Human multidrug and toxin extrusion 1 (hMATE1) is a major candidate for being the molecular identity of organic cation/proton (OC/H+) exchange activity in the luminal membrane of renal proximal tubules (RPT). Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, I measured the time course of hMATE1-mediated efflux of the prototypic MATE1-substrate, [3H]1-methyl-4-phenylpyridinium ([3H]MPP), under a variety of conditions, including different values for intra- and extracellular pH, from CHO cells that stably expressed hMATE1. I showed that an IC50/Ki for interaction between extracellular H+ and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] were not significantly different (P = 0.6). To test a hypothesis that H+ interacts symmetrically with each face of hMATE1, kinetics of interaction between intracellular H+ and inward-facing hMATE1 were determined using the efflux protocol. The IC50 for interaction with H+ was 11.5 nM (pH 7.91), consistent with symmetrical interactions of H+ with the inward-facing and outward-facing aspects of hMATE1. The efflux protocols demonstrated in this study are a potential means to examine kinetics at cytoplasmic face of hMATE1 and also a practical tool to screen uptake of substrates at extracellular face of hMATE1.

This research sought to elucidate the molecular mechanism by which a recently described family of ion transport proteins, the cation diffusion facilitators (CDFs), transfer ions across biomembranes. Using the Escherichia coli homologs ZitB, and FieF, as well as CzcD from Cupriavidus metallidurans CH34 as models, the amino acids essential to CDF function were identified, and the transport behavior of ZitB and its homolog FieF, were described.Site-directed ZitB mutants were used to determine the necessity of individual amino acids. The mutation of several well-conserved acidic residues resulted in the loss of a ZitB-mediated zinc-resistant phenotype in the zinc-sensitive E. coli strain GG48. ZitB also complemented the potassium uptake deficient strain TK2420, suggesting that ZitB works as an antiporter, possibly allowing potassium into the cell while effluxing zinc.This result was further investigated in experiments using everted membrane vesicles(EMVs). Vesicles bearing ZitB accumulated 65Zn(II) in a NADH-dependent manner, with an apparent KM of 1 micromolar. This accumulation was inhibitable by the protonophore FCCP, suggesting CDF dependence upon the proton motive force. Similar results were obtained using both EMVs and proteoliposomes containing the CDF homologs CzcD, from C. metallidurans CH34, and FieF from E. coli. Despite facilitating 65Zn(II) uptakeinto EMVs, fieF transcription was zinc and iron-dependent, but independent of the ironuptake regulator Fur. FieF expression in trans complemented the iron-sensitive phenotypeof a strain lacking fur, causing it to accumulate less 55Fe than wild type. Reconstituted proteoliposomes containing FieF also accumulated less 55Fe than those without.This research confirms that CDF proteins likely depend on the cell's proton gradient, effluxing substrate in a metal:proton antiport arrangement. Substrate acquisition and throughput is facilitated by a set of acidic amino acids and histidines. The relatively lowapparent KM of ZitB suggests a homeostatic role for the protein; however, the iron-inducibilityof fieF hints at a role in iron detoxification, so the cellular functions of the CDF family may be quite diverse, even within the bacteria.

