Nerve Stimulation and Electrical Properties of Frog Skin

The suitability of frog skin glands as a model for the study of secretory mechanisms in exocrine glands was explored. Periodic voltage clamp was used to determine continually the short-circuit current, chord conductance, and electromotive force of frog skin during neural and pharmacological activation of the skin glands. Both the chord conductance and the short-circuit current increased with glandular activation; the temporal dissociation of these increases suggests that there are at least two separate components to the secretory response. The sensitivity of the secretory electrical changes to changes in the ionic composition of the bathing solutions supports the notion of electrogenic chloride active transport as being basic to the activity of the exocrine glands.     

Chloride Electrochemical Potentials and Membrane Resistances in Nitella translucens

The chloride electrochemical potential difference between theinside of cells of Nitella translucens and the bathing mediumhas been measured by a direct electrical method employing Ag/AgClelectrodes. The membrane potential has been measured by meansof conventional salt bridge microelectrodes. These data havebeen used to calculate the internal chloride concentration ofthe cells; the mean value obtained was 39 mM. This chlorideelectrochemical potential difference has been short-circuitedthus causing an outward (depolarizing) electric current to flowthrough the cell membrane. The resulting membrane depolarizationhas been measured at two points along the length of the cellenabling the membrane resistance and space constant to be deduced;the respective values obtained were 24.8 Kcm² and 3.0 cm. Itis suggested that these experiments lend additional supportto the hypothesis that during the action potential in the Characeaethere occurs a transient increase in the chloride conductanceof the plasmalemma.     

ENZYME-LABELED ANTIBODIES FOR THE LIGHT AND ELECTRON MICROSCOPIC LOCALIZATION OF TISSUE ANTIGENS

Enzymes, either acid phosphatase or horseradish peroxidase, were conjugated to antibodies with bifunctional reagents. The conjugates, enzymatically and immunologically active, were employed in the immunohistochemical localization of tissue antigens utilizing the reaction product of the enzymatic reaction as the marker. Tissues reacted with acid phosphatase-labeled antibodies directed against basement membrane were stained for the enzyme with Gomori's method, and those reacted with peroxidase-labeled antibody were stained with Karnovsky's method. The reaction products of the enzymes localized in the basement membrane. Unlike the preparations of the fluorescent antibody technique, enzyme-labeled antibody preparations were permanent, could be observed with an ordinary microscope, and could be examined with the electron microscope. In the latter, specific localization of antibody occurred in the basement membrane and in the endoplasmic reticulum of cells known to synthesize basement membrane antigens. The method is sensitive because of the amplifying effect of the enzymatic activity. The ultrastructural preservation and localization were better with acid phosphatase-labeled antibody than with peroxidase-labeled antibody, but acid phosphatase conjugated antibody was unstable and difficult to prepare. Peroxidase-antibody conjugates were stable and could be stored for several months at 4°C, or indefiniely in a frozen state.     

Electrical Potentials and Na, K, and Cl Concentrations in the Vacuole and Cytoplasm of Nitella translucens

The potential differences across the tonoplast and plasmalemmamembranes have been measured in the single cells of Nitellatranslucens, the cells being immersed in an artificial pondwater (composition: NaCl _1.0 mM., KC1 _0.1 mM., CaCl₂, _0.1 mM.).The potential of the cytoplasm is –138 m V with respectto the bathing medium and –18 mV with respect to the vacuole.The concentrations of Na, K, and Cl have been measured in thetwo cell fractions. The concentrations in the flowing cytoplasmare: Na 14 mM., K 119 mM., and Cl 65 mM.; the vacuolar concentrationsare: Na 65 mM., K 75 mM.,and Cl 160 mM. The observed potential differences across the two membranesare compared with the Nernst potentials for all three ions.This analysis shows that all three ions are actively transportedat the plasmalemma: Na is pumped outwards while K and Cl arepumped inwards. At the tonoplast Na is pumped into the vacuolewhile K and Cl are close to electrochemical equilibrium. The inhibitor, ouabain, has no effect on the cell resting potential.     

The Effects of Alkali Metal Cations and Common Anions on the Frog Skin Potential

The effects on the potential difference across isolated frog skin (R. catesbeiana, R. pipiens) of changing the ionic composition of the bathing solutions have been examined. Estimates of mean values and precision are presented for the potential changes produced by substituting other alkali metal cations for Na at the outside border and for K at the inside border. In terms of ability to mimic Na at the outside border of bullfrog skin, the selectivity order is Li > Rb, K, Cs; at the outside border of leopard frog skin, Li > Cs, K, Rb. In terms of ability to mimic K at the inside border of bullfrog and leopard frog skin: Rb > Cs > Li > Na. Orders of anion selectivity in terms of sensitivity of the potential for the outside border of bullfrog skin are Br > Cl > NO₃ > I > SO₄, isethionate and of leopard frog skin are Br, Cl > I, NO₃, SO₄. An effect of the solution composition (ionic strength?) on the apparent Na-K selectivity of the outside border is described. The results of the investigation have been interpreted and discussed in terms of the application of the constant field equation to the Koefoed-Johnsen-Ussing frog skin model. These observations may be useful in constructing and testing models of biological ionic selectivity.     

THE EFFECT OF CERTAIN SOIL TREATMENTS ON DIDYMELLA STEM-ROT OF TOMATOES

About 93% of Didymella lycopersici spores were destroyed after 4 weeks incubation in unsterilized soil. A survey of the microflora of glasshouse soil receiving different treatments and inoculated with D. lycopersici showed no clear relation between numbers of any group of organisms and the incidence of stem rot. Sterilized soil was not made toxic to D. lycopersici by the growth of a number of soil microorganisms even after 9 months incubation, but addition of unsterilized soil or of a suspension of unsterilized soil quickly restored toxicity. Direct observations of spores in soil on slides showed that their fate varied with the treatment of the soil before inoculation. With fresh soil or air-dry soil moistened 2 or more days before inoculation, lysis of spores occurred. With air-dry soil moistened and inoculated simultaneously, some spores germinated but growth of germ tubes soon ceased. No direct connexion could be seen between the fate of the spores and soil microorganisms. Addition of glucose to unsterilized soil reduced its toxicity to D. lycopersici. Soils steamed for 1 min. or longer were not toxic to D. lycopersici , but soils steamed for very short periods were as toxic as unsterilized soils although the soil microflora was much reduced.