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Type II NADH:quinone oxidoreductase (NDH-2) plays a crucial role in the respiratory chains of many organisms. Its absence in mammalian cells makes NDH-2 an attractive new target for developing antimicrobials and antiprotozoal agents. We established a novel bioelectrochemical platform to characterize the catalytic behavior of NDH-2 from Caldalkalibacillus thermarum and Listeria monocytogenes strain EGD-e while bound to native-like lipid membranes. Catalysis of both NADH oxidation and lipophilic quinone reduction by membrane-bound NDH-2 followed the Michaelis-Menten model; however, the maximum turnover was only achieved when a high concentration of quinone (>3 mM) was present in the membrane, suggesting that quinone availability regulates NADH-coupled respiration activity. The quinone analogue 2-heptyl-4-hydroxyquinoline- N -oxide inhibited C. thermarum NDH-2 activity, and its potency is higher in a membrane environment compared to assays performed with water-soluble quinone analogues, demonstrating the importance of testing compounds under physiologically relevant conditions. Furthermore, when phenothiazines, one of the most commonly identified NDH-2 inhibitors, were tested, they did not inhibit membrane-bound NDH-2. Instead, our assay platform unexpectedly suggests a novel mode of phenothiazine action where chlorpromazine, a promising antitubercular agent and key medicine used to treat psychotic disorders, is able to disrupt pH gradients across bacterial membranes.

A comprehensive geochemical study of the Chelyabinsk meteorite reveals further details regarding its history of impact-related fragmentation and melting, and later aqueous alteration, during its transit toward Earth. We support an ∼30 Ma age obtained by Ar-Ar method (Beard et al., 2014) for the impact-related melting, based on Rb-Sr isotope analyses of a melt domain. An irregularly shaped olivine with a distinct O isotope composition in a melt domain appears to be a fragment of a silicate-rich impactor. Hydrogen and Li concentrations and isotopic compositions, textures of Fe oxyhydroxides, and the presence of organic materials located in fractures, are together consistent with aqueous alteration, and this alteration could have pre-dated interaction with the Earth's atmosphere. As one model, we suggest that hypervelocity capture of the impact-related debris by a comet nucleus could have led to shock-wave-induced supercritical aqueous fluids dissolving the silicate, metallic, and organic matter, with later ice sublimation yielding a rocky rubble pile sampled by the meteorite.

Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ 1 ) interactions in the yeast Ndi1-UQ 1 complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Type II NADH:quinone oxidoreductase (NDH-2) is a respiratory enzyme found in the electron-transport chain of many species, with the exception of mammals. It is a 40-70 kDa single-subunit monotopic membrane protein that catalyses the oxidation of NADH and the reduction of quinone molecules via the cofactor FAD. NDH-2 is a promising new target for drug development given its essential role in many bacterial species and intracellular parasites. Only two bacterial NDH-2 structures have been reported and these structures are at moderate resolution (2.3-2.5 Å). In this communication, a new crystallization platform is reported that produced high-quality NDH-2 crystals that diffracted to high resolution (2.15 Å). The high-resolution NDH-2 structure was used for in silico quinone substrate-docking studies to investigate the binding poses of menadione and ubiquinone molecules. These studies revealed that a very limited number of molecular interactions occur at the quinone-binding site of NDH-2. Given that the conformation of the active site is well defined, this high-resolution structure is potentially suitable for in silico inhibitor-compound screening and ligand-docking applications.

Frameshifting during translation of viral or in rare cases cellular mRNA results in the synthesis of proteins from two overlapping reading frames within the same mRNA. In HIV-1 the protease, reverse transcriptase, and integrase enzymes are in a second reading frame relative to the structural group-specific antigen (gag), and their synthesis is dependent upon frameshifting. This ensures that a strictly regulated ratio of structural proteins and enzymes, which is critical for HIV-1 replication and viral infectivity, is maintained during protein synthesis. The frameshift element in HIV-1 RNA is an attractive target for the development of a new class of anti HIV-1 drugs. However, a number of examples are now emerging of human genes using -1 frameshifting, such as PEG10 and CCR5. In this study we have compared the HIV-1 and PEG10 frameshift elements and shown they have distinct functional characteristics. Frameshifting occurs at several points within each element. Moreover, frameshift modulators that were isolated by high-throughput screening of a library of 114,000 lead-like compounds behaved differently with the PEG10 frameshift element. The most effective compounds affecting the HIV-1 element enhanced frameshifting by 2.5-fold at 10 μM in two different frameshift reporter assay systems. HIV-1 protease:gag protein ratio was affected by a similar amount in a specific assay of virally-infected cultured cell, but the modulation of frameshifting of the first-iteration compounds was not sufficient to show significant effects on viral infectivity. Importantly, two compounds did not affect frameshifting with the human PEG10 element, while one modestly inhibited rather than enhanced frameshifting at the human element. These studies indicate that frameshift elements have unique characteristics that may allow targeting of HIV-1 and of other viruses specifically for development of antiviral therapeutic molecules without effect on human genes like PEG10 that use the same generic mechanism.

Effects of zinc (Zn) on cadmium (Cd) distribution in the mouse body were investigated after intranasal administration. The amounts of Cd (2 x 10(-6) M/0.02 ml) reaching respiratory organs increased as the ip dose of pentobarbital increased. Administration of Zn with Cd (2 x 10(-6) M Zn + 2 x 10(-6) M Cd /0.02 ml) further increased the Cd amounts reaching respiratory organs and increased mouse mortality. A single administration of Cd increased Cd levels in blood, liver, kidney and respiratory organs. High levels of Cd were maintained for at least 90 days in these organs after single administrations of Cd. The administration of Cd plus Zn further increased the Cd content in these organs during 0-60 min after administration. However, simultaneous administration of Zn with Cd inhibited prolonged Cd accumulation in kidney and liver. These results suggest that intranasally administered Zn (2 x 10(-6) M) has dual effects on Cd movement. Zn further increases the Cd levels in the lung and mouse mortality increases. On the contrary, Zn inhibits prolonged Cd accumulation in mice and decreases the chronic toxicity in mice that survive the acute phase.