that the complex exhibits a trans-geometry. Cyclic voltammetry carried out in water:acetone 1:1 solution revealed a quasi-reversible process centered on the Ru(II) atom, as well as a dependence of the redox potential, E1/2, on pH. An analysis of the electronic spectra revealed that the MLCT (metal ligand charge transfer) band underwent a hypsochromic shift as the pH increased. Spectroelectrochemical analysis indicated that the visible region band progressively faded out upon oxidation. The equilibrium constants for the eight protons of the complex were determined by potentiometric titration. The complex neither inhibits the activity of nitrogen monoxide synthase nor acts as a scavenger for nitrogen monoxide. Nevertheless, the complex shows antinociceptive properties and acts as a scavenger for hydroxyl radicals.

The mechanisms underlying the relaxant effect of methyl and ethyl gallates in the guinea pig trachea in vitro: contribution of potassium channels.

The relaxant response and the possible contribution of K+ channels to the relaxation caused by both methyl and ethyl gallates, two compounds isolated from the Brazilian medicinal plant Phyllanthus urinaria, were investigated in the guinea pig trachea in vitro. Both methyl and ethyl gallate (0.01-30 microM) caused graded and complete relaxation of the guinea pig trachea without epithelium, pre-contracted by histamine, with mean EC50 values of 1.8 (1.2-2.2) microM and 0.7 (0.6-0.8) microM, respectively, and Emax of both 100+/-0%. Response to ethyl, but not methyl gallate, was significantly shifted to the right, with no change in the maximum effect when the epithelium was removed. The increase in K+ concentration in the medium to 80 mM completely abolished the relaxant response caused by both methyl and ethyl gallate. In addition, tetraethylammonium (10 mM) reduced by 50+/-6% and 43+/-4% the relaxation caused by methyl and ethyl gallates. In contrast, glibenclamide (3 microM) shifted (by about two- and fourfold) the concentration-response curves for both methyl and ethyl gallates, with no changes in the maximum effect. Charybdotoxin (100 nM), but not apamin (100 nM), significantly blocked by 54+/-5% and 59+/-4% the relaxation of both methyl and ethyl gallates. In contrast, SQ 22536 (10 microM; a selective adenylyl cyclase inhibitor), methylene blue (10 microM) or ODQ (1 microM; a guanylyl cyclase inhibitor) did not significantly affect the relaxant response caused by either of the compounds. These results provide evidence that the relaxation caused by both methyl and ethyl gallates in the guinea pig trachea in vitro may involve the activation of large-conductance Ca2+-activated K+ channels, and, to a lesser extent, ATP-sensitive K+ channels. Such results extend our previous observations and are consistent with the notion that methyl and ethyl gallates are mainly responsible for the relaxant action previously demonstrated in the extract of this plant.

Antinociceptive properties and nitric oxide synthase inhibitory action of new ruthenium complexes.

This study evaluates the actions of the new ruthenium complexes trans-[RuCl2(nic)4] (Complex I) and trans-[RuCl2(i-nic)4] (Complex II) as antinociceptives, and their interaction with nitric oxide isoenzymes and with acetylcholine-induced relaxation of rat and rabbit aorta. Complex II inhibited, in a graded manner, neuronal and inducible nitric oxide (NO) synthase, and was about two fold more effective in inhibiting the neuronal NO synthase than the inducible form of the enzyme. Complex I was inactive. Both complexes failed to interfere with constitutive endothelial nitric oxide synthase because they did not change the mean arterial blood pressure of rats. The vasorelaxant effect of acetylcholine was markedly antagonised by the Complexes I and II in rings of both rat and rabbit aorta. Complexes I and II, given intraperitoneally, like N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(G)-nitro-L-arginine (L-NOARG), inhibited, in a graded manner, both phases of the pain response induced by formalin. The actions of L-NAME, L-NOARG and Complex II, but not that of Complex I, were largely reversed by L-arginine. Both complexes failed to affect the motor response of animals in the rota-rod test and had no effect in the hot-plate assay. Together, these findings provide indications that the new ruthenium complexes, especially Complex II and its derivatives, might be of potential therapeutic benefit in the management of pain disorders.

Spinal and supraspinal antinociceptive action of dipyrone in formalin, capsaicin and glutamate tests. Study of the mechanism of action.

