The Escherichia coli vitamin B12 transporter BtuCD-F is a type II importer belonging to the ABC transporter superfamily. Available data suggest both exporters and type I importers in the ABC superfamily employ similar transport mechanism in which the transmembrane (TMDs) are open to cytoplasm in the resting state, and ATP binding induces a major conformational change resulting in opening of the TMDs instead to the periplasm. However, the crystal structures of BtuCD from E. coli and recent EPR spectroscopy studies indicate that this type II importer employs a substantially different mechanism in which the TMDs are open to the periplasm in the resting state and to the cytoplasm after ATP binding. We have developed robust methods to study the conformation and transport mechanism of BtuCD-F reconstituted into lipid bilayers using single molecule fluorescence resonance energy transfer (smFRET) measurements. Fluorescent probes have been introduced at a variety of diagnostic sites, enabling smFRET to be used to measure distance changes in different conformational states as well as to observe the transitions between these states in real time. These data suggest that thermal fluctuations enable the transporter to explore different functional conformational states in the absence of ATP or other ligands. They also suggest that the ATP-bound state is indeed open to the cytoplasm and ATP binding/hydrolysis increases the rate of transition between open and closed states. Efforts are currently underway to observe the transport of vitamin B12 through a single BtuCD-F oligomer in real-time.

Cholecystokinin (CCK) secreting enteroendocrine (EEC) I cells which distribute in gastrointestinal tract play an important role in lipid sensing, digestion and fatty acids uptake. Although a lot of research has been performed, the whole mechanism of fat sensing and fatty acid uptake and hormone expression in the CCK cells is still unclear. Global analysis to characterise the CCK cells is essential. CCK cells have an indistinct morphology, a diffuse distribution and a small percentage of population in the small intestine. However, the generation of genetic fluorescence tagged animal model facilitates the study of these cells. In this thesis, single cell dissociation methods and RT-PCR methodologies for detecting nutrient sensing receptors and fatty acids transporters were established and optimised. Expression of mRNAs for fat sensing GPCRs were detected in mouse duodenal epithelium. Expression of FATP family and CD36 in CCK cells and enterocytes was studied by RT-PCR. FATP2, FATP4 and CD36 mRNA were found in both CCK cells and enterocytes. Cell culture methodologies enabling the study of function (calcium imaging and FACS analysis) were established and optimised by checking the cell viability as a criterion. The methodology combining the immunochemistry and FACS analysis to study the hormone was established but requires further optimisation.

The production of recombinant glycoproteins from mammalian cell cultures requires robust processes that can achieve high protein yield while ensuring the efficacy of these proteins as human therapeutics. We describe two approaches currently being developed in our group to genetically engineer cell lines with desirable characteristics for recombinant protein production. To enhance the degree of sialylation in the glycoprotein product, we propose to increase intracellular sialic acid availability by overexpressing the CMP-sialic acid transporters. We are also interested in engineering mammalian cells that can proliferate at reduced cultivation temperatures. Low temperature cultivation of mammalian cells has been shown to enhance glycoprotein production but reduces cell growth. It is hypothesized that a mutant cell line that can proliferate at low temperatures may be coupled with low temperature cultivation to improve recombinant protein production. / Singapore-MIT Alliance (SMA)

