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The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na+ and glucose. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the first project, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Charged and polar residues and glucose/galactose malabsorption (GGM) missense mutations in TM IV were replaced with cysteine. Mutants exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. The results from mutants T156C and K157C suggest that TM IV participates in sugar interaction with SGLT1. This work has been published in Am J Physiol Cell Physiol 295 (1), C64-72, 2008. The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) was recently published (1) and showed discrepancy with the predicted topology of mammalian SGLT1 in the region surrounding transmembrane segments IV-V. Therefore, in the second project, we investigated the topology in this region, thirty-eight residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were individually replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. Based on the results from biotinylation of mutants in intact COS-7 cells, MTSES accessibility of cysteine mutants expressed in COS-7 cells, effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells, and characterization of cysteine mutants in TM V expressed in Xenopus laevis oocytes, we suggest that the region including residues 143-180 forms part of the Na+- and sugar substrate-binding cavity. Our results also suggest that TM IV of mammalian SGLT1 extends from residue 143-171 and support the crystal structure of vSGLT. This work has been published in Biochem Biophys Res Commun 378 (1), 133-138, 2009 Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C following reaction with methanethiosulfonate derivatives, but Na+ and sugar binding remain intact. Therefore, in the third project, we explored the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 expressed in Xenopus laevis oocytes were studied using chemical modification, the two-electrode voltage-clamp technique and computer model simulations. Our results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. This work has been published in Biophysical Journal 96 (2), 748-760, 2009. Taken together our study along with previous biochemical characterization of SGLT1 and crystal structure of vSGLT, we propose a limited structural model that attempts to bring together the functions of substrate binding (Na+ and sugar), coupling, and translocation. We propose that both Na+ and sugar enter a hydrophilic cavity formed by multiple transmembrane helices from both N-terminal half of SGLT1 and C-terminal half of SGLT1, analogous to all of the known crystal structures of ion-coupled transporters (the Na+/leucine transporter, Na+/aspartate transporter and lactose permease). The functionally important residues in SGLT1 (T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites.

Osteopontin (OPN) is a matricellular cytokine found in most tissues and body fluids. It is involved in a variety of cell processes by binding to integrins and CD44 receptors, and it modulates immune responses. To investigate the functions of OPN during colitis the DSS acute colitis model in OPN-/- and WT control mice was utilized. OPN-/- mice were more susceptible to DSS-induced colitis than the DSS-treated WT control mice. The increased susceptibility of the OPN-/- mice was characterized by greater intestinal crypt destruction; high myeloperoxidase activity of infiltrating neutrophils; lack of differentiation of inflammatory cells such as lymphocytes subsets (CD4+, CD8+) and macrophages (F4/80); reduced production of certain cytokines, especially TNF-alpha; and non-programmed cell death of enterocytes. It is postulated that the hyperactivity of neutrophils may explain the increased tissue destruction during experimental colitis in the absence of OPN. Analysis of OPN’s impact on neutrophil function showed that while OPN may be important for the recruitment and migration of neutrophils, the expression of OPN by neutrophils is not required for manifestation of their destructive capabilities. This would suggest that OPN administration may protect the intestines from the adverse effects of colitis. Exogenous bovine milk OPN (20 μg/ml), administered for 8 days dissolved in the drinking water, ameliorated DSS-induced colitis. It diminished signs of disease, with a greater impact in the WT than the OPN-/- mice. It reduced the levels of neutrophils, macrophages, and pro-inflammatory mediators in the colon tissue. Recombinant OPN failed to reproduce the beneficial effects of milk OPN, which suggests that post-translational modifications of OPN are crucial to ameliorate experimental colitis. Collectively, these studies demonstrate that OPN has a protective effect during experimental colitis and that the oral administration of bovine milk OPN (20 μg/ml) ameliorates acute DSS-induced colitis. The results of this study also imply that the protective effect probably depends on a post-translationally modified form of OPN, and may require intracellular-OPN as a cofactor for significant attenuation of colitis. Future research could concentrate on more detailed investigation of these latter findings to determine OPN’s mechanism of action.

In this thesis, the discharge products formed at the cathode and the performance and cell chemistry of sodium-oxygen batteries have been studied. This was carried out using different NaOTf salt concentrations. The influence of different salt concentrations on sodium-oxygen batteries was investigated since it has been shown that increasing the salt concentration beyond conventional concentrations could result in advantages such as increased stability of the electrolytes towards decomposition, higher thermal stability and lower volatility. An increase in salt concentration has also been shown to influence the electrochemical potential window. The solubility of NaOTf was investigated in two different solvents, DME and diglyme. NaOTf was found to be more soluble in DME compared to diglyme but due to the volatile nature of DME, three different concentrations of NaOTf were prepared with diglyme as solvent. Experimentation involved discharging the batteries to either maximum or limited capacity. The discharge products were examined and characterized using XRD and SEM. The main discharge product was identified as sodium superoxide although sodium peroxide dihydrate was also identified in one battery. A trend of higher capacity and voltage plateaus with higher salt concentration was also found. The influence of trace amounts of water was suggested as one explanation as it works as a catalyst, promoting sodium superoxide cube growth due to improved transportation of superoxide. Another or contributing explanation could be a possible change in donor number with increased salt concentration, resulting in higher solubility and longer lifetime of superoxide, promoting the growth of sodium superoxide cubes.

Objectives: To explore salt content in bread and to evaluate the feasibility of reducing salt contained in ‘pan francés’ bread. Design: The study had two phases. Phase 1, an exploratory phase, involved the estimation of salt contained in bread as well as a triangle taste test to establish the amount of salt to be reduced in ‘pan francés’ bread without detection by consumers. In Phase 2, a quasi-experimental, pre–post intervention study assessed the effects of the introduction of low-salt bread on bakery sales. Setting: A municipal bakery in Miraflores, Lima, Peru. Subjects: Sixty-five clients of the bakery in Phase 1 of the study; sales to usual costumers in Phase 2. Results: On average, there was 1·25 g of salt per 100 g of bread. Sixty-five consumers were enrolled in the triangle taste test: fifty-four (83·1 %) females, mean age 58·9 (SD 13·7) years. Based on taste, bread samples prepared with salt reductions of 10 % (P = 0·82) and 20 % (P =0·37) were not discernible from regular bread. The introduction of bread with 20 % of salt reduction, which contained 1 g of salt per 100 g of bread, did not change sales of ‘pan francés’ (P=0·70) or other types of bread (P =0·36). Results were consistent when using different statistical techniques. Conclusions: The introduction of bread with a 20 % reduction in salt is feasible without affecting taste or bakery sales. Results suggest that these interventions are easily implementable, with the potential to contribute to larger sodium reduction strategies impacting the population’s cardiovascular health.

The purpose of this study was to see if any soil bacteria were able to use pentachlorophenol or sodium pentachlorophenate either aerobically or anaerobically as a sole carbon source, to see if any soil bacteria could survive in high concentrations of sodium pentachlorophenate, to determine the maximum concentration of sodium pentachlorophenate which permitted the growth of some soil bacteria, to see the effects of varying concentrations of sodium pentachlorophenate on the growth curves of soil bacteria capable of growing in its presence, and to see if any soil bacteria could degrade sodium pentachlorophenate.