High-resolution solid-state 13C-NMR study of carbons C-5 and C-12 of the chromophore of bovine rhodopsin. Evidence for a 6-S-cis conformation with negative-charge perturbation near C-12

Solid-state 13C magic-angle spinning NMR spectroscopy has been employed to study the conformation of the 11-cis-retinylidene Schiff base chromophore in bovine rhodopsin. Spectra were obtained from lyophilized samples of bovine rhodopsin selectively 13C-labeled at position C-5 or C-12 of the retinyl moiety, and reconstituted in the fully saturated branched-chain phospholipid diphytanoyl glycerophosphocholine. Comparison of the NMR parameters for carbon C-5 presented in this paper with those published for retinyl Schiff base model compounds and bacteriorhodopsin by Harbison and coworkers [Harbison et al. (1985) Biochemistry 24, 6955-6962], indicate that in bovine rhodopsin the C-6-C-7 single bond has the unperturbed cis conformation. This is in contrast to the 6-S-trans conformation found in bacteriorhodopsin. The NMR parameters for bovine [12-13C]rhodopsin present evidence for the presence of a negative charge interacting with the retinyl moiety near C-12, in agreement with the model for the opsin shift presented by Honig and Nakanishi and coworkers [Kakitani et al. (1985) Photochem. Photobiol. 41, 471-479].     

Glutathione oxidase activity of selenocystamine: a mechanistic study.

Selenocystamine (RSe-SeR) was shown to catalyze the oxygen-mediated oxidation of excess GSH to glutathione disulfide, at neutral pH and ambient PO2. This glutathione oxidase activity required the heterolytic reduction of the diselenide bond, which produced two equivalents of the selenolate derivative selenocysteamine (RSe-), via the transient formation of a selenenylsulfide intermediate (RSe-SG). Formation of RSe- was the only reaction observed in anaerobic conditions. At ambient PO2, the kinetics and stoichiometry of GSSG production as well as that of GSH and oxygen consumptions demonstrated that RSe- performed a three-step reduction of oxygen to water. The first step was a one-electron transfer from RSe- to dioxygen, yielding superoxide and a putative selenyl radical RSe., which decayed very rapidly to RSe-SeR. In the second step, RSe- reduced superoxide to hydrogen peroxide through a much faster one-electron transfer, also associated with the decay of RSe. to RSe-SeR. The third step was a two-electron transfer from RSe- to hydrogen peroxide, again much faster than oxygen reduction, which resulted in the production of RSe-SG, presumably via a selenenic acid intermediate (RSeOH) which was trapped by excess GSH. This third step was studied on exogenous hydroperoxide in anaerobic conditions, and it could be eliminated from the glutathione oxidase cycle in the presence of excess catalase. The role of RSe- as a one- and two-electron reductant was confirmed by competitive carboxymethylation with iodoacetate. RSe- was able to rapidly reduce ferric cytochrome c to its ferrous derivative. The overall rate of catalytic glutathione oxidation was GSH concentration dependent and oxygen concentration independent. Excess glutathione reductase and NADPH increased the catalytic oxidation of GSH, probably by switching the rate-limiting step from selenylsulfide to diselenide cleavage. When GSH was substituted for dithiothreitol, it was shown to reduce RSe-SeR to RSe- in a fast and quantitative reaction, and selenocystamine behaved as a dithiothreitol oxidase, whose catalytic cycle was dependent on oxygen concentration. The oxidase cycle of glutathione was inhibited by mercaptosuccinate, while that of dithiothreitol was not affected. When mercaptosuccinate was substituted for GSH, a stable selenenylsulfide was formed. These observations suggest that electrostatic interactions affect the reductive cleavage of diselenide and selenenylsulfide linkages. This study illustrates the ease of one-electron transfers from RSe- to a variety of reducible substrates. Such free radical mechanisms may explain much of the cytotoxicity of alkylselenols, and they demonstrate that selenocystamine is a poor catalytic model of the enzyme glutathione peroxidase.     

Reduction of lactoperoxidase-H2O2 compounds by ferrocyanide: indirect evidence of an apoprotein site for one of the two oxidizing equivalents

The titration by ferrocyanide and the localization of the oxidizing equivalents of lactoperoxidase "compound II" were studied as a function of pH. It was demonstrated that 1) whatever the pH, the structure of lactoperoxidase "compound II" was compatible with a Fe IV R degree state, 2) at acidic pH, ferrocyanide preferentially reduced the oxidizing equivalent localized on the heme iron to give an Fe III R degree compound, 3) at pH 4.2 only the Fe III R degree form was obtained after reduction of lactoperoxidase "compound II" with one mole of ferrocyanide and whereas at pH greater than 4.2, a mixture of both Fe III R degree and Fe IV R forms was present, 4) lowering the pH from 7.2 to 4.0 induced a transition of Fe IV R state to Fe III R degree state, but increasing the pH from 4.0 to 7.2 did not permit the formation of Fe IV R compound from Fe III R degree compound.     