Positron emission tomography (PET) tracer 3-fluoro-3-deoxythymidine (FLT) is used for imaging tumor proliferation. Prior to this work, human equilibrative nucleoside transporter 1 (hENT1) was the only known human nucleoside transporter (hNT) capable of FLT transport. The aim of this research was to determine if other hNTs, including hENT2, human concentrative nucleoside transporter 1 (hCNT1), hCNT2 and hCNT3, were capable/important of/for FLT transport in mammalian cells. Transport assays performed in Xenopus laevis oocytes producing recombinant hNTs demonstrated that hENT1/2 and hCNT1/3 were capable of FLT transport. FLT uptake assays with or without hENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) in various cultured cancer cell lines demonstrated that hENT1 was responsible for the majority of mediated FLT uptake in all tested cell lines, suggesting that hENT1 was important for FLT uptake. The in vivo role of hENT1 in FLT uptake was determined by performing [18F]FLT PET on wild-type and ENT1 knockout mice. One hour after [18F]FLT injection, ENT1 knockout mice displayed significantly reduced [18F]FLT accumulation in the blood, heart, brain, kidney, liver, and lungs compared to wild-type mice. Interestingly, ENT1 knockout mice displayed increased [18F]FLT accumulation in the bone marrow and spleen which both have high CNT expression, suggesting that loss of ENT1 significantly alters FLT biodistribution in mice. hENT1 is a predictive marker of gemcitabine response in pancreatic cancers. Since FLT uptake and gemcitabine toxicity are dependent on hENT1, FLT uptake may predict gemcitabine response in pancreatic cancers. To test this hypothesis, six different pancreatic cancer cell lines were analyzed for FLT uptake and gemcitabine toxicity. hENT1/2 inhibition in cells decreased FLT uptake and gemcitabine sensitivity. In five of six cell lines, a positive correlation was observed between FLT uptake and gemcitabine toxicity, suggesting that FLT PET may be clinically useful for predicting gemcitabine response in pancreatic cancers. The results from this research suggest that hNTs, especially hENT1, are important for FLT uptake in mammalian cells and that FLT uptake can predict gemcitabine response in most cultured pancreatic cancer cells. The results warrant FLT PET clinical trials in pancreatic cancer patients to determine the potential of FLT PET in predicting gemcitabine response.

Intravenous feeding of patients with essential and balanced nutrition is required when enteral feeding is not tolerated, therefore indicating the need for Total Parenteral Nutrition (TPN). This life-saving therapy is also associated with the increase risk of intrahepatic cholestasis. The incidence of TPN-related hepatobiliary complications is common in both adults and infants on TPN. Previous work in in vivo models suggested that one of the potential contributing factors is the aluminum contamination of TPN solutions. The mechanism by which aluminum contributes to the PNAC development, though, was unknown. Aluminum as a risk factor may influence a number of hepatocellular functions to lead to cholestasis but one possible mechanism is the potential for aluminum to cause dysfunction of those transporters responsible in the maintenance of bile flow. To provide some initial information regarding the role of aluminum as a contributing factor to cholestasis and the possible underlying mechanism, cytotoxicity studies were conducted to determine whether aluminum causes direct toxicity of HepG2 cells. Furthermore, the influence of aluminum on the mRNA expression of hepatic biliary transporters (BSEP, MRP2, MATE1, NTCP) and nuclear transcription factor (FXR) in HepG2 cells using real-time RT-PCR analysis was assessed. Since inflammation is a component of cholestasis, these investigations also involved the use of an inflammatory stimulus, lipopolysaccharide (LPS), to determine whether the effects of aluminum were exacerbated by underlying inflammation. My data suggest that for the canalicular hepatic transporters MATE1 and BSEP, aluminum at higher concentration alone as well as with LPS caused increased mRNA expression levels. In addition to this, BSEP mRNA expression was preserved and that of MATE1 was increased on LPS exposure. Given the particular importance of BSEP in the maintenance of bile flow and reported effects of drug-induced inhibition of BSEP to cause hepatic cholestasis, the influence of aluminum on BSEP (and MATE1) protein expression and activity warrant investigation. Further studies may identify that inhibition of BSEP function (and possibly MATE1) by aluminum contamination of total parenteral nutrition formulations may explain, in part, the intrahepatic cholestasis associated with parenteral nutrition.

The bacterium Streptococcus pyogenes requires heme, which is taken up via an ABC transporter. An understanding of this pathway may result in new approaches to antibacterial agents. Both SiaA and NEAT2 (NEAr Transporter 2) are proteins involved in heme binding. One of the axial ligands of SiaA, His 229, was purified to study how mutagenesis affects heme binding. UV-visible studies showed a small band at 420 nm with respect to the protein band at 288 nm which probably indicates that heme was lost easily from this mutant. We have also worked to optimize the yield of Shr-NEAT2 by changing different variables. For each of the batches, the yield of holoNEAT2 was calculated by UV-visible spectroscopy. Increasing oxygen during growth did not improve holoNEAT2 yield. On the other hand, lower temperature, decrease in time after induction, and addition of ALA all increased the protein production.

