Fish nodavirus causes the death of several high economic fish. For studying the package and stability of dragon grouper necrosis virus (DGNNV), wild type virus-like particles (wt-VLPs) and £GN25-VLPs were employed. After experiments of disassembly and assembly, the result showed that DNA of 608 bp was able to be packaged by wt-VLPs. The sedimentation of VLPs was affected by different pH buffers during the process of purification: the VLPs at alkaline conditions behaved faster than those at the acidic. The stability of pure VLPs was pH-independent. The micrographs of the wt-VLPs in alkaline buffers showed that particles were rough and irregular in shapes, some of which were stain-permeable. However, the wt-VLPs in acidic buffers were morphologically indistinguishable from the untreated VLPs. The Western blotting for gradiently purified miture of VLPs and lysozyme revealed that lysozyme was co-precipitated with wt-VLPs.

The Wingless (Wnt) signaling pathway is one of the critical developmental pathways for control of cell differentiation, proliferation, and cell growth. The DEP domain, located on the C-terminus of Dishevelled (Dvl), plays a role in cytoplasm-membrane association, which branches the canonical and non-canonical Wnt signaling pathway within the cell. It has been suggested that the DEP domain requires the recruitment of ionic lipids, such as phosphatidic acid, to regulate its localization to the plasma membrane and association to the frizzle receptor. However, the physical mechanism for DEP association to the plasma membrane is still unknown. We show that mDvl2-DEP interacts with phosphatidic acid at a distinct patch on the surface formed by a positively charged surface area by NMR spectroscopy. The binding of this interaction was also found at physiologically relevant concentration using fluorescence spectroscopy. We also determined that the interaction is pH-dependent and regulated through a 'histidine switch' mechanism at His464 and His465 where there is increased association of mDvl2-DEP to the plasma membrane at higher pH values (7.5). This association is based on tertiary structure conformational changes with rearrangement of the loop regions by a change in local pH, not its interaction with phosphatidic acid. Overall, our work will contribute to elucidate how cells regulate their developmental pathways through localized molecular interactions. / Master of Science

Cerium oxide nanoparticles have been widely used for various applications such as catalytic converters for automobile exhaust, ultraviolet absorber, and electrolyte in fuel cells. Most recently, cerium oxide nanoparticles (nanoceria) have been employed as potent free-radical scavengers with neuroprotective, radioprotective, and anti-inflammatory properties. These properties of cerium oxide nanoparticles can open new vistas in medicine and biotechnology. The present study utilizes the water-based-wet-chemical method to synthesize biocompatible,stable and highly monodisperse polymer coated cerium oxide nanoparticles. Polymer coated cerium oxide nanoparticles possess all the characteristics of the uncoated cerium oxide nanoparticles. These nanoparticles were found to be effective as pH-dependent antioxidant giving cytoprotection to normal cell lines against hydrogen peroxide and nitric oxide radical but not to cancer cells. Moreover, cerium oxide nanoparticle also exhibits unique oxidase-like activity at acidic pH oxidizing a series of organic compound without the need of hydrogen peroxide. Based on these results, we have designed an immunoassay in which folate-conjugated cerium oxide nanoparticles provide dual functionality by binding to folate expressing cancer cells and facilitating detection by catalytic oxidation of sensitive colorimetric substrates (dyes). Finally, we have shown that the polymer coated cerium oxide nanoparticles shows distinct toxicity depending upon their subcellular localization based on uptake studies using DiI loaded cerium oxide nanoparticles. In these results, we have found that cerium oxide nanoparticles entrapped into lysosomes are more toxic as opposed to when they are localized in the cytoplasm.Overall we propose that the polymer coated cerium oxide nanoparticles displays selective antioxidant property, oxidase-like activity, and cytotoxicity to biological systems depending upon its pH environment.

