Metabolism and toxicity of two new benzodiazepine-type antileishmanial agents in hepatocytes and macrophages

With increasing reports of resistance of Leishmania to antimonials (Thakur et al., 2004) and other traditional antileishmanial drugs, the need for the discovery of new antileishmanial agents is rising. In an attempt to find new antileishmanial agents, two new benzodiazepine (BNZ) analogues (7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benodiazepine-2,5-dione (BNZ-1) and 4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2)) were synthesised, and found to be effective against leishmaniasis in mice. This study investigates the metabolism of these two drugs together with the prototype BNZ, flurazepam (FZP), using rat hepatocytes, and investigates their interaction with glutathione in macrophages. Hepatocytes (>80% viability by Trypan Blue exclusion isolated by liver perfusion with collagenase) were prepared from male Sprague-Dawley rats (200-250 g). Drugs (100 μM) were incubated with 2 × 106 viable cells/ml in Krebs-Hepes buffer, pH 7.4, in rotating round bottomed flasks under an atmosphere of 95% O2/5% CO2, at 37 °C for 3 h, and timed samples taken for metabolite measurement. Samples were extracted twice with ethyl acetate at pH 10, the combined organic phases evaporated to dryness and stored at −20 °C until analysis. Metabolites were separated by HPLC using an ACE C18 column (50 mm × 3.0 mm i.d., 5 μm packing), and a solvent gradient consisting of 0.1% formic acid: acetonitrile (starting composition 95:5%, and composition after 25 min 65:35% for FZP and 70:30% for both BNZ 1 and 2). Flow rate was 0.5 ml/min, and detection was at 230 nm. Identification of the metabolites was by mass spectrometry with both positive and negative ion electronspray ionization. The effects of 24 h exposure to the compounds (100 μM) was investigated in the macrophage cell line J774.1 in terms of reduced glutathione content (GSH) and the activity of glutathione reductase (GR). There was no evidence of significant cytotoxicity with any of the compounds at the concentration used. FZP (m/z 388) was metabolised by dealkylation of the two N-1 ethyl substituents (m/z 360 and m/z 332), followed by hydroxylation on the BNZ ring. There was no evidence for either N- or O-glucuronidation of the resulting metabolites. BNZ-1 (m/z 321) was metabolised by N-demethylation (m/z 307) followed by hydroxylation (m/z 323), mono- and di-hydroxylation of the parent (m/z 337 and m/z 353) and by glucuronidation of the mono-hydroxylated metabolite (m/z 513). BNZ-2 (m/z 287) was transformed by N-demethylation (m/z 273) and hydroxylation of the parent (m/z 303), with the latter further metabolised by O-glucuronidation (m/z 479). In addition, the hydroxylated N-demethylated product (m/z 289) was also formed. Macrophages did not produce detectable metabolism of any of the drugs. All the drugs depleted macrophage GSH significantly (p < 0.05 by ANOVA followed by Dunnett's test) with BNZ-1 and BNZ-2 causing greater depletion than FZP (40.6 ± 4.0 and 45.8 ± 8.4, respectively, compared with 55.5 ± 4.9 nmol/mg protein with FZP, n = 3). Control macrophage GSH was 74.1 ± 6.6 nmol/mg protein. The depletion in GSH was not due to inhibition of GR: only FZP caused a significant decrease in macrophage GR activity (28.0 ± 5.9 compared with 42.1 ± 8.0 nmol/ml/min in control cells, p < 0.05 by ANOVA followed by Dunnett's test, n = 3). The marked depletion of macrophage GSH indicates a potential toxic interaction in mammalian cells. The new BNZ analogues were rapidly metabolised by hepatocytes, producing N-dealkylated and multiple hydroxylated phase I metabolites, followed by glucuronidation. This rapid metabolism may limit the therapeutic effect of BNZ 1 and 2 if their metabolites are inactive against leishmaniasis and suggest the need to optimise these lead structures further to obtain compounds with reduced rates and extent of metabolism

Periodic orbit resonances in layered metals in tilted magnetic fields

The frequency dependence of the interlayer conductivity of a layered Fermi liquid in a magnetic field which is tilted away from the normal to the layers is considered. For both quasi-one- and quasi-two-dimensional systems resonances occur when the frequency is a harmonic of the frequency at which the magnetic field causes the electrons to oscillate on the Fermi surface within the layers. The intensity of the different harmonic resonances varies significantly with the direction of the field. The resonances occur for both coherent and weakly incoherent interlayer transport and so their observation does not imply the existence of a three-dimensional Fermi surface.Comment: 4 pages, RevTeX + epsf, 2 figures. Discussion of other work revised. To appear in Phys. Rev. B, Rapid Commun., October 1

