No description available.

The cultured cell lines HeLa (human cervical carcinoma) and MDCK (renal epithelial) possess a trans-membrane K+ transport that can be pharmacologically divided into ouabain-sensitive, diuretic-sensitive and ouabain- and diuretic-insensitive components. The K+ transport of primary interest in this thesis is that sensitive to "loop" diuretic. Analysis of the cation and anion dependencies and "loop" diuretic sensitivity of this K+ transport indicates that it is mediated via a Na+ K+ Cl- "cotransporter" as has already been reported in other cell types. Cell shrinkage stimulated the diuretic-sensitive K+ transport in both HeLa and MDCK cells. Analysis of the Na+ K+ and Cl- dependency of this K+ flux demonstrated that this stimulation is principally due to an increase in the maximal velocity (Vmax) of this K+ transport. This stimulated K+ flux was not regulated by cAMP nor was the response modified by elevated cellular cAMP or by low extracellular Ca2+ (+ EGTA) media. The increased maximal velocity for K+ transport cannot simply be considered to be an absolute increase in the number of Na+ K+ Cl- cotransport units in the membrane. However, a possible regulation of the Na+ K+ Cl- "cotransport" turnover rate (molecules/site. second) may be Involved. In this study the Na+ K+ Cl- "cotransport" of both HeLa and MDCK cells was inhibited in metabolically depleted cells (metabolic inhibition induced by the limited-metabolisable sugar 2 deoxy-D-glucose). This effect may be considered a secondary response of cell swelling however, since exposure of metabolically depleted cells to hyperosmolar media re-activates the Na+ K+ C1- "cotransport" in both HeLa and MDCK cells.

A cellular protein, EBP1, was identified, which bound to the "core" region of the SV40 enhancer. The DNA sequences requirements for specific EBP1 binding distinguished this protein from a number of previously reported enhancer binding proteins, Multiple copies of the EBP1 binding site could serve, in vivo, to enhance transcription. The in vivo transcriptional activity of wild type and mutant SV40 enhancers correlated with EBP1 binding. Mutations that abolish EBP1 binding also severely reduced transcription, indicating that this protein may be important for SV40 enhancer activity. Nuclease protection and chemical probing studies identified purine bases and backbone phosphate groups which participate in the formation of a specific EBP1/DNA complex. "Footprinting" with hydroxyl radicals revealed deoxyribose residues in the binding site which are protected from cleavage by EBP1. Computer graphics were used to display this information indicating that EBP1 made specific base and backbone contacts over one complete turn of the DNA double helix, supporting a model whereby the protein makes sequence-specific contacts in the major groove, although binding may also be influenced by interactions in the minor groove. Competition and contact point analyses revealed that EBP1 bound similarly to sites present in several other viral and cellular enhancers. Mutational analysis of EBP1 binding sites identified base pairs important for specific EBP1/DNA complex formation. All high affinity binding sites contained the sequence 5'-GG(N)6CC-3'. Although single base pair changes in the region between the conserved guanines and cytosines can generally be tolerated, it is clear that the central six base pairs contribute to binding affinity. Mutations in the recognition site which could lead to gross structural changes in the DNA abolish EPB1 binding.

