Thesis--Wisconsin. / Vita. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--Bowling Green State University, 2006. / Document formatted into pages; contains xiii, 186 p. : ill. Includes bibliographical references.

The first part of this thesis consists of a study of the transport of alkali metal cations across model biological membranes mediated by ionophoric antibiotic cationomycin. The transport rates were measured by 23Na and 39K NMR using large unilamellar vesicles (LUVs) prepared from phosphatidylcholine and were compared with results obtained using red blood cells. These kinetic studies have established that there is a 1:1 complex between alkali cations and the cationomycin during the transport process. The transport rate measured are comparable to those of other ionophoric antibiotics studied previously. Cationomycin transports potassium cation more rapidly than sodium cation, and forms a more stable complex with potassium cation. The rate limiting step in the transport process is release of the cation at the membrane surface. The second part involves the study of spirotetrahydrofuran analogues as potential alkali cation transporters. A set of new synthetic ionophoric materials designed to resemble to some degree the naturally occurring ionophoric antibiotics was investigated. The kinetic rates of these compounds were measured in phosphatidylcholine LUVs by an NMR study of 7Li/23Na exchange. However, these compounds are much poorer transporters than the ionophoric antibiotics which are 10 times faster under similar conditions. The third part of this project deals with the study of the transport of halide anions by steroid-based anionophores. The transport rates of these compounds were measured by an NMR study of 35Cl/81Br exchange in phosphatidylcholine LUVs. This preliminary study indicates a close relationship between the lipophilicity of the different anionophores and the transport rates of different halides anions through the model membrane. The final part of this thesis involves the total assignment of the 1H and 13C NMR spectra of the ionophoric antibiotic tetronasin by utilising two dimensional NMR techniques such as COSY 45, Relay COSY, DQF COSY, TOCSY and HSQC.

The crnA gene of A.nidulans is confirmed to encode a transport protein, possibly for nitrate. The single open reading frame of 1449 nt encodes a polypeptide of 483 AAs with a molecular weight of 51,769 Da. The high percentage of clustered apolar AAs suggests a membrane protein with ten membrane spanning domains. No similarity between the crnA encoded polypeptide and those of other transport genes or any other gene was observed. The regulation of the A.nidulans nitrate transporter was investigated by studying levels of mRNA transcribed from the crnA gene. The crnA cDNA hybridised to two mRNAs on Northern blots. The 1.8 kb message corresponding to the size of the crnA gene is inducible with nitrate and nitrite. The 1.1 kb, constitutively synthesized message is believed to be a second transcript from the crnA gene rather than from a gene elsewhere in the genome. The expression of the crnA gene, determined from Northern blotting experiments, is shown to be at the level of mRNA accumulation. A requirement for both positive acting regulatory genes, areA and nirA is observed. The expression of the niiA and niaD genes, encoding nitrite reductase and nitrate reductase respectively, is shown to be regulated at the level of mRNA accumulation by a mechanism involving both the areA and nirA gene products. In addition, the nitrate reductase apoenzyme influences the expression of all three structural genes crnA, niiA and niaD. This effect is also exerted at the level of mRNA accumulation. Sequences upstream of the niaD and niiA genes from Aspergillus oryzae, A.nidulans and A.niger were compared, in addition to a similar region of the crnA gene. In particular, one sequence of ten nucleotides was found to share substantial similarity between each gene. It is possible that this motif is a cis-acting transcription factor recognised by a regulatory protein. The consensus TATCTA, a possible receptor site for the areA gene product, was identified several times upstream of each gene. The nit-4: gene of Neurospora crassa is shown to complement the A.nidulans nirA loss of function mutant via genetic transformation. The three transformant strains each have multiple integrates of the nit-4 gene, although none are present at the homologous site. The regulation of A.nidulans nitrate reductase by the N.crassa nit-4 gene is observed to be essentially normal, i.e. wild type.

