MAPPING CELL WALL POLYSACCHARIDES OF LIVING MICROBIAL CELLS USING ATOMIC FORCE MICROSCOPY

Functionalized atomic force microscope tips were used to sense specific forces of interaction between ligand—receptor pairs and to map the positions of polysaccharides on a living microbial cell surface. Gold-coated tips were functionalized with concanavalin A using a cross-linker with a spacer arm of 15.6Å. It was possible to measure the binding force between concanavalin A and mannan polymers on the yeast (Saccharomyces cerevisiae) cell surface. This force ranged from 75 to 200pN. The shape of the force curve indicated that the polymers were pulled away from the cell surface for a fairly long distance that sometimes reached several hundred nanometres. The distribution of mannan on the cell surface was mapped by carrying out the force measurement in the force volume mode of atomic force microscopy (AFM). During the measurement, the maximum cantilever deflection after contact between the tip and the sample was kept constant at 10nm using trigger mode to keep the pressing force on the sample surface as gently as possible at a force of 180pN. This regime was used to minimize the non-specific adhesion between the tip and the cell surface. Specific molecular recognition events took place on specific areas of the cell surface that could be interpreted as reflecting a non-uniform distribution of mannan on the cell surface.     

Studies of fluid exchanges between rat liver slices and simple media

1. The exchange of fluid between slices of rat liver and solutions of monosaccharides, disaccharides, and sodium chloride has been studied in relation to the temperature of incubation. The point of apparent isotonicity (P.A.I.) of the tissue was defined as the concentration of the solution in which the slices neither gained nor lost weight after immersion for a period of 10 minutes. 2. In solutions of glucose, the P.A.I. of the slices was significantly lower at 4°C. than at 20°C. but similar at 20°C. and 37°C. Upon immersion for 15 to 60 minutes in 0.66 molar glucose the slices always swelled more at 37°C. than at 20°C. In solutions of sucrose change in the temperature of incubation was without effect on the hydration of the tissues. 3. In solutions of sodium chloride, the P.A.I. and the content of water, chloride, and sodium plus potassium were lower at 37°C. than at 20°C. 4. These findings emphasize the role of translocation of solute in providing an osmotic gradient for the movement of water between the tissue slices and the media.