Phosphorous in its inorganic form, orthophosphate (Pi), is found in every compartment of the plant cell and serves as a substrate, product or effector for a wide range of metabolic processes. Several Pi transporters exist in plants and these help regulate Pi homeostasis within different cellular compartments. The PHT4 family of organellar Pi transporters consists of six members in the model plant Arabidopsis thaliana, and five of these are localized to plastids. I used gene expression analyses and reverse genetics to demonstrate functional specialization for the PHT4 family members with a focus on PHT4;1 and PHT4;2. The PHT4;1 Pi transporter is localized to chloroplast thylakoid membranes and it is expressed in a circadian manner. Plants that lack a functional copy of the PHT4;1 gene have reduced rosette size and altered responses to photooxidative stress. The PHT4;2 transporter is localized to heterotrophic plastids in roots and other sink organs and pht4;2 mutants exhibit decreased starch accumulation, which is consistent with a defect in Pi export, and increased rosette size, which is caused by increased cell proliferation. These results confirm that PHT4;1 and PHT4;2 have specialized functions and that plastidic Pi homeostasis influences broad aspects of plant metabolism, including abiotic stress response and control of lateral organ growth.

<p>Copper (Cu) is a co-factor that is essential for oxidative phosphorylation, protection from oxidative stress, angiogenesis, signaling, iron acquisition, peptide hormone maturation, and a number of other cellular processes. However, excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. The basic components of these regulatory systems are remarkably conserved in most eukaryotes, and this has allowed the use of a variety of model organisms to further our understanding of how Cu is taken into the cell and utilized.</p><p>While the components involved in Cu uptake, distribution, and storage are similar in many eukaryotes, evolution has led to differences in how these processes are regulated. For instance, fungi regulate the components involved in Cu uptake and detoxification primarily at the level of transcription while mammals employ a host of post-translational homeostatic mechanisms. In <italic>Saccharomyces cerevisiae</italic>, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. In the first part of this work, we show that the copper-dependent enzyme Sod1 (Cu,Zn superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either <italic>CCS1</italic> or <italic>SOD1</italic> results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. </p><p>Higher eukaryotes including mice and humans regulate Cu uptake predominately by means of post-translational control of the localization and stability of the Cu transport proteins. One of these proteins, Ctr1, is the primary means of Cu uptake into the cell, and members of the highly conserved Ctr family of Cu ion channels have been shown to mediate high affinity Cu(I) uptake into cells. In yeast and cultured human cells, Ctr1 functions as a homo-trimer with each monomer harboring an amino-terminal extracellular domain, three membrane spanning domains, a cytoplasmic loop, and a cytoplasmic tail. In addition to the highly conserved Ctr1 Cu ion importer, the baker's yeast <italic>S. cerevisiae</italic> expresses a related protein called Ctr2. Experimental evidence demonstrates that unlike yeast and mammalian Ctr1, yeast Ctr2 is localized to the vacuolar membrane where it mobilizes Cu stores to the cytoplasm under conditions of Cu limitation. </p><p>In mice and humans a gene encoding a protein with significant similarity to the <italic>Ctr</italic> family has been identified, denoted <italic>Ctr2</italic>. Publications from others suggest that mammalian Ctr2 may either be a low affinity Cu importer at the plasma membrane or, similar to yeast Ctr2, may mobilize Cu from intracellular organelles such as the lysosome to the cytosol. In agreement with a previous report we found that a fraction of mouse Ctr2 is localized to the plasma membrane and that its membrane topology is the same as Ctr1. Interestingly, over-expression of Ctr2 by stable transfection results in decreased intracellular bioavailable Cu. To begin to understand the physiological role of Ctr2, mice bearing a systemic deletion of the <italic>Ctr2</italic> gene were generated. The <italic>Ctr2-/-</italic> mice are viable but hyper-accumulate Cu in all tissues analyzed. Moreover, protein levels of the Ctr1 Cu importer are dramatically altered in tissues from the <italic>Ctr2</italic> knock out mice, and over-expression of Ctr2 in cultured mammalian cells enhances processing of the Ctr1 protein into a less active form. Taken together these results suggest that mammalian Ctr2 functions in the cell as a negative regulator of Cu import via Ctr1.</p> / Dissertation