Planococcus C.C.M. 316, a gram-positive facultative marine halophile, was studied with respect to growth and membrane composition of cells grown in media containing 0.5%, 3% and 10% sea salt. Membranes were prepared from cells grown in the three sea salt concentrations and analysed to determine any changes which may have been caused by the increasing concentrations of salt in the growth media. The three membrane preparations were found to have similar compositions to those reported for other gram-positive cocci. Cells grown in the 3% sea salt concentration contained membranes with a higher protein:lipid ratio and RNA content than the membranes from cells grown in the 0.5% and 10% concentrations. Amino acid analysis of the membrane proteins showed that the composition remained virtually unchanged in the three membrane preparations. The ratio of acidic: basic amino acid residues was nearer to the figures reported for non-halophiles than for those of the extreme halophiles. Examination of the lipids showed that phospholipids predominated to the extent of about 70% of the total lipids. Cardiolipin and lysocardiolipin were the major phospholipids, with phosphatidyl ethanolamine, phosphatidyl glycerol and phosphatidyl serine present as minor components. Glyco-lipids were found to decrease with increasing sea salt concentration in the medium, and in all three membrane preparations constituted only a very small proportion of the total lipids. Neutral lipids contained long chain alcohols, mono-, di- and tri-glycerides, as well as relatively large amounts of the isoprenoid compound squalene. The major fatty acid associated with the lipids was a branched saturated C15 acid which constituted 50 - 7% of the total fatty acids in most fractions. Although increasing salt in the medium produced changes within the proteins and lipids in the membranes, these changes were not such that they could be interpreted as an increase in the halophilic nature of the membrane. The carotenoids were shown to be derived from beta carotene and to consist mainly of 3'hydroxy 4' oxo compounds, although the extent of polar substitutions was dependent on both culture age and the concentration of salt in the medium.

The transporting properties of MDCK cells have been investigated upon epithelial monolayers grown upon permeable filter supports and mounted in Ussing chambers. When mounted without edge damage epithelial monolayers of MDCK cells (strain I) exhibited a transepithelial electrical resistance of 7.9 kO.cm2 and supported a small open-circuit potential (5.9 mV, basal-lateral surface positive) and a small short circuit current in agreement with the small magnitude of the net Na+, K+ and Cl- fluxes. Addition of adrenaline to the basal-lateral, but not the apical bathing solution stimulated a net basal-lateral to apical Cl- secretion, the magnitude of which accounted for the adrenaline stimulated short circuit current response. The Cl- secretion in MDCK cells exhibited many of the features of Cl- secretion in natural epithelia including sensitivity to the loop diuretics: furosemide, bumetamide and piretanide. In a number of secretory epithelia Cl- is accumulated across the basal-lateral cell border by a diuretic sensitive Na+K+Cl cotransport system. A comparison of the actions of the loop diuretics upon adrenaline stimulated Cl secretion and upon the cotransport system in MDCK cells provided strong but indirect evidence for a central role of the cotransport system in Cl secretion across MDCK cell monolayers. Measurement of influx and efflux across the apical and basal-lateral cell border demonstrated that the apical cell border was relatively impermeable to K. Influx across the basal-lateral border consisted of three major compenents; a ouabain sensitive , pump mediated, component; a diuretic sensitive component and a ouabain and diuretic insensitive flux component. K efflux across the basal-lateral cell border also consisted of these three components. Addition of adrenaline, ATP or A23187 to MDGK cells resulted in a transient stimulation of K+ efflux across both cell borders, although flux across the basal-lateral cell border was of greatest quantitative importance. Stimulation of K efflux was dependent upon the presence of Ca2+ in the external media and exhibited a similar pharmacology; sensitive to quinine and TEA, but insensitive to inhibition by apamin, as Ca2+-dependent K permeability?s in other, natural epithelia. Identification of an adrenaline stimulated Cl- secretory mechanism, a coupled Na + K + Cl cotransport system and a Ca2+-dependent K permeability in high resistance MDCK cells supports their use a model epithelium for the study of epithelial cell function.

The living organisms are varied when viewed from a macroscopic perspective, but on the molecular level they function based on the same fundamental principles. All living organisms are compartmented into cells via cellular walls made of lipid membranes. Through the wall the cell needs to transport nutrients, waste, genetic information and signaling molecules. The cell achieves this task via passive and active transport. In this thesis we focus on passive transport processes. In Chapter 1 of this thesis I introduce the topic of passive transport and its importance for biological systems. In Chapter 2 I use fluorescence methods to determine the passive transport of molecules with self intrinsic fluorescence through lipid membranes. We have built a unique fluorescence microscope which is capable of visualizing the fluorescence of molecules excited with deep UV light. With this new tool we monitored passive transport through the lipid membrane for several biologically significant molecules like for example the bacterial signal indole. Indole is an organic compound linked to important cellular processes like bacterial growth rates and cellular morphology. It is believed that indole is actively transported through the membrane of Escherichia coli via influx and efflux pumps. Here we give an unambiguous proof that indole can freely diffuse through intact bacterial lipid membranes. We extend this study to other molecules with self intrinsic fluorescence , like for example the antibiotic norfloxacin and the fluorescent dye fluorescein. We show that both these molecules can undergo passive transport through the lipid membrane.