Coherent vs incoherent interlayer transport in layered metals

The magnetic-field, temperature, and angular dependence of the interlayer magnetoresistance of two different quasi-two-dimensional (2D) organic superconductors is reported. For $\kappa$-(BEDT-TTF)$_2$I$_3$ we find a well-resolved peak in the angle-dependent magnetoresistance at $\Theta = 90^\circ$ (field parallel to the layers). This clear-cut proof for the coherent nature of the interlayer transport is absent for $\beta$''-(BEDT-TTF)$_2$SF$_5$CH$_2$CF$_2$SO$_3$. This and the non-metallic behavior of the magnetoresistance suggest an incoherent quasiparticle motion for the latter 2D metal.Comment: 4 pages, 4 figures. Phys. Rev. B, in pres

Larkin-Ovchinnikov-Fulde-Ferrell state in quasi-one-dimensional superconductors

The properties of a quasi-one-dimensional (quasi-1D) superconductor with {\it an open Fermi surface} are expected to be unusual in a magnetic field. On the one hand, the quasi-1D structure of the Fermi surface strongly favors the formation of a non-uniform state (Larkin-Ovchinnikov-Fulde-Ferrell (LOFF) state) in the presence of a magnetic field acting on the electron spins. On the other hand, a magnetic field acting on an open Fermi surface induces a dimensional crossover by confining the electronic wave-functions wave-functions along the chains of highest conductivity, which results in a divergence of the orbital critical field and in a stabilization at low temperature of a cascade of superconducting phases separated by first order transistions. In this paper, we study the phase diagram as a function of the anisotropy. We discuss in details the experimental situation in the quasi-1D organic conductors of the Bechgaard salts family and argue that they appear as good candidates for the observation of the LOFF state, provided that their anisotropy is large enough. Recent experiments on the organic quasi-1D superconductor (TMTSF)$_2$ClO$_4$ are in agreement with the results obtained in this paper and could be interpreted as a signature of a high-field superconducting phase. We also point out the possibility to observe a LOFF state in some quasi-2D organic superconductors.Comment: 24 pages+17 figures (upon request), RevTex, ORSAY-LPS-24109

Magnetic field-dependent interplay between incoherent and Fermi liquid transport mechanisms in low-dimensional tau phase organic conductors

We present an electrical transport study of the 2-dimensional (2D) organic conductor tau-(P-(S,S)-DMEDT-TTF)_2(AuBr)_2(AuBr_2)_y (y = 0.75) at low temperatures and high magnetic fields. The inter-plane resistivity rho_zz increases with decreasing temperature, with the exception of a slight anomaly at 12 K. Under a magnetic field B, both rho_zz and the in-plane resistivity plane rho_xx show a pronounced negative and hysteretic magnetoresistance with Shubnikov de Haas (SdH)oscillations being observed in some (high quality)samples above 15 T. Contrary to the predicted single, star-shaped, closed orbit Fermi surface from band structure calculations (with an expected approximate area of 12.5% of A_FBZ), two fundamental frequencies F_l and F_h are detected in the SdH signal. These orbits correspond to 2.4% and 6.8% of the area of the first Brillouin zone(A_FBZ), with effective masses F_l = 4.0 +/- 0.5 and F_h = 7.3 +/- 0.1. The angular dependence, in tilted magnetic fields of F_l and F_h, reveals the 2D character of the FS and Angular dependent magnetoresistance (AMRO) further suggests a FS which is strictly 2-D where the inter-plane hopping t_c is virtually absent or incoherent. The Hall constant R_xy is field independent, and the Hall mobility increases by a factor of 3 under moderate magnetic fields. Our observations suggest a unique physical situation where a stable 2D Fermi liquid state in the molecular layers are incoherently coupled along the least conducting direction. The magnetic field not only reduces the inelastic scattering between the 2D metallic layers, but it also reveals the incoherent nature of interplane transport in the AMRO spectrum. The apparent ferromagnetism of the hysteretic magnetoresistance remains an unsolved problem.Comment: 33 pages, 11 figure

State-Dependent Network Connectivity Determines Gating in a K+ Channel

YesX-ray crystallography has provided tremendous insight into the different structural states of membrane proteins and, in particular, of ion channels. However, the molecular forces that determine the thermodynamic stability of a particular state are poorly understood. Here we analyze the different X-ray structures of an inwardly rectifying potassium channel (Kir1.1) in relation to functional data we obtained for over 190 mutants in Kir1.1. This mutagenic perturbation analysis uncovered an extensive, state-dependent network of physically interacting residues that stabilizes the pre-open and open states of the channel, but fragments upon channel closure. We demonstrate that this gating network is an important structural determinant of the thermodynamic stability of these different gating states and determines the impact of individual mutations on channel function. These results have important implications for our understanding of not only K+ channel gating but also the more general nature of conformational transitions that occur in other allosteric proteins.Wellcome Trus

