Genome sequence of Enterococcus hirae (Streptococcus faecalis) ATCC 9790, a model organism for the study of ion transport, bioenergetics, and copper homeostasis

Enterococcus hirae ATCC 9790 is a Gram-positive lactic acid bacterium that has been used in basic research for over 4 decades. Here we report the sequence and annotation of the 2.8-Mb genome of E. hirae and its endemic 29-kb plasmid pTG9790.

The sodium-dependent ascorbic acid transporter family SLC23

Transporters for vitamin C and its oxidized form dehydroascorbic acid (DHA) are crucial to maintain physiological concentrations of this important vitamin that is used in a variety of biochemical processes. The human SLC23 family consists of the Na(+)-dependent vitamin C transporters SVCT1 (encoded by the SLC23A1 gene) and SVCT2 (SLC23A2) as well as an orphan transporter SVCT3 (SLC23A3). Phylogenetically, the SLC23 family belongs to the nucleobase-ascorbate transporter (NAT) family, although no nucleobase transport has yet been demonstrated for the human members of this family. The SVCT1 and SVCT2 transporters are rather specific for ascorbic acid, which is an important antioxidant and plays a crucial role in a many metal-containing enzymes. SVCT1 is expressed predominantly in epithelial tissues such as intestine where it contributes to the supply and maintenance of whole-body ascorbic acid levels. In contrast to various other mammals, humans are not capable of synthesizing ascorbic acid from glucose and therefore the uptake of ascorbic acid from the diet via SVCT1 is essential for maintaining appropriate concentrations of vitamin C in the human body. The expression of SVCT2 is relatively widespread, where it serves to either deliver ascorbic acid to tissues with high demand of the vitamin for enzymatic reactions or to protect metabolically highly active cells or specialized tissues from oxidative stress. The murine Slc23a3 gene encoding the orphan transporter SVCT3 was originally cloned from mouse yolk sac, and subsequent studies showed that it is expressed in the kidney. However, the function of SVCT3 has not been reported and it remains speculative as to whether SVCT3 is a nucleobase transporter.

Inhibition of indoleamine 2,3-dioxygenase in mixed lymphocyte reaction affects glucose influx and enzymes involved in aerobic glycolysis and glutaminolysis in alloreactive T-cells

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. T-cell proliferation and differentiation to effector cells require increased glucose consumption, aerobic glycolysis and glutaminolysis. The effect of IDO on the above metabolic pathways was evaluated in alloreactive T-cells. Mixed lymphocyte reaction (MLR) in the presence or not of the IDO inhibitor, 1-DL-methyl-tryptophane (1-MT), was used. In MLRs, 1-MT decreased tryptophan consumption, increased cell proliferation, glucose influx and lactate production, whereas it decreased tricarboxylic acid cycle activity. In T-cells, from the two pathways that could sense tryptophan depletion, i.e. general control nonrepressed 2 (GCN2) kinase and mammalian target of rapamycin complex 1, 1-MT reduced only the activity of the GCN2 kinase. Additionally 1-MT treatment of MLRs altered the expression and/or the phosphorylation state of glucose transporter-1 and of key enzymes involved in glucose metabolism and glutaminolysis in alloreactive T-cells in a way that favors glucose influx, aerobic glycolysis and glutaminolysis. Thus in alloreactive T-cells, IDO through activation of the GCN2 kinase, decreases glucose influx and alters key enzymes involved in metabolism, decreasing aerobic glycolysis and glutaminolysis. Acting in such a way, IDO could be considered as a constraining factor for alloreactive T-cell proliferation and differentiation to effector T-cell subtypes.

Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

The H(+) -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H(+) electrochemical gradient. At low extracellular pH (pH(o)), H(+) binding preceded binding of Fe(2+) and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pH(o), and at pH(o) 7.4 we observed Fe(2+) transport that was not associated with H(+) influx. His(272) --> Ala substitution uncoupled the Fe(2+) and H(+) fluxes. At low pH(o), H272A mediated H(+) uniport that was inhibited by Fe(2+). Meanwhile H272A-mediated Fe(2+) transport was independent of pH(o). Our data indicate (i) that H(+) coupling in DMT1 serves to increase affinity for Fe(2+) and provide a thermodynamic driving force for Fe(2+) transport and (ii) that His-272 is critical in transducing the effects of H(+) coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe(2+) transport in the absence of a H(+) gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H(+) -uncoupled facilitative Fe(2+) transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.

