Compartmentation of the inulin space in mouse brain slices

(1) Mouse cerebrum slices swell in tris-buffered Krebs-Ringer medium. Swelling is rapid at first, then slows to a more or less constant rate. Even after 3 hr incubation, water content/g of tissue dry wt. shows no sign of an asymptotic limit. Swelling is the same at 37 degrees and at 0 degree. (2) Tissue water measured by incubation with tritiated water is equal to total tissue water measured by drying slices. Equilibration between tritiated water and tissue water is complete within 2 min. (3) Tissue liquid can be divided into three phenomenologically distinguishable compartments: first inulin space, which is the compartment permeable to inulin at both 0 degree and 37 degrees; second inulin space, which is the compartment permeable to inulin at 37 degrees but not at 0 degree; and 37 degrees non-inulin space, which is the compartment impermeable to inulin at both 0 degree and 37 degrees. The evidence for this is: (a) Penetration of inulin into tissue is greater at 37 degrees than at 0 degree. After the first 20 min the rate of penetration at 0 degree is approximately equal to the rate of penetration at 37 degrees, and only slightly less than the rate of increase of total tissue water. Therefore the smaller inulin space observed at 0 degree cannot be due to slower entry of inulin. (b) The inulin content of slices incubated in inulin-containing medium at 37 degrees and cooled to 0 degree in the same medium is the same as the inulin content of tissue incubated at 37 degrees without subsequent cooling. In contrast, the inulin content of tissues preincubated in inulin-free medium at 37 degrees and then incubated in inulin-containing medium at 0 degree is the same as the inulin content of tissues incubated in inulin-containing medium at 0 degree without preincubation at 37 degrees. Therefore the smaller inulin space at 0 degree than at 37 degrees can be due neither to a reversible temperature-dependent change in the size of one single inulin space nor to an irreversible, greater swelling of a single inulin space at the higher temperature, but is due to some portion of the 37 degrees inulin space becoming impermeable to inulin at 0 degree. (c) Some inulin is retained by tissue incubated with inulin at 37 degrees, then transferred to inulin-free medium at 0 degree; the amount of retained inulin is equal to the difference between inulin content of tissue incubated with inulin at 37 degrees and tissue incubated with inulin at 0 degree This confirms 3b above and in addition shows that inulin which has entered the second inulin space at 37 degrees is trapped there when this space becomes impermeable to inulin at 0 degree. (4) The penetration of the amino acids, L-lysine and D-glutamate at 0 degree is equal to the penetration of inulin at 37 degrees. This confirms the real existence of the 37 degrees inulin space at 0 degree, and shows that the barrier at 0 degree between the first and second inulin spaces does not exist for these substances. (5) The amino acids L-leucine and glycine penetrate total tissue water at 0 degree. L-leucine is actively transported at this temperature. (6) The amino acids alpha-aminoisobutyric acid, L-leucine, and L-lysine at 2 mM have no effect at 37 degrees on either the inulin space or the non-inulin space. (7) The inulin space is insensitive at 37 degrees to physiologically significant changes in the medium. In contrast, the non-inulin space is quite sensitive to these changes. Addition of D-glutamate greatly increases the non-inulin space; addition of ouabain or cyanide, or omission of glucose, increases the non-inulin space slightly; and replacement of Na+ ion by choline+ ion greatly decreases this space. These changes are independent and roughly additive.     

THE PATHOLOGY OF HYPODERMELLA LARICIS ON LARCH,LARIX OCCIDENTALS

A study of the larch needle disease caused by Hypodermella laricis provided information on the mode of infection, the manner of spread, and the relationship between the parasite and its host, Larix occidentalis. Infection by spores occurs in early spring as soon as the leaves emerge, but there is no evidence that hyphae invade the dwarf shoots. The fungus disrupts the normal abscission mechanism, and the fructifications and spores of H. laricis develop on the leaves which remain attached to the dwarf shoots. The time of precipitation is a decisive factor in the initiation of the disease, i.e., infection occurs only when precipitation coincides with the emergence of young leaves. However, vulnerability to infection diminishes rapidly as the leaves mature. By observing leaf size and noting the time of rainfall, the severity of infection can be accurately predicted. Several adaptive mechanisms related to successful parasitism in H. laricis are discussed.     

Polyamines and the Accumulation of Ribonucleic Acid in Some Polyauxotrophic Strains of Escherichia coli

The cellular accumulations of polyamines and ribonucleic acid (RNA) were compared in the polyauxotrophic mutants of Escherichia coli strain 15 TAU and E. coli K-12 RC(re1) met(-) leu(-). Putrescine, spermidine, and their monoacetyl derivatives were the main polyamines in both strains, when grown in glucose-mineral medium. No significant degradation of either (14)C-putrescine or (14)C-spermidine was found in growing cultures of strain 15 TAU, which requires thymine, arginine, and uracil for growth. Experiments with this organism showed that in a variety of different incubation conditions, which included normal growth, amino acid starvation, inhibition by chloramphenicol or streptomycin, or thymine deprivation, a close correlation was seen between the intracellular accumulation of unconjugated spermidine and RNA. In the presence of arginine, the antibiotics stimulated the production of putrescine and spermidine per unit of bacterial mass. Deprivation of arginine also resulted in an increase in the production of putrescine per unit of bacterial mass, most of which was excreted into the growth medium. However, in this system the antibiotics reduced the synthesis of putrescine. Furthermore, streptomycin caused a rapid loss of cellular putrescine into the medium. The latter effect was not seen in anaerobic conditions or in a streptomycin-resistant mutant of 15 TAU. Methionine added to the growth medium of growing TAU not only markedly increased the total production of spermidine, but also increased both the intracellular concentration of spermidine and the accumulation of RNA. Exogenous spermidine extensively relaxed RNA synthesis in amino acid-starved cultures of 15 TAU. Analysis in sucrose density gradients showed that the RNA accumulated in the presence of spermidine was ribosomal RNA.Cells of E. coli K-12 RC(rel) met(-) leu(-), grown in a complete medium, had approximately the same ratio of free spermidine to RNA as did strain 15 TAU. However, the relaxed strain showed a much lower ratio of putrescine to spermidine than the stringent 15 TAU. Omission of methionine stopped spermidine synthesis and markedly increased both the intracellular accumulation and the total production of putrescine. It seems that a high intracellular level of spermidine acts as a feedback inhibitor in the biosynthesis of putrescine in this strain. The hypothesis that the intracellular concentration of polyamines may participate in the control of the synthesis of ribosomal RNA in bacteria is discussed.     

Specific Binding of Rubidium in Chlorella

Specific binding sites for potassium, which may be components of the carriers for active transport for K in Chlorella, were characterized by their capacity to bind rubidium. A dense suspension was allowed to take up Rb⁸⁶ from a low concentration of Rb⁸⁶ and a high concentration of ions which saturate non-specific sites. The amount bound was derived from the increase in the external concentration of Rb⁸⁶ following addition of excess potassium. The sites were heterogeneous. The average affinity of Rb and various other ions for the sites was determined by plotting the degree of displacement of Rb⁸⁶ against log molar concentration of the individual ions. Interpolation gave the concentration for 50 per cent displacement of Rb, which is inversely related to affinity. The order of affinity was not changed when the cells were frozen, or boiled either in water or in 70 per cent ethanol. The affinity is maximal for ions with a crystalline radius of 1.3 to 1.5 A and a high polarizability, and is not related to the hydrated radius or valency. It is suggested that binding groups in a site are rigidly arranged, the irregular space between them being 2.6 to 3.0 A across, so that affinity is high for ions of this diameter and high polarizability.