Alternative promoters in the pst operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes pstS, pstC, pstA, pstB and phoU, that encode a high-affinity phosphate transport system and a negative regulator of the PHO regulon. Transcription of pst is induced under phosphate shortage and is initiated at the promoter located upstream of the first gene of the operon, pstS. Here, we show by four different technical approaches the existence of additional internal promoters upstream of pstC, pstB and phoU. These promoters are not induced by Pi-limitation and do not possess PHO-box sequences. Plasmids carrying the pst internal genes partially complement chromosomal mutations in their corresponding genes, indicating that they are translated into functional proteins.

Immobilization of Alcohol Dehydrogenase in Phospholipid Langmuir-Blodgett Films To Detect Ethanol

Enzyme immobilization in nanostructured films may be useful for a number of biomimetic systems, particularly if suitable matrixes are identified. Here we show that alcohol dehydrogenase (ADH) has high affinity toward a negatively charged phospholipid, dimyristoylphosphatidic acid (DMPA), which forms a Langmuir monolayer at an air-water interface. Incorporation of ADH into the DMPA monolayer was monitored with Surface pressure measurements; and polarization-modulation infrared reflection absorption spectroscopy, with the alpha-helices from ADH being mainly oriented parallel to the water surface. ADH remained at the interface even at high surface pressures, thus allowing deposition of Langmuir-Blodgett (LB) films from the DMPA-ADH film. Indeed, interaction with DMPA enhances the transfer of ADH, where the mass transferred onto a solid support increased from 134 ng for ADH on a Gibbs monolayer to 178 ng for an LB film with DMPA. With fluorescence spectroscopy it was possible to confirm that the ADH structure was preserved even after one month of the LB deposition. ADH-containing films deposited onto gold-interdigitated electrodes were employed in a sensor array capable of detecting ethanol at concentrations down to 10 ppb (in volume), using impedance spectroscopy as the method of detection.

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

This paper describes the preparation of a biomimetic Langmuir-Blodgett film of tyrosinase incorporated in a lipidic layer and the use of lutetium bisphthalocyanine as an electron mediator for the voltammetric detection of phenol derivatives, which include one monophenol (vanillic acid), two diphenols (catechol and caffeic acid) and two triphenols (gallic acid and pyrogallol). The first redox process of the voltammetric responses is associated with the reduction of the enzymatically formed o-quinone and is favoured by the lutetium bisphthalocyanine because significant signal amplification is observed, while the second is associated with the electrochemical oxidation of the antioxidant and occurs at lower potentials in the presence of an electron mediator. The biosensor shows low detection limit (1.98 x 10(-6)-27.49 x 10(-6) M), good reproducibility, and high affinity to antioxidants (Km in the range of 62.31-144.87 mu M). The excellent functionality of the enzyme obtained using a biomimetic immobilisation method, the selectivity afforded by enzyme catalysis, the signal enhancement caused by the lutetium bisphthalocyanine mediator and the increased selectivity of the curves due to the occurrence of two redox processes make these sensors exceptionally suitable for the detection of phenolic compounds. (C) 2010 Elsevier B.V. All rights reserved.

Identification of a novel ligand binding motif in the transthyretin channel

The design of therapeutic compounds targeting transthyretin (TTR) is challenging due to the low specificity of interaction in the hormone binding site. Such feature is highlighted by the interactions of TTR with diclofenac, a compound with high affinity for TTR, in two dissimilar modes, as evidenced by crystal structure of the complex. We report here structural analysis of the interactions of TTR with two small molecules, 1-amino-5-naphthalene sulfonate (1,5-AmNS) and 1-anilino-8-naphthalene sulfonate (1,8-ANS). Crystal structure of TTR: 1,8-ANS complex reveals a peculiar interaction, through the stacking of the naphthalene ring between the side-chain of Lys15 and Leu17. The sulfonate moiety provides additional interaction with Lys15` and a water-mediated hydrogen bond with Thr119`. The uniqueness of this mode of ligand recognition is corroborated by the crystal structure of TTR in complex with the weak analogue 1,5-AmNS, the binding of which is driven mainly by hydrophobic partition and one electrostatic interaction between the sulfonate group and the Lys15. The ligand binding motif unraveled by 1,8-ANS may open new possibilities to treat TTR amyloid diseases by the elucidation of novel candidates for a more specific pharmacophoric pattern. (C) 2009 Published by Elsevier Ltd.

pH-Sensitive Binding of Cytochrome c to the Inner Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pK(a) values and site L containing ionizable groups with pK(aobs),values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, We demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pi-I-independent binding (microscopic dissociation constant K(sapp2), similar to 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pK(a) of similar to 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on K(sapp1), was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed K(sapp1) values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site Lionization influences the participation of cytochrome c in the respiratory chain and apoptosis.

Stereoselective synthesis of azetidine-derived glutamate and aspartate analogues from chiral azetidin-3-ones

The stereoselective syntheses of cis conformationally constrained glutamate and aspartate analogues, containing an azetidine framework were accomplished from (S)-N-tosyl-2-phenylglycine in moderate overall yields. The key steps in these syntheses involved an efficient Wittig olefination of an azetidin-3-one, followed by a highly stereoselective rhodium catalyzed hydrogenation. The route could also be applied to the synthesis of a trans glutamate analogue, since epimerization of cis to trans isomer could be performed using DBU in toluene at reflux. (C) 2008 Elsevier Ltd. All rights reserved.

