The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate and stimulation of 3H-choline incorporation into endoplasmic reticulum membranes and other subcellular fractions of Krebs II ascites cells during in vitro incubation

Summary After transfer of Krebs II ascites cells from the mouse peritoneum to suspension culture addition of the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) causes an early stimulation of ³H-choline incorporation into phosphatidylcholine (PC). Choline transport into the treated cells, however, was unaffected. Within 30 min of TPA treatment ³H-choline incorporation was almost 300% above the control level. During a 5 hr period of suspension culture the overall patterns of ³H-choline incorporation were similar in TPA-treated and control cultures though the rate was greatly accentuated by the presence of the phorbol ester. Incubation of cells with cycloheximide prior to incubation with TPA did not result in an inhibition of the TPA-directed ³H-choline incorporation. After 3 hr incubation with TPA there were large increases in radioactivity in all subcellular fractions. At 20 hr, however, the values were not far from those of the control. During the first 3 hr of incubation with TPA the incorporation of ³H-choline into light rough (LR) and smooth (S) membranes was stimulated to levels of 400% and 320% respectively above control values. At later times the profiles of radioactivity in membrane subfractions in TPA-treated and control cultures were similar. The results illustrate an early effect of TPA on PC biosynthesis in Krebs II ascites cells while at later times of incubation the stimulatory effect was virtually abolished.     

Purification and partial characterization of four proteins from human parotid saliva

Four proteins, which have been designated A, B, C and D, have been purified from human parotid saliva. These proteins are the major constituents of parotid saliva which migrate rapidly to the anode in polyacrylamide electrophoresis at pH9.5. Gel filtration and polyacrylamide electrophoresis were employed in the purification procedures. After purification all four preparations were tested for homogeneity by electrophoresis at pH2.8 and 9.5, by isoelectric focusing in the pH range 3-10, by immunodiffusion, and by sedimentation in the analytical ultracentrifuge. None of the proteins showed significant activity in assays for amylase, acid and alkaline phosphatase, protease, lysozyme, ribonuclease, peroxidase, beta-glucuronidase, beta-galactosidase, iron-binding activity and esterase. No cross-reactions were detected with antisera specific for lactoferrin and 15 serum proteins. All four proteins were rich in glutamic acid, proline and glycine and were lacking completely the sulphur-containing amino acids. Proteins A and C contained no threonine or tyrosine. Carbohydrate could be demonstrated only in protein A at a concentration of 4% of the total protein.     

Preparative thin-layer and column chromatography of prostaglandins

Chromatographic methods are used to identify various monounsaturated PGs (prostaglandins). TLC (thin-layer chromatography) and column chromatography are described in detail. These systems were developed specifically for separating epimers of PGE1 and PGF1, but they are useful in separating some of the known natural PGs as well. A table presents information on identification of the various PGs with neutral silica and acidic silica. The results of these laboratory experiments indicate that 1 ug-1g of PG can be purified by chromatographic methods with little or no loss due to irreversible adsorption or rearrangement if proper precautions and solvent systems are used. TLC seems to be useful in distinguishing diastereomers of PGs. Relative mobilities of the various hydroxy diastereomers are not changed by the solvent systems or by esterification.     

Electron microscopic observations on iron-labelled secondary lysosomes in renal tubules in mice

Summary Labelling of renal tubule cytosomes with electron dense iron granules can be attained by daily intramuscular injections to mice of an iron sorbitol citric-acid compound in a total of approximately 50 mg Fe⁺⁺⁺/100 g of body weight. The labelled cytosomes correspond to secondary lysosomes and represent heterolysosomes or ambilysosomes. The evidence suggests that tubule and lysosome function are undisturbed by the labelling procedure. The use of this method for fine structural studies of the interaction between secondary lysosomes and other cytoplasmic organelles and elements is indicated.Microbodies do not incorporate administered Fe⁺⁺⁺. The morphological observations support the opinion that these bodies are formed in specialized portions of the smooth surfaced endoplasmic reticulum of proximal tubule cells.Supported in part by grants from the Swedish Medical Research Council (Projects No. K 67-12x-1006-2, B 67-12x-1006-02K, K 68-12x-1006-03, and B 69-12x-1006-04A). The assistance of Miss Silwa Mengarelli and Miss Britt-Marie Pettersson is gratefully acknowledged.     

Application of a Theory for Circumnutations to Geotropic Movements

From a previously published theory (Israelsson and Johnsson 1967) for circumnutations in Helianthus annuus it is possible to predict the geotropical curvatures of the hypocotyls. This extension of the theory is given in the present paper and some geotropical experiments are performed and discussed. The agreement between the theory and the experiments has been verified in the case of gravitational stimulation during relatively short stimulation periods, in the case of continuous gravitational stimulation, etc. Restrictions in the proposed model are discussed.     

Adrenergic nerves in the pars intermedia of the pituitary in the toad,Bufo arenarum

Summary By means of a highly sensitive and specific histochemical method for the demonstration of certain biogenic monoamines a plexus of nerves containing a primary catecholamine has been demonstrated in the pars intermedia of the toad, Bufo arenarum. These nerves are restricted in distribution to the part of the gland which contains colloid vesicles (stored MSH ?) in the cells. The view is put forward, based on the results of pharmacological and surgical experiments, that the adrenergic nerves inhibit the release of the MSH from the pars intermedia. The origin of the nerves in the brain is unknown, but experiments with lesions have shown that it is not to be found in the nucleus periventricularis arcuatus.The research reported in this document has been sponsored by the Swedish Medical Research Council and by the Swedish Natural Science Research Council.Research fellow of the Commission of Scientific Research of the Province of Buenos Aires, Argentina.