Thesis (Ph. D.)--Lehigh University, 2006. / Includes vita. Includes bibliographical references (leaves 117-125).

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Thesis (Ph. D.)--Lehigh University, 2000. / Includes vita. Includes bibliographical references (leaves 146-152).

The initial aim of this thesis was to study the transmembrane and intracellular distribution of Cl⁻ in single striated muscle fibers of the giant barnacle, Balanus nubilus. The study was expanded to include the transmembrane distribution of K⁺ and a consideration of the Donnan relation. Chloride sensitive Ag-AgCl microelectrodes were inserted into single fibers to measure the activity of Cl⁻ in the myoplasm. The myoplasmic Cl⁻ activity was just slightly higher than predicted from the Nernst equation, indicating that Cl⁻ is probably distributed passively across the membrane of the resting fiber. Comparison of the electrode results with analytic results suggested that less than half of the fiber Cl⁻ is free in the myoplasm. Most of this extra Cl⁻ was located in an extracellular space, determined, by ¹⁴C-sorbitol penetration and a Cl⁻ washout method, to occupy about 5% of the fiber volume. Electron micrographs indicate that this space is comprised of large clefts and smaller tubules which penetrate deeply into the fiber. In a second series of experiments, the kinetics of Cl⁻ exchange and the outflow of intracellular Cl⁻ into solutions with reduced Cl⁻ concentrations were studied. The results indicate that the intracellular Cl⁻ can be divided about equally between a rapidly exchanging fraction which is free in the myoplasm and a slowly exchanging fraction which is either bound or compartmentalized. Furthermore, the free myoplasmic Cl⁻ appears to be excluded from about 45% of the intracellular water. In the third series of experiments, the activities of K⁺ and Cl⁻ in the myoplasm of single fibers at differing states of equilibrium were measured by means of ion sensitive micro-electrodes. Fibers were equilibrated in Ringer solution which was modified in three ways: [K]₀ and [C1]₀ were varied but [K]₀ x [Cl]₀ and [K]₀ + [Na]₀ kept constant; [K]₀ was varied but [Cl]₀ and [K]₀ + [Na]₀ kept constant; [K]₀, [Cl]₀, and osmolarity were increased. The results provide rigorous proof for the widely held theory that, under most equilibrium conditions, K⁺ and Cl⁻ are distributed across the membrane of a muscle fiber according to the Donnan relation. The results also confirm the finding.that Cl⁻ is apparently excluded from 45% of the intracellular water and earlier findings from this laboratory that K⁺ is excluded from a smaller fraction of the intracellular water. Finally, two experiments are described which show that the intracellular distribution of K⁺ and Na⁺ is not the same in fibers from barnacles collected in two different locations. It is suggested that although the sum of the K⁺ plus Na⁺ that is bound or compartmentalized may be constant, the differences in the results could be due to an inverse variation in the amount of each cation that is bound or compartmentalized. The results of this study are in accord with recent findings in this and other laboratories which indicate that monovalent inorganic ions and water are distributed in a heterogeneous manner in the striated muscle fiber. The intracellular Cl⁻of the barnacle muscle that is not free in the myoplasm must be either bound to proteins or accumulated in the sarcoplasmic reticulum; at present there is not sufficient evidence to select one or the other (or both) of these possibilities. It is proposed that the free myoplasmic Cl⁻ is excluded from water that is ordered by the cell proteins and partially excluded from water that lies within the electrical field that surrounds the negatively charged proteins. / Medicine, Faculty of / Graduate