Les différentes études entreprises ont porté sur la détermination du potentiel accumulateur du peuplier à une fin d’utilisation en phytoremédiation. Chez les végétaux, les éléments traces comme le Zn, Fe, Cu, Co, Mn, Mo et Ni sont nécessaires aux processus biologiques mais leur accumulation excessive peut être toxique. D’autres éléments non essentiels comme le Cd et le Pb peuvent également être absorbés par les végétaux, et donc constituer un danger potentiel puisqu’ils entrent ainsi dans la chaîne alimentaire. La phytoremédiation est l’utilisation de plantes et de leurs microbes associés pour la dépollution de l’environnement. Dans le cadre de cette étude, il a été démontré que le peuplier (Populus spp.) pouvait accumuler de relativement fortes concentrations de certains métaux (Cd, Zn et Ni) par rapport à d’autres espèces ligneuses, par exemple 30-50 µg de Cd /g de matière sèche foliaire, soit 20 fois plus que les autres espèces. Après avoir mis en évidence le potentiel accumulateur du peuplier, nous avons testé différents cultivars en culture hydroponique et avons ainsi mis en évidence des potentiels accumulateurs variés entre cultivars. Nous avons montré que les hybrides de P. trichocarpa accumulaient davantage que les autres hybrides. Parallèlement à cette étude, nous avons implanté ces mêmes cultivars sur deux sites pollués dans le cadre du projet ANR PHYTOPOP. Combiné à la production d’une forte biomasse qui peut être utilisée pour produire de l’énergie, ce caractère accumulateur fait du peuplier un bon candidat pour la phytoremédiation. Les membres de la famille des CDF (Cation Diffusion Facilitator) sont impliqués dans le transport des métaux. Après une étude phylogénétique des membres de cette famille (démonstration d’un nombre supérieur de CDF chez le peuplier par rapport aux autres espèces), leur expression dans différents tissus et dans différentes conditions physiologiques (sur sol témoin et sur sol pollué) a été étudiée. De plus, la caractérisation fonctionnelle a permis de mettre en évidence un transporteur de Zn (PtMTP2) et un transporteur de Mn (PtMTP9). Une tentative de localisation de ces transporteurs a également été entreprise. Dans le but de localiser les métaux dans les tissus de la plante, nous avons effectué des analyses en spectrométrie de masse à ionisation secondaire (SIMS) sur des feuilles de peupliers soumis à un stress métallique. Cette dernière technique a permis de montrer que le Zn était essentiellement concentré au niveau du mésophylle. / The present study beared on the capacity of poplar to be used in phytoremediation programmes. In plants, trace elements such as Zn, Fe, Cu, Co, Mn, Mo and Ni are necessary to biological processes but they also can be toxic when present at high concentrations. Other non essential elements, such as Cd and Pb also can be taken up by plants and therefore constitute a potential risk for health via accumulation into the food chain. Phytoremediation is the use of plants and associated microorganisms to clean up the environment. First, we have shown that poplar (Populus) species could relatively accumulate high concentrations of metals (Cd, Zn and Ni) compared to other woody plants. For instance, poplar is able to accumulate 30 times more Cd than other woody species. Second we have measured in hydroponic cultures, the potential of metal accumulation by different poplar cultivars. We found that P. trichocarpa hybrids accumulated more metals than the other hybrids. As part of the PHYTOPOP programme, we have also grown the same cultivars on two different polluted sites. Finally, by secondary ion mass spectrometry, we were able to localize Zn in poplar leaf mesophyll. The high biomass, which can also be used for energetic processes, and the heavy metals accumulation properties of poplar lead to the conclusion that poplar can constitute a good candidate for phytoremediation. The CDF (Cation Diffusion Facilitator) members are proteins involved in the transport of heavy metals. Phylogenetic analyses of the CDF family have shown that poplar contains a higher number of CDF members compared with other species. Expression levels of CDF members were studied in different tissues and in different physiological conditions. Moreover, heterologous expression in yeast has shown that PtMTP2 and PtMTP9 transport Zn and Mn respectively. Localization experiments in planta were also realized.

Thesis advisor: Mary F. Roberts / Amphitropic proteins, such as the virulence factor phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis, often depend on lipid-specific recognition of target membranes. However, the recognition mechanisms for zwitterionic lipids such as phosphatidylcholine (PC), which is enriched in the outer leaflet of eukaryotic cell membranes, are not well understood. Molecular dynamics (MD) simulation and mutagenesis results strongly indicate that PI-PLC interacts with PC head groups via cation-π interactions with aromatic tyrosine residues, and suggest that cation-π interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins. Aromatic amino acids can not only form cation-π interactions at the interface but also insert into membranes and have hydrophobic interactions with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side-chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring while their enhanced hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis PI-PLC identifies those involved in cation-π interactions with PC. Cation-π interactions provide a likely molecular mechanism for BtPI-PLC PC specificity but do not account for its preference for bilayers containing a small fraction of anionic lipids. MD simulations and fluorescence correlation spectroscopy (FCS) vesicle binding measurements of positively charged amino acids as well as surface tyrosine residues are used to formulate a complete model of BtPI-PLC specific binding to mixed anionic phospholipid/PC membrane. DNF2, a new plant protein with homology to bacterial PI-PLC, is confirmed to be the first plant small PI-PLC enzyme that can cleave both PI and glycosylphosphatidylinositol (GPI) anchored proteins. GPI-anchored protein cleavage also confirms that DNF2 plays an important role in symbiosome, the intracellular compartment formed by the plant that contains nitrogen fixing bacteria. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Wong, Ching On. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 150-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Bentonite clays have long been used as additives in animal feed, aiming to improve pellet quality and prevent caking. Certain bentonites are also capable of deactivating aflatoxin B_(1) (AfB_(1)) in feed by adsorption, therefore, detoxifying the feed. However, a 10–fold difference in adsorption capacity has been observed among selected bentonites. The major mineralogical and chemical properties of smectites in determining their adsorption capacities for AfB_(1) are still poorly understood. Improved knowledge of the key controlling factors of aflatoxin adsorption to bentonite clays is needed to guide the selection, modification, and application of the clays as aflatoxin binders. The objective of this study was to test a hypothesis that a smectite's selectivity and adsorption capacity for aflatoxin was mainly determined by the size matching requirement on interlayer surface domains and the aflatoxin molecules. Three approaches were used to vary the size of nanometer-scaled nonpolar domains in the interlayer of smectites: 1) exchanging interlayer cations, 2) selecting natural bentonites with different cation exchange capacities (CEC), and 3) reducing charge density of a high CEC smectite. Six bentonites were fractionated, with their major mineralogical and chemical properties determined. Clay suspensions saturated with different cations were tested for aflatoxin adsorption. Some aflatoxin-smectite complexes were prepared and analyzed with FTIR and XRD. AfB_(1) adsorption isotherms were fitted with Langmuir, modified Langmuir with adsorption dependent affinity, and exponential Langmuir models. Divalent exchange cations with low hydration energy in general resulted in a much higher adsorption capacity and affinity for all six natural bentonite clays. Cations with smaller hydration radii tended to further enhance the adsorption process for aflatoxin on smectites. Charge density of smectite had shown significant effects on the adsorption capacity, affinity, and the isotherm shape. Aflatoxin adsorption isotherms on the six natural smectites and the CEC-reduced 5OK samples by Hofmann and Klemen effects suggested that there is an optimal CEC range between 80~110 cmol(+)/kg for the best aflatoxin binding smectites. When the smectite has a CEC within this range, the mineral has the highest affinity and adsorption capacity for AfB_(1). The aflatoxin adsorption results after cation exchange treatment, selection of different CEC smectites, and the CEC reduction on 5OK confirmed the importance of size and polarity matching on the nanometer scale in smectites’ adsorption for AfB_(1). All clay samples tested in this study were capable of adsorbing aflatoxin into interlayers, and the charge density seemed to have no effect on bonding strength.