Salt glands are important structural adaptations in some plant and animal species that are involved in the excretion of excess salts. Zoysia matrella is a highly salt tolerant turf grass that has salt glands. Two cultivars of Z. matrella, ‘Diamond’ and ‘Cavalier’, were examined in this study to look for salt gland related factors responsible for the differences in their degree of salt tolerance. In addition to the adaxial salt gland density being higher in ‘Diamond’, the salt glands in salt treated (300 mM NaCl) plants of this cultivar were bigger than the ones in ‘Cavalier’. ‘Diamond’, as well as some of the ‘Diamond’ x ‘Cavalier’ hybrid lines, showed a significant induction in salt gland density in response to salt treatment. Examination of salt gland density in ‘Diamond’ x ‘Cavalier’ hybrid lines showed that salt gland density was a highly heritable trait in the salt-treated population. Ultrastructural modifications in the salt glands observed with Transmission Electron Microscopy (TEM), coupled with Cl- localization studies, suggested a preference for symplastic transport of saline ions in Z. matrella. Salt glands have been studied in several plant species; however, no studies have tried to associate the role of ion transporters with the functioning of salt glands in plants. RNA in situ studies with Na+ transporters showed localization of ZmatHKT1 transcripts in the adaxial salt glands, leaf mesophyll and bundle sheath cells for both cultivars. ZmatSOS1 expression was observed in the xylem parenchyma cells for leaves from both cultivars, but the expression was markedly different around the cells bordering the vascular tissue. The strongest expression of ZmatSOS1 for ‘Diamond’ was seen in the bundle sheath cells and the phloem, while for ‘Cavalier’ the signal was strongest in the mestome sheath cells and in cells surrounding the phloem. No expression of ZmatSOS1 was seen in the salt glands for either cultivars. ZmatNHX1 expression in both cultivars was very low, and observed in the salt glands and neighboring epidermal cells. Three alleles of ZmatNHX1 were identified in Z. matrella, along with three alternatively-spliced forms of ZmatNHX1, the expression of which were cultivar and treatment specific. Together, these results provide a model for salt transport in Z. matrella and signify potential roles of salt glands and select ion transporters in the salt tolerance of this species.

Correct dosing in pharmacotherapeutics is based on the idea that too much of a drug will cause toxicity, while too little will result in failure to elicit the desired response. A major factor in the ability of a patient to handle any dose of a drug is the capacity to metabolize and eliminate that drug from the body. For the vast majority of drugs, the liver plays a key role in determining the rate at which drugs are eliminated. First, drugs must be taken up across the cell membrane into hepatocytes by uptake transporters. Once inside the hepatocyte, biotransformation enzymes metabolize and conjugate the drug to a more water-soluble compound, the distribution of which is more easily controlled. These water-soluble metabolites are then transported out of the hepatocyte by additional drug transporters either into bile for elimination, or back into the blood.More than 2 million severe adverse drug reactions occur in the US each year and often result from interindividual variation in the ability to metabolize and eliminate drugs. This number does not include medical errors, but rather circumstances where an individual is unable to handle the standard dose of the correctly prescribed drug. Although genetics plays an important role, the greatest source of variation comes from other environmental factors such as disease states. Nonalcoholic fatty liver disease (NAFLD) is a chronic condition that comprises a spectrum of histopathologies that range from simple steatosis to the more severe steatohepatitis. Specifically, nonalcoholic steatohepatitis (NASH) has become one of the leading causes for liver transplantation in the United States, and thus clearly become a considerable burden to the U.S. healthcare system.It is not known whether the capacity of the liver to metabolize and excrete drugs is altered in patients with NASH. Because the liver plays such a critical role in drug metabolism and disposition, any disease state that disrupts or modifies these functions will alter the fate of a given drug within the body. It is therefore very likely that the ability of the liver to metabolize and excrete clinically relevant drugs is compromised in NASH patients.