Functional properties of multiple isoforms of human divalent metal-ion transporter 1 (DMT1)

DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5'-ends (starting in exon 1A or exon 1B) and at their 3'-ends giving rise to mRNAs containing (+) or lacking (-) the 3'-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE+-DMT1 mediated Fe2+-evoked currents that were saturable (K(0.5)(Fe) approximately 1-2 microM), temperature-dependent (Q10 approximately 2), H+-dependent (K(0.5)(H) approximately 1 muM) and voltage-dependent. 1A/IRE+-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+>>Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl- conductances and was not associated with significant Zn2+ transport. 1B/IRE+-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).

Loss of the high-affinity pentamidine transporter is responsible for high levels of cross-resistance between arsenical and diamidine drugs in African trypanosomes

Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable cross-resistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [(3)H]pentamidine. In particular, the high-affinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1(-/-) line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4',6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 muM pentamidine. No overexpression of the Trypanosoma brucei brucei ATP-binding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance.

Inherited epithelial transporter disorders-an overview

In the late 1990s, the identification of transporters and transporter-associated genes progressed substantially due to the development of new cloning approaches such as expression cloning and, subsequently, to the implementation of the human genome project. Since then, the role of many transporter genes in human diseases has been elucidated. In this overview, we focus on inherited disorders of epithelial transporters. In particular, we review genetic defects of the genes encoding glucose transporters (SLC2 and SLC5 families) and amino acid transporters (SLC1, SLC3, SLC6 and SLC7 families).

Transport model of the human Na+-coupled L-ascorbic acid (vitamin C) transporter SVCT1

Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-ascorbic acid transport was saturable (K(0.5) approximately 70 microM), temperature dependent (Q(10) approximately 5), and energized by the Na(+) electrochemical potential gradient. We obtained a Na(+)-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-ascorbic acid and Na(+) saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na(+), L-ascorbic acid, Na(+). In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na(+), maximal charge translocation (Q(max)) was approximately 25 nC, around a midpoint (V(0.5)) at -9 mV, and with apparent valence approximately -1. Q(max) was conserved upon progressive removal of Na(+), whereas V(0.5) shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na(+) partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease.

Price risk management in the copper market using commodity derivatives and options strategies