The period of industrialization after the second World War in Europe released SO2 and NOx by combustion of fossil fuels and contributed the formation of S and N compounds in the forest ecosystem. The Swedish forest soil systems were influenced by emissions of SO2 followed by H2SO4 deposition, consequently the pool of SO42- had increased in the forest ecosystem. This thesis studied SO42- adsorption in a podzolic Bs horizon soils taken from a Swedish forest soil system. The soil samples from five different sampling sites were collected and the results revealed different amounts of adsorbed SO42- in response to changes in equilibrium concentration and pH. This study found that the amount of adsorbed SO42- (mmol/kg) increased with an added equilibrium concentration of SO42- (mmol/l) and with a decreasing pH. This was determined by equilibration experiments. Based on the results a Freundlich-based model was developed to predict the pool of adsorbed SO42- in the soil samples. The model predicted the pool of adsorbed SO42- (mmol/kg) as a function of pH and the equilibrium concentration of SO42- (mmol/l) in the soil solution system. The extended Freundlich model was optimized in three different ways: by use of unconstrained, constrained and simplified two-point calibration. The results showed that the adsorption of sulfate in the Kloten Bs1 and Risbergshöjden B soils was higher as compared to the Tärnsjo B, Österström B, and Risfallet B soils. The coefficient of determination (R2) determined from an unconstrained fit of the extended Freundlich model (with three adjustable parameters) for Risbergshöjden B and Kloten Bs1 were R2 =0.998 and R2=0.993. Nearly as good fits were found in a constrained fit with two adjustable parameters when it was assumed that nearly 2 protons (2 H+) are co-adsorbed with one SO42- ion (Risbergshöjden B; R2=0.997 and Kloten Bs; R2=0.992). The simplified two-point calibration with two adjustable parameters showed similar parameter values for all most soils and was considered the best optimization method of extended Freundlich model, especially as it requires only limited input data.

No description available.

Yes / Fluorescence spectroscopy on a series of aqueous solutions of poly(acrylic acid) containing a luminescent label showed that polymers with molar mass, Mn < 16.5 kDa did not exhibit a pH responsive conformational change, which is typical of higher molar mass poly(acrylic acid). Below this molar mass, polymers remained in an extended conformation, regardless of pH. Above this molar mass, a pH-dependent conformational change was observed. Diffusion-ordered nuclear magnetic resonance spectroscopy confirmed that low molar mass polymers did not undergo a conformational transition, although large molar mass polymers did exhibit pH-dependent diffusion. / Engineering and Physical Sciences Research Council (EPSRC) funded CASE award PhD. Part funded by flocculant manufacturer SNF (UK) Ltd.

The structure of the voltage-gated proton (H+) channel Hv1 is homologous to the voltage sensor domain (VSD) of tetrameric voltage-gated Na+, K+ and Ca2+ channels (VGCs), but lacks a pore domain and instead forms a homodimer. Similar to other VSD proteins, Hv1 is gated by changes in membrane potential (V), but unlike VGCs, voltage-dependent gating in Hv1 is modulated by changes in the transmembrane pH gradient (DpH = pHo - pHi). In Hv1, pHo or pHi changes shift the open probability (POPEN)-V relation by ~40 mV per pH unit. To better understand the structural basis of pHo-dependent gating in Hv1, we constructed new resting- and activated-state Hv1 VSD homology models using physical constraints determined from experimental data measured under voltage clamp and conducted all-atom molecular dynamics (MD) simulations. Analyses of salt bridges and calculated pKas at conserved side chains suggests the existence of intracellular and extracellular electrostatic networks (ICEN and ECEN, respectively) that stabilize resting- or activated-state conformations of the Hv1 VSD. Structural analyses led to a novel hypothesis: two ECEN residues (E119 and D185) with coupled pKas coordinately interact with two S4 ‘gating charge’ Arg residues to modulate activated-state pHo sensitivity. Experimental data confirm that pH-dependent gating is compromised at acidic pHo in Hv1 E119A-D185A mutants, indicating that specific ECEN residue interactions are critical components of the ∆pH-dependent gating mechanism. E119 and D185 are known to participate in extracellular Zn2+ coordination, suggesting that H+ and Zn2+ utilize similar mechanisms to allosterically modulate the activated/resting state equilibrium in Hv1.