Chitosan, sisal cellulose, and biocomposite chitosan/sisal cellulose films prepared from thiourea/NaOH aqueous solution

Environmentally friendly biocomposites were successfully prepared by dissolving chitosan and cellulose in a NaOH/thiourea solvent with subsequent heating and film casting. Under the considered conditions, NaOH/thiourea led to chain depolymerization of both biopolymers without a dramatic loss of film forming capacities. Compatibility of both biopolymers in the biocomposite was firstly assessed through scanning electron microscopy, revealing an intermediate organization between cellulose fiber network and smoothness of pure chitosan. DSC analyses led to exothermic peaks close to 285 and 315 degrees C for the biocomposite, compared to the exothermic peaks of chitosan (275 degrees C) and cellulose (265 and 305 degrees C), suggesting interactions between chitosan and cellulose. Contact angle analyses pointed out the deformation that can occur at the surface due to the high affinity of the;e materials with water. T(2) NMR relaxometry behavior of biocomposites appeared to be dominated by chitosan. Other properties of films, as crystallinity, water sorption isotherms, among others, are also discussed. (C) 2010 Published by Elsevier Ltd.

Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions

Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions. with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following Michaelian kinetics with a specific activity of 36.7 U/mg and K-0.5 = 88 mu M. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited Michaclian kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed. K-m values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites (K-0.5 = 40 and 90 mu M, for magnesium and calcium, respectively), high-affinity sites were also observed with K-0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 mu M), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed. (C) 1998 Published by Elsevier B.V. Ltd. All rights reserved.

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

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

Comparative transcriptome analysis of Paracoccidioides brasiliensis during in vitro adhesion to type I collagen and fibronectin: identification of potential adhesins

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Allosteric water and phosphate effects in Hoplosternum littorale hemoglobins

This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfish Hoplosternum littorale, a species that displays facultative accessorial air oxygenation. We demonstrate that water potential affects the oxygen affinity of H. littorale Hbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize the high-affinity state of the Hb. This effect is the same as that observed in tetrameric vertebrate Hbs. We show that both anodic and cathodic Hbs show conformational substrates similar to other vertebrate Hbs. For both Hbs, addition of anionic effectors, especially chloride, strongly increases the number of water molecules bound, although anodic Hb did not exhibit sensitivity to saturating levels of ATP. Accordingly, for both Hbs, we propose that the deoxy conformations coexist in at least two anion-dependent allosteric states, T-o and T-x, as occurs for human Hb. We found a single phosphate binding site for the cathodic Hb.

Crotoxin-induced behavioral effects in rats

Crotoxin is the major component of Crotalus durissus terrificus venom. In view of the presence of high-affinity specific binding sites for crotoxin in the brain, the objective of this work was to investigate whether crotoxin induces behavioral effects in the open-field and hole-board tests. Adult male Wistar rats (180-220 g) treated with crotoxin, 100, 250 and 500 mu g/kg, ip, administered 2 h before the test, presented statistically significant behavioral alterations (ANOVA for one-way classification complemented with Dunnet test, P<0.05). In the open-field test, 250 and 500 mu g/kg of crotoxin increased freezing (from 3.22 sec to 10.75 sec and 11.2 sec) and grooming (from 13.44 sec to 22.75 sec and 21.22 sec) and decreased ambulation (from 64.8 to 39.38 and 45.8). The dose of 500 mu g/kg also decreased rearing (from 24.9 to 17.5). In the hole-board test, 500 mu g/kg of crotoxin decreased head-dip count (from 6.33 to 4.00). All the crotoxin-induced behavioral effects were antagonized by an anxiolytic dose of diazepam (1.5 mg/kg, ip, 30 min before the tests). These results show that crotoxin reduced open-field activity and exploratory behavior as well. We suggest that these effects express an increased emotional state induced by this toxin.

2-Mercaptoimidazole covalently bonded to a silica gel surface for the selective separation of mercury(II) from an aqueous solution

Silica gel with a specific surface area of 365 m(2).g(-1) and an average pore diameter of 60 Angstrom was chemically modified with 2-mercaptoimidazole. The degree of functionalization of the covalently attached molecule, (drop SiO)(3)(CH2)(3) - MI, where MI is the 2-mercaptoimidazole bound to the silica surface by a propyl group, was 0.58 mmolg.(-1). In individual metal adsorption experiments from aqueous solutions by the batch procedure, the affinity order was Hg(II)much greater than Cd-II > Cu-II approximate to Zn-II approximate to Pb-II > Mn-II at solution pHs between 4 and 7. Due to the high affinity by the sulfur atom, Hg-II is strongly bound to the functional groups. When solution containing a mixture of Hg-II, Cd-II, Cu-II, Zn-II, Pb-II, and Mn-II ions was passed through a column packed with the adsorbent, Hg-II was the only one whose adsorption and elution was not affected by the presence of other ions.

The role of hydration on the mechanism of allosteric regulation: In situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98%, relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.

Nickel chloride: potential accumulation from the airway. Toxicokinetics in rats.

The rate removal of nickel from the airway was measured in vivo. Removal in vivo was studied by intratracheal injection of nickel chloride solutions. Regardless of time after injection, the lungs and heart retained the greatest concentration of nickel and 40 days after 1.68 mumol administration they were the organs where nickel was still significantly measurable. The slow removal of nickel may indicate the presence of high affinity binding sites in the lung. Nickel can interact with others metals, such as copper and zinc, so that nickel exposure may have public health implications.