Modeling magnetospheric fields in the Jupiter system

The various processes which generate magnetic fields within the Jupiter system are exemplary for a large class of similar processes occurring at other planets in the solar system, but also around extrasolar planets. Jupiter's large internal dynamo magnetic field generates a gigantic magnetosphere, which is strongly rotational driven and possesses large plasma sources located deeply within the magnetosphere. The combination of the latter two effects is the primary reason for Jupiter's main auroral ovals. Jupiter's moon Ganymede is the only known moon with an intrinsic dynamo magnetic field, which generates a mini-magnetosphere located within Jupiter's larger magnetosphere including two auroral ovals. Ganymede's magnetosphere is qualitatively different compared to the one from Jupiter. It possesses no bow shock but develops Alfv\'en wings similar to most of the extrasolar planets which orbit their host stars within 0.1 AU. New numerical models of Jupiter's and Ganymede's magnetospheres presented here provide quantitative insight into the processes that maintain these magnetospheres. Jupiter's magnetospheric field is approximately time-periodic at the locations of Jupiter's moons and induces secondary magnetic fields in electrically conductive layers such as subsurface oceans. In the case of Ganymede, these secondary magnetic fields influence the oscillation of the location of its auroral ovals. Based on dedicated Hubble Space Telescope observations, an analysis of the amplitudes of the auroral oscillations provides evidence that Ganymede harbors a subsurface ocean. Callisto in contrast does not possess a mini-magnetosphere, but still shows a perturbed magnetic field environment. Callisto's ionosphere and atmospheric UV emission is different compared to the other Galilean satellites as it is primarily been generated by solar photons compared to magnetospheric electrons.Comment: Chapter for Book: Planetary Magnetis

Influence of storm surge on tidal range energy

The regular and predictable nature of the tide makes the generation of electricity with a tidal lagoon or barrage an attractive form of renewable energy, yet storm surges affect the total water-level. Here, we present the first assessment of the potential impact of storm surges on tidal-range power. Water-level data (2000–2012) at nine UK tide gauges, where tidal-range energy is suitable for development (e.g. Bristol Channel), was used to predict power. Storm surge affected annual resource estimates −5% to +3%, due to inter-annual variability, which is lower than other sources of uncertainty (e.g. lagoon design); therefore, annual resource estimation from astronomical tides alone appears sufficient. However, instantaneous power output was often significantly affected (Normalised Root Mean Squared Error: 3%–8%, Scatter Index: 15%–41%) and so a storm surge prediction system may be required for any future electricity generation scenario that includes large amounts of tidal-range generation. The storm surge influence to tidal-range power varied with the electricity generation strategy considered (flooding tide only, ebb-only or dual; both flood and ebb), but with some spatial and temporal variability. The flood-only strategy was most affected by storm surge, mostly likely because tide-surge interaction increases the chance of higher water-levels on the flooding tide

Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

In vivo and in vitro proinflammatory effects of particulate air pollution (PM10).

Epidemiologic studies have reported associations between fine particulate air pollution, especially particles less than 10 mm in diameter (PM10), and the development of exacerbations of asthma and chronic obstructive pulmonary disease. However, the mechanism is unknown. We tested our hypothesis that PM10 induces oxidant stress, causing inflammation and injury to airway epithelium. We assessed the effects of intratracheal instillation of PM10 in rat lungs. The influx of inflammatory cells was measured in bronchoalveolar lavage (BAL). Airspace epithelial permeability was assessed as total protein in bronchoalveolar lavage fluid (BALF) in vivo. The oxidant properties of PM10 were determined by their ability to cause changes in reduced glutathione (GSH) and oxidized glutathione (GSSG). We also compared the effects of PM10 with those of fine (CB) and ultrafine (ufCB) carbon black particles. Six hours after intratracheal instillation of PM10, we noted an influx of neutrophils (up to 15% of total BAL cells) in the alveolar space, increased epithelial permeability, an increase in total protein in BALF from 0.39 +/- 0.01 to 0.62 +/- 0.01 mg/ml (mean +/- SEM) and increased lactate dehydrogenase concentrations in BALF. An even greater inflammatory response was observed after intratracheal instillation of ufCB, but not after CB instillation. PM10 had oxidant activity in vivo, as shown by decreased GSH in BALF (from 0.36 +/- 0.05 to 0.25 +/- 0.01 nmol/ml) after instillation. BAL leukocytes from rats treated with PM10 produced greater amounts of nitric oxide, measured as nitrite (control 3.07 +/- 0.33, treated 4.45 +/- 0.23 mM/1 x 10(6) cells) and tumor necrosis factor alpha (control 21.0 +/- 3.1, treated 179.2 +/- 29.4 unit/1 x 10(6) cells) in culture than BAL leukocytes obtained from control animals. These studies provide evidence that PM10 has free radical activity and causes lung inflammation and epithelial injury. These data support our hypothesis concerning the mechanism for the adverse effects of particulate air pollution on patients with airway diseases