<p>Drug transport across the intestinal epithelium is roughly predicted from permeability values obtained from Caco-2 cell monolayers. This thesis examines the important role of <i>pH</i> and extracellular additives for increasing the reliability and predictivity of the <i>in vitro</i> screening system, Caco-2.</p><p>It was shown that the passive transport of ionizable compounds may be biased by a false efflux or uptake component, when applying a physiological <i>pH</i>-gradient across the membrane. <i>pH</i> also affected the amount of compound available at the transporter-binding site. Therefore, <i>pH</i> dependence should be considered in studies of such compounds and of drug-drug interactions involving efflux transporters. It was also shown that proton-dependent apical uptake or basolateral efflux should be studied both with and without a <i>pH</i> gradient over the whole monolayers. </p><p>The two extracellular additives, bovine serum albumin (BSA) and the solubilizing agent, Cremophor^® EL, also influenced Caco-2 permeabilities. BSA applied to the receiver side increases, and to the donor side decreases drug permeation according to the drug’s protein binding capacity. Thus, the absorptive transport for both passive and active compounds is favoured, giving a physiologically sound improvement of the Caco-2 cell model. Inclusion of BSA increased both the predictivity and quality of permeability studies, particularly of highly lipophilic, BCS class II compounds. Passive and active transport processes could also be distinguished after accounting for unbound concentrations. The overall effect of Cremophor^® EL on the permeability to a drug was compound-specific and probably dependent on micellar incorporation. Cremophor^® EL can therefore not be recommended. </p><p>Neither <i>pH</i> nor BSA affect the functionality of transporters such as P-glycoprotein. However, efflux ratios of ionizable or protein bound drugs are altered in the presence of a <i>pH</i>-gradient or BSA, indicating that an experimental system without protein or <i>pH</i> gradient can over- or underestimate active and passive efflux in drug transport.</p>

Ligand-gated ion channels couple intra- and extracellular chemical signals to cellular excitability. In response to a specific ligand, these channels change their permeability to certain ions by opening or closing their ion conductive pathway, a controlling mechanism known as channel gating. Although recent studies with X-ray crystallography and site-directed mutagenesis have revealed several structures potentially important for channel gating, the gating mechanism is still elusive. Ligand-dependent channel gating involves a series of transient events and asymmetric movements of individual subunits. Understanding of these events appears to be a challenge to current approaches in gating studies by using the homomeric wild-type or mutant channels. I therefore took an alternative approach by constructing heteromeric channels. Subunit stoichiometric studies of the Kir1.1 channel showed that a minimum of one functional subunit was required for the pH-dependent gating of the channel. Four subunits in this channel were coordinated as dynamic functional dimers. In Kir6.2 channel, stoichiometry for proton-binding was almost identical to that for channel gating in the M2 helix, suggesting a one-to-one direct coupling of proton binding in C-terminus to channel gating in M2 helix. Positive cooperativity was suggested among subunits in both the proton binding and channel gating. Ligand binding can be differentiated from channel gating by studying the ATP-dependent gating of Kir6.2 channel. Disruptions in ATP binding were found to change both the potency and efficacy of the concentration-dependent curves, while the baseline activity instead of maximum inhibition was affected by disruptions of channel gating. Four subunits in the Kir6.2 channel undergo negative cooperativity in ATP binding and positive cooperativity in channel gating. The ligand binding was coupled to the gating mechanism in the same subunit and neighboring subunits, although the intrasubunit coupling was more effective. These results are well described with the operational model which we have applied to ion channel studies for the first time. By manipulating the relative distance and the interaction of two transmembrane helices, the inner helix bundle of crossing was found to not only serve as a gate but also determine the consequence of ligand binding.

Drug transport across the intestinal epithelium is roughly predicted from permeability values obtained from Caco-2 cell monolayers. This thesis examines the important role of pH and extracellular additives for increasing the reliability and predictivity of the in vitro screening system, Caco-2. It was shown that the passive transport of ionizable compounds may be biased by a false efflux or uptake component, when applying a physiological pH-gradient across the membrane. pH also affected the amount of compound available at the transporter-binding site. Therefore, pH dependence should be considered in studies of such compounds and of drug-drug interactions involving efflux transporters. It was also shown that proton-dependent apical uptake or basolateral efflux should be studied both with and without a pH gradient over the whole monolayers. The two extracellular additives, bovine serum albumin (BSA) and the solubilizing agent, Cremophor® EL, also influenced Caco-2 permeabilities. BSA applied to the receiver side increases, and to the donor side decreases drug permeation according to the drug’s protein binding capacity. Thus, the absorptive transport for both passive and active compounds is favoured, giving a physiologically sound improvement of the Caco-2 cell model. Inclusion of BSA increased both the predictivity and quality of permeability studies, particularly of highly lipophilic, BCS class II compounds. Passive and active transport processes could also be distinguished after accounting for unbound concentrations. The overall effect of Cremophor® EL on the permeability to a drug was compound-specific and probably dependent on micellar incorporation. Cremophor® EL can therefore not be recommended. Neither pH nor BSA affect the functionality of transporters such as P-glycoprotein. However, efflux ratios of ionizable or protein bound drugs are altered in the presence of a pH-gradient or BSA, indicating that an experimental system without protein or pH gradient can over- or underestimate active and passive efflux in drug transport.