Cell surfaces of aggregation, adherence or hydrophilic variants of Streptococcus sanguis were compared to cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with SDS-BME, urea, lithium chloride and boiling water did not solubilize any material which stained with silver nitrate in an SDS-polyacrylamide gel. The parent organism S. sanguis 12 which aggregates in saliva, adheres to saliva coated hydroxyapatite (S-HA) and is hydrophobic was found to possess a prominently staining 160,000 MW protein. This protein was almost completely absent from strain I2na, a hydrophobic non-aggregating variant, and was completely absent from the hydrophilic non-aggregating, non-adherent strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000 MW protein and the ability to aggregate in saliva. Trypsin treatment reduced, but did not eliminate the hydrophobic character of the cells. Boiling destroyed the ability to aggregate but did not alter hydrophobicity. Cell wall digests of strain 12 contained a large number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of the hydrophilic strains contained no material that was visible in a silver stained SDS-polyacrylamide gel. Electron microscopy of phosphotungstic acid stained cells showed a thick capsular material spread evenly over the cell surface of the parent strain 12. This layer was thinner around the cells of strain 12na and appeared patchy on hydrophilic strains. Electron microscopy of uranyl acetate stained cells revealed that strain 12 had short fibrillar structures evenly distributed over the cell surface, and long fibrils which were more concentrated at the end of the cell. The hydrophilic strain 12L lacked both types of fibrils. Crossed immunoelectrophoresis confirmed that the major cell surface antigens were located in the 160,000 MW region. Cell wall digests of strain 12 and 12na inhibited adherence of strain 12 to S-HA by 36% and 19% respectively. The digests of hydrophilic strain 12L were not inhibitory. The inhibitory activity was sensitive to heat and SDS. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The cell surface glycoproteins of mouse neuroblastoma cells were visualized using fluorescent lectins in combination with fluorescent light microscopy and SDS-gel electrophoresis, lectin-microsphere conjugates in combination with scanning electron microscopy, and iodinated lectins. The lectins used in these studies were: Con-canavalin A (ConA) which is specific for α-D-mannose and α-D-glucose groups, wheat germ agglutinin (WGA) which binds to N-acetylglucosa-mine oligomers and nicinus communis agglutinin (RCAI) which is specific for D-galactose residues. All three lectins were found to have over 10⁷ high affinity (K[sub d]= 2 x 10⁻⁷ M) binding sites on the neuroblastoma cell surface. These sites were found to be densely and randomly distributed over the surfaces of cells which were fixed with glutaraldehyde before labelling or labeled at 4°C. However, when the cells were labeled at 23°C or 37°C, the lectin receptors were found to undergo redistribution. All three lectins were internalized by the cells in an energy- dependent process when the cells were treated with the lectins for 60 min or more at 37°C. However, the patterns of redistribution for the different lectin receptors were not the same. When cells were labeled with ConA, the receptors were shown to patch and then clear from the surface of the cells. Once the label had completely cleared from the cell surface, the cells could not be labeled with additional ConA, even when the first labelling was done using ConA at concentrations well below saturation. However, when cells were treated in the same way with WGA or RCAI, a heavy, uniform display of marker was seen on the cell surface at all times. It was only when the cells were briefly labeled with these lectins and then incubated in buffer that it became apparent that these receptors could also patch and clear from the cell surface. In addition, in order to clear all the receptors for these two lectins from the cell surface, it was necessary to label the cells with saturating concentrations of lectin. Thus, labeled and unlabeled ConA receptors on the neuroblastoma cells undergo co-ordinate redistribution whereas labeled WGA and RCAI receptors redistribute independently of their unlabeled counterparts. Studies with drugs which disrupt the various cytoskeletal elements suggested that the microfilament system played a role in the coordinate redistribution of ConA receptors. Double labelling studies with both different iodinated and fluorescent lectins indicated that the binding sites for WGA were not associated with those for ConA. WGA binding sites did appear to be directly associated with many of the binding sites for RCAI. Studies with ConA and RCAI were not carried out because it was shown that ConA binds to RCAI. Using the fluorescent lectins in combination with SDS-gel electrophoresis, the lectin-binding polypeptides from the neuroblastoma cell membranes were identified and characterized. Plasma membranes were purified from the neuroblastoma cells using hypotonic disruption followed by differential and isopycnic gradient centifugation. The membrane preparation showed a 10-fold increase in the specific activity of two plasma membrane markers and little contamination by other cell components. The membranes were dissociated in SDS and run on SDS-polyacrylamide gels to separate out the different polypeptides. The gels were fixed and stained with fluorescent lectins. WGA and RCAI were both found to bind almost exclusively to a single polypeptide with a molecular weight of 30,000 daltons. This polypeptide also stained strongly for carbohydrate after periodate oxidation. ConA, on the other hand, bound to over 20 polypeptides, most of which had molecular weights between 60,000 and 120,000 daltons. However, when the cells were made to internalize all their ConA receptors and their membranes isolated and run on gels, four of the ConA-staining polypeptides were found to be absent as compared to membranes from untreated cells. Thus, it appears that only four of the ConA-binding polypeptides seen on the gels were available for ConA binding at the cell sur- face. These polypeptides also labeled with I¹²⁵ when intact cells were treated with I¹²⁵ and lactoperoxidase. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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