Metals price risk management is a key issue related to financial risk in metal markets because of uncertainty of commodity price fluctuation, exchange rate, interest rate changes and huge price risk either to metals’ producers or consumers. Thus, it has been taken into account by all participants in metal markets including metals’ producers, consumers, merchants, banks, investment funds, speculators, traders and so on. Managing price risk provides stable income for both metals’ producers and consumers, so it increases the chance that a firm will invest in attractive projects. The purpose of this research is to evaluate risk management strategies in the copper market. The main tools and strategies of price risk management are hedging and other derivatives such as futures contracts, swaps and options contracts. Hedging is a transaction designed to reduce or eliminate price risk. Derivatives are financial instruments, whose returns are derived from other financial instruments and they are commonly used for managing financial risks. Although derivatives have been around in some form for centuries, their growth has accelerated rapidly during the last 20 years. Nowadays, they are widely used by financial institutions, corporations, professional investors, and individuals. This project is focused on the over-the-counter (OTC) market and its products such as exotic options, particularly Asian options. The first part of the project is a description of basic derivatives and risk management strategies. In addition, this part discusses basic concepts of spot and futures (forward) markets, benefits and costs of risk management and risks and rewards of positions in the derivative markets. The second part considers valuations of commodity derivatives. In this part, the options pricing model DerivaGem is applied to Asian call and put options on London Metal Exchange (LME) copper because it is important to understand how Asian options are valued and to compare theoretical values of the options with their market observed values. Predicting future trends of copper prices is important and would be essential to manage market price risk successfully. Therefore, the third part is a discussion about econometric commodity models. Based on this literature review, the fourth part of the project reports the construction and testing of an econometric model designed to forecast the monthly average price of copper on the LME. More specifically, this part aims at showing how LME copper prices can be explained by means of a simultaneous equation structural model (two-stage least squares regression) connecting supply and demand variables. A simultaneous econometric model for the copper industry is built: {█(Q_t^D=e^((-5.0485))∙P_((t-1))^((-0.1868) )∙〖GDP〗_t^((1.7151) )∙e^((0.0158)∙〖IP〗_t ) @Q_t^S=e^((-3.0785))∙P_((t-1))^((0.5960))∙T_t^((0.1408))∙P_(OIL(t))^((-0.1559))∙〖USDI〗_t^((1.2432))∙〖LIBOR〗_((t-6))^((-0.0561))@Q_t^D=Q_t^S )┤ P_((t-1))^CU=e^((-2.5165))∙〖GDP〗_t^((2.1910))∙e^((0.0202)∙〖IP〗_t )∙T_t^((-0.1799))∙P_(OIL(t))^((0.1991))∙〖USDI〗_t^((-1.5881))∙〖LIBOR〗_((t-6))^((0.0717) Where, Q_t^D and Q_t^Sare world demand for and supply of copper at time t respectively. P(t-1) is the lagged price of copper, which is the focus of the analysis in this part. GDPt is world gross domestic product at time t, which represents aggregate economic activity. In addition, industrial production should be considered here, so the global industrial production growth that is noted as IPt is included in the model. Tt is the time variable, which is a useful proxy for technological change. A proxy variable for the cost of energy in producing copper is the price of oil at time t, which is noted as POIL(t ) . USDIt is the U.S. dollar index variable at time t, which is an important variable for explaining the copper supply and copper prices. At last, LIBOR(t-6) is the 6-month lagged 1-year London Inter bank offering rate of interest. Although, the model can be applicable for different base metals' industries, the omitted exogenous variables such as the price of substitute or a combined variable related to the price of substitutes have not been considered in this study. Based on this econometric model and using a Monte-Carlo simulation analysis, the probabilities that the monthly average copper prices in 2006 and 2007 will be greater than specific strike price of an option are defined. The final part evaluates risk management strategies including options strategies, metal swaps and simple options in relation to the simulation results. The basic options strategies such as bull spreads, bear spreads and butterfly spreads, which are created by using both call and put options in 2006 and 2007 are evaluated. Consequently, each risk management strategy in 2006 and 2007 is analyzed based on the day of data and the price prediction model. As a result, applications stemming from this project include valuing Asian options, developing a copper price prediction model, forecasting and planning, and decision making for price risk management in the copper market.

An investigation of phenolic glycoside and condensed tannin homeostasis in Populus by salicyl alcohol feeding to cell cultures and by transgenic manipulation of the sucrose transporter, PTSUT4, in planta

Secondary metabolites play an important role in plant protection against biotic and abiotic stress. In Populus, phenolic glycosides (PGs) and condensed tannins (CTs) are two such groups of compounds derived from the common phenylpropanoid pathway. The basal levels and the inducibility of PGs and CTs depend on genetic as well as environmental factors, such as soil nitrogen (N) level. Carbohydrate allocation, transport and sink strength also affect PG and CT levels. A negative correlation between the levels of PGs and CTs was observed in several studies. However, the molecular mechanism underlying such relation is not known. We used a cell culture system to understand negative correlation of PGs and CTs. Under normal culture conditions, neither salicin nor higher-order PGs accumulated in cell cultures. Several factors, such as hormones, light, organelles and precursors were discussed in the context of aspen suspension cells’ inability to synthesize PGs. Salicin and its isomer, isosalicin, were detected in cell cultures fed with salicyl alcohol, salicylaldehyde and helicin. At higher levels (5 mM) of salicyl alcohol feeding, accumulation of salicins led to reduced CT production in the cells. Based on metabolic and gene expression data, the CT reduction in salicin-accumulating cells is partly a result of regulatory changes at the transcriptional level affecting carbon partitioning between growth processes, and phenylpropanoid CT biosynthesis. Based on molecular studies, the glycosyltransferases, GT1-2 and GT1-246, may function in glycosylation of simple phenolics, such as salicyl alcohol in cell cultures. The uptake of such glycosides into vacuole may be mediated to some extent by tonoplast localized multidrug-resistance associated protein transporters, PtMRP1 and PtMRP6. In Populus, sucrose is the common transported carbohydrate and its transport is possibly regulated by sucrose transporters (SUTs). SUTs are also capable of transporting simple PGs, such as salicin. Therefore, we characterized the SUT gene family in Populus and investigated, by transgenic analysis, the possible role of the most abundantly expressed member, PtSUT4, in PG-CT homeostasis using plants grown under varying nitrogen regimes. PtSUT4 transgenic plants were phenotypically similar to the wildtype plants except that the leaf area-to-stem volume ratio was higher for transgenic plants. In SUT4 transgenics, levels of non-structural carbohydrates, such as sucrose and starch, were altered in mature leaves. The levels of PGs and CTs were lower in green tissues of transgenic plants under N-replete, but were higher under N-depleted conditions, compared to the levels in wildtype plants. Based on our results, SUT4 partly regulates N-level dependent PG-CT homeostasis by differential carbohydrate allocation.