A review of organic, π-electron donor molecules is given. The focus is on non- tetrathiafulvalene based systems. Polycyclic arenes, thioalkyl substituted arenes, chalcogenated fulvalenes, peri-dichalcogen bridged polycyclic arenes and heteroarenes are covered. Various π-electron donor molecules based on acenaphtho[ 1,2-b][ 1,4]-dithin have been synthesised via various methodologies. The redox properties of these molecules, as studied by cyclic voltammetry, provide evidence that these species are efficient π-electron donors. A selection of these compounds have also been studied by Electron Spin Resonance. Two ring expansion methodologies have been utilised in the synthesis of acenaphtho[l,2-b][l,4]-dithin based systems from 1,2-dithiols. Complexation of 7,12- dithia-benzo[k]fluoranthene, thus synthesised, with 2,5-dibromo-7,7,8,8-tetracyano-p- quinodimethane and iodine (I(_4) counter ion) yielded highly crystalline but poorly conducting salts.1,2-Dibromoacenaphthylene and benzo-l,2-dithiolate species have been reacted to form new 7,12-dithia-benzo[k]fluoranthene derivatives, two of which have been studied by X-ray diffraction. The versatile oligo(l,3-dithiole-2,4,5-trithione) compound has been used to generate various compounds containing the 1,4-dithiin ring including 8,9- di(methylsulfanyl)acenaphthyleno[l,2-b][l,4]dithine, which forms complexes with 7, 7,8,8-tetracyano-p-quinodimethane, 2,5-dibromo-7,7,8,8-tetracyano-p-quinodimethane and iodine (I(_7) counter ion) all three of which have been studied by X-ray diffraction. A novel 1,2,4-trithiolane has also been synthesised and characterised by X-ray diffraction.

No description available.

The 2-norbornyl cation is an old topic in physical-organic chemistry. Whether in classical or non-classical form (partial bridged form) it has been one of the focus of discussion. Currently the experimental data and theoretical calculations favorably support the idea that 2-norbornyl cation is not in the classical form in the stable-ion condition. In this paper, first, we will show that a 3-center-2-electron π-complex is formed by the collapse of 2-norbornyl cation. Further, using different theoretical methods (B3LYP, MP2) with different basis sets (6-31+G, 6-31G(d, p), 6-311G(d, p), 6-311G(2d, p)), we find that there is a trend for the 3-center-2-electron π-complex to approach the Cs symmetry, and this π-complex oscillates within the numerical limits of the perfect Cs symmetrical configuration. The stabilization energies of the π-complex are 13.87 Kcal/mol and 19.47 Kcal/mol by B3LYP/6-31+G and MP2/6-31+G, respectively. Second, our calculations also show that the transition state between 2-norbornyl cation and 3-norbornyl cation is formed by a 3, 2-proton shift, not the generally accepted 3, 2-hydride shift. The activation energy of this 3, 2-proton shift is 10.9 Kcal/mol. Detailed structural changes in the optimization process and the formation of transition state (also a 3-center-2-electron π-complex) between 2-norbornyl cation and 3-norbornyl cation will also be included.

No description available.