A generalized mathematical model is developed to describe the transport of fluid and plasma proteins or other macromolecules within the interstitium. To account for the effects of plasma protein exclusion and interstitial swelling, the interstitium is treated as a multiphase deformable porous medium. Fluid flow is assumed proportional to the gradient in fluid chemical potential and therefore depends not only on the local hydrostatic pressure but also on the local plasma protein concentrations through appropriate colloid osmotic pressure relationships. Plasma protein transport is assumed to occur by restricted convection, molecular diffusion, and convective dispersion. A simplified version of the model is used to investigate microvascular exchange of fluid and a single 'aggregate' plasma protein species in mesenteric tissue. The interstitium is approximated by a rigid, rectangular, porous slab displaying two fluid pathways, only one of which is available to plasma proteins. The model is first used to explore the effects the interstitial plasma protein diffusivity, the tissue hydraulic conductivity, the restricted convection of plasma proteins, and the mesothelial transport characteristics have on the steady-state distribution and transport of plasma proteins and flow of fluid in the tissue. The simulations predict significant convective plasma protein transport and complex fluid flow patterns within the interstitium. These flow patterns can produce local regions of high fluid and plasma protein exchange along the mesothelium which might be erroneously identified as 'leaky sites'. Further, the model predicts significant interstitial osmotic gradients in some instances, suggesting that the Darcy expression invoked in a number of previous models appearing in the literature, in which fluid flow is assumed to be driven by hydrostatic pressure gradients alone, may be inadequate. Subsequent transient simulations of hypoproteinemia within the model tissue indicate that the interstitial plasma protein content decreases following this upset. The simulations therefore support (qualitatively, at least) clinical observations of hypoproteinemia. Simulations of venous congestion, however, demonstrate that changes in the interstitial plasma protein content following this upset depends, in part, on the relative sieving properties of the filtering and draining vessels. For example, when the reflection coefficients of these two sets of boundaries are similar, the interstitial plasma protein content increases with time due to an increased plasma protein exchange rate across the filtering boundaries and sieving of interstitial plasma proteins at the draining boundaries. (This effect is supported by the clinical observation that interstitial plasma protein content in liver increases during venous congestion.) As the reflection coefficient of the draining boundaries decreases relative to that of the filtering boundaries, there is a net loss of plasma proteins from the interstitium, resulting in a decrease in the total interstitial plasma protein content over time (i.e., the familiar 'plasma protein washout'). Further, the model predicts increased fluid transfer from the interstitium to the peritoneum during venous congestion, supporting the clinical observation of ascites. Finally, the model is used to study the effects of interstitial plasma protein convection and diffusion, plasma protein exclusion, and the capillary transport properties on the transit times of two macromolecular tracers representative of albumin and γ-globulin within a hypothetical, one-dimensional tissue. As was expected, the transit times of each of the tracers through the model tissue varied inversely with the degree of convective transport. Increasing the interstitial diffusivity of the albumin tracer also led to a moderate decrease in the transit time for that tracer. The capillary wall transport properties, meanwhile, had only a marginal effect on the transit time for the range of capillary permeabilities and reflection coefficients considered. However, these properties (and, in particular, the reflection coefficient) had a more pronounced effect on the ultimate steady-state concentration of the tracer in the outlet stream. It was the interstitial distribution volume of a given tracer that had the greatest impact on the time required for the outlet tracer concentration to reach 50 % of its steady-state value. This was attributed to the increased filling times associated with larger interstitial distribution volumes. These findings suggest that the 'gel chromatographic effect' observed in some tissues could possibly be explained on the basis of varying distribution volumes. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (leaves: 49-51)

It is generally accepted that biological membranes consist of a lipid bilayer matrix with proteins incorporated into the lipid bilayer. Typically, these membranes are negatively charged due to the presence of negatively charged lipids in the bilayer as well as negatively charged molecular groups on proteins. Biologically active molecules, such as environmental pollutants, enter the membrane from the aqueous phase by adsorption or partitioning into the lipid bilayer. The thesis consists of two parts. Part I is a computational study of spatial distribution of electric potential in the aqueous portion of the membrane-water interface using two models of charge distribution: (i) the discrete charge model, in which charges are located on a square lattice either on the surface or embedded in the membrane: (ii) the continuous charge density (smeared charge), Gouy-Chapman, model in which the charge is assumed to be evenly spread on the membrane surface. The computed distributions of electric potential are used to predict spatial distributions of positively charged hexavalent cation of Ruthenium Red (RuR) at the membranewater interface. It was found that anomalous behavior of RuR cannot be explained by this version of the discrete charge theory. Part II is concerned with the distribution of ionized and un-ionized species of 2,4,6-Trichlorophenol (2,4,6-TrCP) in octanol-water system, which is often used as an experimental model for predicting the distribution of toxic chemicals in the environment. In this experimental study we obtained the pH dependence of the total distribution coefficient of 2,4,6-TrCP from which the octanol water partition coefficients of the un-ionized and ionized species were determined. We compared the octanol-water partition coefficient of several chlorophenols with experimental data on adsorption of ionized chlorophenols to lipid membranes. It was found that the membrane-water partition coefficient of ionized 2,4,6-TrCP is about 240 greater than that predicted from the octanol-water system. This finding supports the hypothesis that octanol-water partition coefficients cannot be used for predicting concentrations of ionized species of chlorophenols in lipid membranes.

No description available.

Includes bibliographical references (leaves 169-184) / xiv, 184 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the concept of membrane homeostatis, which implies that biological membranes tend to maintain a constant level of lipid fluidity in the face of potential exogenous and endogenous pertubations. Manipulations of dietary cholesterol and/or saturated (coconut oil) v/s unsaturated (sunflower seed oil) fatty acids have been used to study the relationship between membrane lipid composition, membrane lipid fluidity and membrane-bound enzymes of lipid metabolism; and, to see whether these enzymes act co-ordinately for the maintenance of a membrane homeostatis under these dietary conditions. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1986

Thesis (M. Phil.)--University of Hong Kong, 1984.

published_or_final_version / Biochemistry / Master / Master of Philosophy