Dipyrone injected intraperitoneally (i.p.) or subplantarly into the mouse paw caused dose-related antinociception against the early and the late phases of formalin-induced licking, with mean ID50 values of 154.5 and 263.7 micromol/kg, and 2.6 and 1.2 micromol/paw, respectively. Given either by intracerebroventricular (i.c.v.) or by intrathecal (i.t.) routes, dipyrone produced a similar inhibition of both phases of the formalin-induced licking, with mean ID50 values of 0.4 and 1.3 micromol/site, and 0.4 and 0.9 micromol/site against the early and the late phase of the formalin response, respectively. Dipyrone, given by i.p., subplantar, i.t. or i.c.v. routes, caused dose-related antinociception of capsaicin-induced licking. The mean ID50 values were: 207.6 micromol/kg, 2.2 micromol/paw, 0.4 micromol/site and 0.14 micromol/site, respectively. In addition, dipyrone given i.p. caused a significant increase of the latency both in the hot-plate and the tail-flick assays. Dipyrone, given i.p., i.t. or i.c.v., reversed significantly the hyperalgesia caused by i.t. injection of glutamate, with mean ID50 values of 9 micromol/kg, 29 nmol/site and 94 nmol/site, respectively. The antinociception caused by dipyrone was not influenced by naloxone, L-arginine, phaclofen, glibenclamide, p-chlorophenylalanine methyl ester, pertussis toxin or by adrenal gland hormones, when assessed against the formalin assay. Dipyrone analgesic action was not secondary to its anti-inflammatory effect, nor was it associated with non-specific effects such as muscle relaxation or sedation actions of animals. Dipyrone at a higher concentration caused significant inhibition of [3H]glutamate binding (37%) in cerebral cortical membranes from both mice and rats. However, dipyrone had no significant effect on brain constitutive neuronal nitric oxide synthase activity. It is concluded that dipyrone produces peripheral, spinal and supraspinal antinociception when assessed on formalin and capsaicin-induced pain as well as in glutamate-induced hyperalgesia in mice. Dipyrone antinociception seems unlikely to involve an interaction with the L-arginine-nitric oxide pathway, serotonin system, activation of Gi protein sensitive to pertussis toxin. interaction of ATP-sensitive K+ channels, GABA(B) receptors, or the release of endogenous glucocorticoids. However, a modulatory effect on glutamate-induced hyperalgesia and, to a lesser extent, an interaction with glutamate binding sites, seems to account for its analgesic action.

Oral anti-inflammatory action of NPC 18884, a novel bradykinin B2 receptor antagonist.

This study describes the anti-inflammatory actions of NPC 18884, a non-peptide bradykinin B2 receptor antagonist in bradykinin and carrageenan-induced inflammation in the mouse model of pleurisy. The selectivity of NPC 18884 was assessed in the pleurisy caused by histamine, substance P and des-Arg9-bradykinin. NPC 18884 given intraperitoneally or orally inhibited bradykinin-induced leukocytes influx (ID50 value of 63 nmol/kg and 141 nmol/kg, respectively). The NPC 18884 also inhibited the exudation induced by bradykinin (P < 0.05). NPC 18884 given either intraperitoneally or orally caused dose-dependent inhibition of the exudation and total and differential cell content caused by intrapleural injection of carrageenan (1%, assessed 4 h after), with mean ID50, values of 132 and 295 nmol/kg, respectively. The NPC 18884 actions installs rapidly (0.5 h), lasted for up to 4 h and were selective for the bradykinin B2 receptors; at similar doses it had no significant effect against the inflammatory responses induced by des-Arg9-bradykinin, histamine or substance P. These results indicate that the novel non-peptide bradykinin B2 receptor antagonist, NPC 18884, exhibited selective intraperitoneal and oral anti-inflammatory properties when assessed in the inflammatory reaction induced by bradykinin and carrageenan in the mice model of pleurisy.

ADP binding and ATP synthesis by reconstituted H(+)-ATPase from chloroplasts.

The H(+)-ATPase from chloroplasts, CF0F1, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid-base transition delta pH (pHin = 5.0, pHout = 8.5) and a K+/valinomycin diffusion potential, delta phi (Kin+ = 0.6 mM, Kout+ = 60 mM). A rate of 250 s-1 was observed with the reduced enzyme (85 s-1 in the absence of delta phi). A rate of 50 s-1 was observed with the oxidized enzyme under the same conditions (15 s-1 in the absence of delta phi). The reconstituted enzyme contained 2 ATPbound per CF0F1 and 1 ADPbound per CF0F1. Upon energisation the enzyme was activated and 0.9 ADP per CF0F1 was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10(5) M-1.s-1 under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.