Syntheses and structures of molybdenum and tungsten complexes capable of epoxidaton and copper coordination polymers and dendrimers

We are interested in the syntheses of new complexes and in their characterization by single crystal X-ray diffraction techniques. Once we understand the structures, studies aimed at understanding uses of these complexes in the field of catalytic epoxidation using complexes soluble in water and syntheses of thin films (not assessed) were conducted. The syntheses, characterization and catalytic properties of a series of mononuclear, dinuclear and tetranuclear molybdenum and tungsten oxo complexes are described. The syntheses and structural characterization of two copper coordination polymers with 3,5-dihydroxylbenzoate ligand, and five paddlewheel shaped copper dendrimers coordinated with Fréchet-type dendrons are also detailed. The background of this dissertation is outlined in Chapter 1. Chapter 2 describes the syntheses, and characterization of two new mononuclear molybdenum(VI) and tungsten(VI) oxo complexes, MoO2Cl2(OPPh2CH2OH)2, and WO2Cl2(OPPh2CH2OH)2, bearing hydrophilic phosphine oxide ligand. The catalytic properties of these complexes for the epoxidation of cis-cyclooctene were also studied. Two new dinuclear molybdenum(VI) and tungsten(VI) oxo complexes Mo2O4Cl2[(HOCH2)PhPOO]2, and (CH3O)2(O)W(μ-O)(μ-O2PPh2)2W(O)(CH3O)2, bearing organophosphinate ligand are described in Chapter 3 and 4. Chapter 4 and 5 describes the syntheses and characterization of tetranuclear molybdenum(V) oxo complexes bearing various organophosphinate ligands. The catalytic abilities of these complexes for the epoxidation of cis-cyclooctene in the presence of hydrogen peroxide as oxidant were explored as well. Various spectroscopic methods, such as IR, UV-vis, and NMR are used to characterize the nature of these complexes. Crystal structures of compounds MoO2Cl2(OPPh2CH2OH)2, WO2Cl2(OPPh2CH2OH)2, Mo2O4Cl2[(HOCH2)PhPOO]2, (CH3O)2(O)W(μ-O)(μ-O2PPh2)2W(O)(CH3O)2, and Mo4(µ3-O)4(µ-O2PR2)4O4 (R=Ph, Me, ClCH2, o-C6H4(CH2)2) are also presented. The syntheses, and structural characterization of three copper(II) coordination polymers bearing 3,5-dihydroxybenzoate ligand are described in Chapter 6. Two copper(II) coordination polymers, [Cu2(3,5-dhb)2(pyridine)4]n, and [Cu2(3,5-dhb)4]n were afforded based on different amount of pyridine used in the reaction. The structures of these complexes are further built into 2D or 3D networks via inter or intra hydrogen bonds. The syntheses and structural characterization of the zinc(II) monomer, Zn(3,5-dhb)2(pyridine)2 is also described in this Chapter. Chapter 7 describes the syntheses, and characterization of five dendronized dicopper complexes bearing different generations of Fréchet-type dendrons. The structures of 3,5- bis(benzoyloxl)benzoic acid, 3,5-(PhCOO)2PhCOOH (G1), Cu2(3,5-dhb)4(THF)2, Cu2(G1)4(pyridine)2, and Cu2(G1)4(CH3OH)2 were characterized unambiguously by single X-ray diffraction. In addition, all compounds were characterized by FT-IR, UV-vis spectroscopy and elemental analyses.