13C magic-angle spinning NMR studies of bathorhodopsin, the primary photoproduct of rhodopsin.

Magic-angle spinning NMR spectra have been obtained of the bathorhodopsin photointermediate trapped at low temperature (less than 130 K) by using isorhodopsin samples regenerated with retinal specifically 13C-labeled at positions 8, 10, 11, 12, 13, 14, and 15. Comparison of the chemical shifts of the bathorhodopsin resonances with those of an all-trans-retinal protonated Schiff base (PSB) chloride salt show the largest difference (6.2 ppm) at position 13 of the protein-bound retinal. Small differences in chemical shift between bathorhodopsin and the all-trans PSB model compound are also observed at positions 10, 11, and 12. The effects are almost equal in magnitude to those previously observed in rhodopsin and isorhodopsin. Consequently, the energy stored in the primary photoproduct bathorhodopsin does not give rise to any substantial change in the average electron density at the labeled positions. The data indicate that the electronic and structural properties of the protein environment are similar to those in rhodopsin and isorhodopsin. In particular, a previously proposed perturbation near position 13 of the retinal appears not to change its position significantly with respect to the chromophore upon isomerization. The data effectively exclude charge separation between the chromophore and a protein residue as the main mechanism for energy storage in the primary photoproduct and argue that the light energy is stored in the form of distortions of the bathorhodopsin chromophore.     

Synthesis of New Pyrrolo[1,2-a]quinoxaline Derivatives as Potential Inhibitors of Akt Kinase

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC₅₀ of 4.5 μM, and 1h that inhibited U937 and MCF7 cell lines with IC₅₀ of 5 and 8 μM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Host genetic requirements for DNA release of lactococcal phage TP901-1

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.     

The cost of tsetse control using ‘Tiny Targets’ in the sleeping sickness endemic forest area of Bonon in Côte d’Ivoire: Implications for comparing costs across different settings

BackgroundWork to control the gambiense form of human African trypanosomiasis (gHAT), or sleeping sickness, is now directed towards ending transmission of the parasite by 2030. In order to supplement gHAT case-finding and treatment, since 2011 tsetse control has been implemented using Tiny Targets in a number of gHAT foci. As this intervention is extended to new foci, it is vital to understand the costs involved. Costs have already been analysed for the foci of Arua in Uganda and Mandoul in Chad. This paper examines the costs of controlling Glossina palpalis palpalis in the focus of Bonon in Côte d’Ivoire from 2016 to 2017.Methodology/Principal findingsSome 2000 targets were placed throughout the main gHAT transmission area of 130 km² at a density of 14.9 per km². The average annual cost was USD 0.5 per person protected, USD 31.6 per target deployed of which 12% was the cost of the target itself, or USD 471.2 per km² protected. Broken down by activity, 54% was for deployment and maintenance of targets, 34% for tsetse surveys/monitoring and 12% for sensitising populations.Conclusions/SignificanceThe cost of tsetse control per km² of the gHAT focus protected in Bonon was more expensive than in Chad or Uganda, while the cost per km² treated, that is the area where the targets were actually deployed, was cheaper. Per person protected, the Bonon cost fell between the two, with Uganda cheaper and Chad more expensive. In Bonon, targets were deployed throughout the protected area, because G. p. palpalis was present everywhere, whereas in Chad and Uganda G. fuscipes fuscipes was found only the riverine fringing vegetation. Thus, differences between gHAT foci, in terms of tsetse ecology and human geography, impact on the cost-effectiveness of tsetse control. It also demonstrates the need to take into account both the area treated and protected alongside other impact indicators, such as the cost per person protected.     

High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.     

A nonlinear compartmental model of Sr metabolism. I. Non-steady-state kinetics and model building

A model of Sr metabolism was developed by using plasma and urinary Sr kinetic data obtained in groups of postmenopausal women who received four different oral doses of Sr and collected during the Sr administration period (25 days) and for 28 days after cessation of treatment. A nonlinear compartmental formalism that is appropriate for study of non-steady-state kinetics and allows dissociation of variables pertaining to Sr metabolism (system 1) from those indirectly operating on it (system 2) was used. At each stage of model development, the dose-dependent model response was fitted to the four sets of data considered simultaneously (1 set per dose). A seven-compartment model with internal Sr distribution and intestinal, urinary, and bone metabolic pathways was selected. It includes two kinds of nonlinearities: those accounting for saturable intestinal and bone processes, which behave as intrinsic nonlinearities because they are directly dependent on Sr, and extrinsic nonlinearities (dependent on system 2), which suggest the cooperative involvement of plasma Sr changes in modulating some intestinal and bone mineral metabolic pathways. With the set of identified parameter values, the initial steady-state model predictions are relevant to known physiology, and some peculiarities of model behavior for long-term Sr administration were simulated.