Steroid Lysis of Protoplasts and Effects of Stabilizers and Steroid Antagonists

Six synthetic antimicrobial steroids were examined for indications of their mechanism of action. Dequadin acetate, cetyl pyridinium chloride (CPC), and sodium deoxycholate were studied for comparison. Aerated cells of Sarcina lutea were washed, suspended in 1.06 M sucrose, and converted to protoplasts with 20 mug/ml of lysozyme. Lysis was measured optically at 650 mmu as a decrease in optical density. Screening tests with 50 mug/ml of each compound showed five steroids and CPC to be lytic. Protoplasts were strongly protected from lysis by pretreatment with 0.001 to 0.004 M spermine tetrahydrochloride. Other polyamines, such as spermidine phosphate, were less protective, and putrescine was ineffective. Uranyl nitrate (5 x 10(-4) M) rapidly agglutinated protoplasts and protected them from rupture by the lytic agents. Similar studies with 0.001 to 0.004 M Mg(++) showed varying degrees of protection, which, in most cases, was only temporary. Steroidal lysis did not appear to be related to chelation, since ethylenediaminetetraacetate did not cause lysis alone and antagonized some lytic compounds. Lecithin, Tween 80, Tween 20, and Span 20 at 0.05% exhibited certain effects on protoplast stability. Span 20 strongly prevented lysis by steroids. Tween 20 alone quickly caused protoplast rupture. Lecithin and Tween 80, which also caused lysis alone, interfered with lytic steroids and CPC. The test compounds were both inhibitory and lethal to cells of Sarcina lutea. The results suggest that direct action on cell membranes may be chiefly responsible for the antimicrobial properties of the steroids.     

Nuclear membranous whorls

Summary Membranous whorls have been seen in the nuclei of peritoneal and testicular cells which had been subjected to various experimental manoeuvres. It seems likely that this is an early manifestation of cell degeneration which is demonstrated readily only by glutaraldehyde fixation, and to that extent can be regarded as a glutaraldehyde artifact. Acknowledgements. This work was supported by grants from the Medical Research Council, and the University of Sheffield Tuberculosis Research Fund, and by a grant to the Department from Unilever Ltd.I am grateful to Professor R. Barer for his advice and criticism, to Dr. G. A. Meek for guidance on electron microscopy, to Dr. E. J. Clegg for permission to use material from joint experiments. Technical and photographic assistance was provided by Messrs. P. GarLick and L. Murgatroyd and by Miss M. Tune.     

Biliary excretion in foreign compounds. Sulphonamide drugs in the rat

1. The extent of biliary excretion in the rat of 15 sulphonamide compounds was studied. 2. Most of the sulphonamides studied, with molecular weights from 172 (sulphanilamide) to 352 (N⁴-acetylsulphadimethoxine) are poorly excreted in the bile (0–4% of the dose), except sulphapyridine, sulphamethoxypyridazine and sulphadimethoxine. The last three are partly metabolized to glucuronides, whose molecular weights and polarities are such as to allow them to be excreted in the bile in appreciable amounts. 3. Succinylsulphathiazole and phthalylsulphathiazole are polar and have molecular weights (355 and 403) of an appropriate order, and are excreted unchanged in the bile in appreciable amounts. 4. Sulphadimethoxine N¹-glucuronide (mol.wt. 487) is extensively excreted in the bile unchanged. 5. The results are examined in the light of the hypotheses put forward in the preceding paper (Millburn, Smith & Williams, 1967).     

Biliary excretion of [C]succinylsulphathiazole in the rat and rabbit

1. After intravenous injection about 30% of the dose (20mg./kg.) of succinylsulphathiazole is excreted unchanged in the bile in 3hr. by the rat, whereas only about 1% is excreted by the rabbit. When the renal pedicles are ligated the biliary excretion of succinylsulphathiazole in the rat increases to about 80% of the dose, but in the rabbit under these conditions the biliary excretion is only 2% of the dose. 2. In the rat, the sulphonamide readily enters the liver and biliary excretion occurs against a concentration gradient from liver to bile; further, the excretory process can be saturated, and can be depressed by the simultaneous administration of phenolphthalein glucuronide or bile salts. 3. In the rabbit, these conditions have not been found; succinylsulphathiazole does not readily enter the liver from the plasma, there is no transfer of the drug from the liver cells to the bile against a concentration gradient, and no saturation or depression of the biliary excretion of succinylsulphathiazole is found. 4. It is suggested that two factors responsible, at least partly, for the low biliary excretion of succinylsulphathiazole in the rabbit are the poor entry of the sulphonamide into the liver in this species and a deficiency of the concentrative mechanism for its excretion in the bile.     

Protein synthesis in the intestine of goldfish acclimatized to different temperatures

1. The incorporation of [¹⁴C]valine and [³H]threonine has been studied in mucosa of goldfish acclimatized to different temperatures. 2. [¹⁴C]Valine was incorporated more rapidly than [³H]threonine at acclimatization temperatures below 15°; this distinction disappeared at higher temperatures. 3. The rate of amino acid incorporation rose as the environmental temperature was increased. 4. The number of valine residues in mucosal protein fell and the number of methionine residues rose as the acclimatization temperature was raised. 5. The concentration of free methionine was closely related to the amount in protein in goldfish mucosa; both increased at higher acclimatization temperatures. 6. An attempt is made to show that these changes are integrated with possible homoeostatic mechanisms in the goldfish intestine.     

The fate of the hydrolysis products of thalidomide in the pregnant rabbit

1. The fate in the pregnant New Zealand White rabbit of oral doses of four ¹⁴C-labelled hydrolysis products of thalidomide, namely α-(o-carboxybenzamido)-glutarimide, 2-phthalimidoglutaramic acid, 2-phthalimidoglutaric acid and 2-(o-carboxybenzamido)glutaramic acid, administered on the 192nd hour of pregnancy has been studied. 2. About 60–95% of the administered ¹⁴C of each compound appears in the urine in 58hr. and the remainder is found in the faeces and in the gut and its contents. 3. Radioactivity is present in the plasma, liver, kidney, brain, muscle, fat and embryo. 4. The ¹⁴C-labelled substances in the plasma and embryo consist of the unchanged compounds and their further hydrolysis products. 5. Since the above four thalidomide hydrolysis products are found in the rabbit conceptus together with their further hydrolysis products after their oral administration to the pregnant rabbit, it appears that the teratogenic activity of thalidomide is due to the compound itself rather than to one or more of its hydrolysis products.     

Growth and gas-vacuole development in vegetative cells of Anabaena flos-aquae

Summary Gas-vacuoles consisting of collections of gas-cylinders, and continuity of the plasma membrane with lamellae, are observed at all stages of growth of Anabaena flos-aquae. In day+4 cells, increased numbers of invaginations of the plasma membrane are observed and the lamellae tend to lie parallel to the cell wall. Some evidence is presented which suggests an associated synthesis of -granules and gas-cylinders by lamellae. Features of older cells are increased numbers of gascylinders, -granules, structured granules, and large intralamellar vesicles which appear to correlate with the presence of pinkish vacuoles as observed with the light microscope. The mean percentage of the volume of cells occupied by gas-vacuoles is about 20% during exponential growth when the doubling time is 56.5 hours, increasing to 34% in day+24 cells when growth is stationary.