The Centrifugal Membrane and Density Separation (CMDS) process is a novel type of membrane process that exploits the action of a centrifuge to generate process pressure for reverse osmosis and nanofiltration. The centrifuge could potentially enhance flux and alleviate fouling of the membrane as a result of the hydrodynamic environment of the centrifuge. All experimental work has been conducted on a prototype model of the CMDS process. The apparatus allows a membrane module to be fixed in space at a specified orientation, with respect to the rotation. This orientation in space is denoted by the terms “pitch, roll and yaw” (p,r,y). Experiments have been done using brine feed solutions at various concentrations to determine if the CMDS process minimizes the effects of concentration polarization. An example of this was illustrated with a 54% flux enhancement relative to a conventional membrane process using a 35000 ppm NaCl feed solution. Colloidal feed solutions were also used to examine how the CMDS process enhances flux in a fouling environment. These feed solutions include 21 g/L silica and 300 mg/L humic acid, with typical relative flux enhancement factors (κ) found to be 0.59 and 0.14, respectively. The final group of experiments examined the use of 50 g/L whey feed solutions with nanofiltration membranes. Results obtained here indicate that the centrifugal action enhanced the flux with an absolute flux enhancement factor (κ′) of 17.5 L/m² hr. These experiments have shown that a given orientation (90,270,0) best enhances the flux of a membrane with respect to colloidal fouling, while showing that another orientation (90,180,0) best reduces the effects of concentration polarization. Scanning electron microscopy (SEM) and an energy dispersive x-ray (EDX) detector have helped to examine the nature of the fouling layers and determine how well the layers adhere to the surface of the membrane. It was determined that in some cases, the fouling layer adhered better to the surface of a membrane used in the CMDS process. However, as the fluxes were typically higher in the dynamic process, it leads to the conclusion that the fouling layer on the CMDS membranes is more permeable. From the experimental work it has been concluded that the forces at work in the CMDS process create sufficient secondary flow instabilities to reduce the effects of fouling and concentration polarization on the membrane surface. The significance of this process with respect to industrial applications is considered, and the process is deemed feasible for such applications. / Graduate

Artificial lipid membranes have been used in biophysical studies as well defined models of biological membranes. In the present work we studied adsorption of ions to artificial lipid membranes, composed of phosphatidylcholine (PC) and phosphatidylserine (PS), and to biological membrane from sarcoplasmic reticulum (SR). The studies of ion adsorption and electrokinetic characterization of membranes were done by means of microelectrophoresis of PC/PS liposomes and SR vesicles. The ions of interest were positively charged potassium (K), calcium (Ca), tetraphenylarsonium (TPAs), tetraphenylphosphonium (TPP) and negatively charged pentachlorophenol (PCP). Electrophoretic mobility of PC/PS liposomes and SR vesicles has been measured as a function of pH, ionic strength and the concentration of adsorbing ions. From the data we have determined the isoelectric point and the density of electric charge of the SR membrane. We have shown that the mobility of PC/PS liposomes and SR vesicles can be understood in terms of electrostatic screening of membrane surface charge by the diffuse double layer of counterions and by ion adsorption. The experimental results, with the exception of Ca and SR membranes, have been found consistent with the adsorption model based on Langmuir adsorption isotherm and Grahame's equation relating the membrane electric potential and the membrane surface charge. The adsorption of ions to membranes have been characterized by the ion association constant and the membrane surface area of adsorption site. These quantities have been obtained from the fit of the adsorption model to the electrophoretic mobility data and the results are reported in the thesis. The following two findings are most interesting and important. First, the adsorption of K and lipophilic ions TPAs, TPP, and PCP to both the SR membrane and the artificial PC/PS membrane can be well characterized by the the adsorption model. The theoretical predictions of the adsorption model agree with the experimental results for a single (although different for PC/PS and SR membranes) set of ion adsorption constant and adsorption site area. This indicates a similarity between the biomembrane and the artificial lipid membrane. In contrast, such similarity was not found for calcium. Adsorption properties of SR membrane and artificial lipid membrane for Ca were found to be very different. Whereas the adsorption of Ca on PC/PS membranes could be described by one set of adsorption sites, the results for the SR vesicles indicate the presence of more than two types of Ca adsorption sites in the SR membrane. This finding is of physiological significance since the SR membrane regulates calcium transport in the muscle cell. Second, the adsorption affinity of SR membrane to all ions has been found to be significantly smaller compared to that of the lipid bilayer. This indicates that membrane proteins in the SR membrane reduce ion adsorption. This effect cannot be due to electrostatic interactions because the artificial lipid membranes had similar surface charge density as the biological membrane and the reduced adsorption was observed for both the positively charged TPAs and TPP as well as for the negatively charged PCP.