Membrane-active Agents Research Articles

Mutants of yeast specifically resistant to petite induction by fluorinated pyrimidines

Induction of the cytoplasmic petite mutation in yeast by 5-fluorouracil (5FU) and 5-fluorocytosine (5FC) is known to depend on the incorporation of 5FU into some species of RNA; 5FC is active only following deamination to 5FU. Several mutants ahve now been isolated which are resistant to petite mutagenesis by 5FU but remain sensitive to growth inhibition by this analogue. They fall into two classes: those in class I are also resistant to mutagenesis by 5FC, while class II mutants retain partial sensitivity to the latter agent. The growth of both classes is sensitive to 5FC. The behavior of class II mutants requires that exogenous 5FU is specifically excluded from the site of synthesis of the target RNA involved in petite mutagenesis, while 5FC has access to it. The most likely explanation is that the RNA concerned is synthesized in the mitochondria, and that the mitochondrial membranes of class II mutants are impermeable to 5FU but not 5FC. This is supported by the finding that the membrane-active agent dimethylsulfoxide restored 5FU sensitivity to this class of mutants. No such effect was observed with class I mutants, and these are thought to have altered mitochondrial RNA-synthesizing systems which are unable to recognize fluorinated nucleotides.

Temperature-sensitive hamster cell line with altered membrane properties

The altered properties of a concanavalin A-resistant Chinese hamster ovary cell line with obvious temperature-sensitive growth properties is described. The variant cell line, C R-7, was shown to have a higher efficiency of colony formation than the parental wild-type population after treatment with various concentrations of concanavalin A (ConA). The variant cells had the properties of a temperature-sensitive cell line as judged by growth studies performed on solid surfaces or in suspension culture at the permissive (34 °C) and non-permissive (39 °C) temperatures; by colony efficiency determinations performed at 34 °C and 39 °C; and by the altered ability to incorporate DNA, RNA, and protein precursors into acid-precipitable material at the non-permissive temperature. Evidence for changes in the membrane properties of C R-7 cells included: a reduced agglutinability in the presence of ConA, an altered cellular morphology on solid surfaces, an enhanced sensitivity to the toxic effects of membrane-active agents, altered and temperature-sensitive adhesiveness properties, and a reduced ability to bind labelled ConA.

Plasma membrane studies on drug sensitive and resistant cell lines: I. Cross resistance and membrane enzyme coordination (Ouabain/cAMP/Na +/K + ATPase/Adenylate Cyclase)

Selection of cell lines resistant to ouabain or 3′, 5′-cyclic adenosine monophosphate (cAMP), leads to a cross resistance in the selected variants which can be observed against each of the other drugs and also to concanavalin A and theophylline. Ouabain elevated the cellular cAMP level in the wild-type cell lines in a cell density and dose-dependent manner, whereas the drug-resistant lines lost this modulation. It is particularly noteworthy that the physiological effects of ouabain are lost in cAMP-resistant cell lines. In addition, the plasma membrane enzymes, adenylate cyclase and Na +/K + ATPase from the resistant lines appear to lose the responsiveness to ouabain exhibited by the wild-type cell lines, i.e. stimulation of adenylate cyclase and inhibition of Na +/K + ATPase activities. The stimulatory effect of ouabain on adenylate cyclase suggests the possibility that the adenylate cyclase and Na +/K + ATPase enzymes may be coordinated in their response to membrane active agents.

Reversible filamentous growth in the psychrophile Bacillus psychrophilus

At the near-maximum growth temperature of 32.5 °C, the psychrophile Bacillus psychrophilus loses the ability to septate and divide, resulting in the formation of filaments, which are four to six times longer than cells grown at 20 °C. DNA synthesis relative to growth occurs at the same rate both in the filaments at 32.5 °C, (which actually become multi-nucleated) and in normal-size cells at 20 °C, showing that the inhibition of DNA synthesis by the elevated temperature is not the cause of the filamentous growth, as has been found for other microorganisms. Similarly, temperature-sensitive cell-wall mucopeptide synthesis does not appear to be responsible. Reversal of filament production occurs when preformed filaments are incubated at 20 °C. Such reversal, i.e., septation of preformed filaments, requires the de novo synthesis of protein, probably throughout the reversal period. Filamentous cells are more nutritionally demanding than cells at 20 °C, with at least one substrate becoming limiting within 8 hr at 32.5 °C but not at 20 °C. However, such variation in nutritional requirement is not the cause of filament formation. KCl and NaCl stimulate cell division in cells growing at 32.5 °C but not in preformed filaments. Other membrane-active agents such as lysolecithin, dimethyl sulfoxide, ethanol, sodium oleate, and pantoyl lactone do not stimulate septum formation in filaments.

Induction of the cortical reaction in hamster eggs by membrane-active agents.

Fusion of capacitated spermatozoa with the vitelline membrane, but not actual penetration, appears to initiate the cortical reaction in hamster eggs. The reaction can be artificially induced by the application of positively charged particles to the vitelline surface, a situation which may normally be prevented by the zona pellucida. Exposure of hamster eggs to neuraminidase, to lectins (concanavalin A and phytohaemagglutinin-P), to a monovalent ionophore (boromycin) and to 1,3-bis(4-chlorocinnamylideneamino)guanidine elicits a cortical granule discharge resulting in a block to fertilization. These agents all appear to act by inducing depolarization of the vitelline membrane.

Bioregulation of lysosomal enzyme secretion from human neutrophils: roles of guanosine 3':5'-monophosphate and calcium in stimulus-secretion coupling.

The biologic role of calcium and guanosine 3':5'-monophosphate (cyclic GMP) in the immunologic secretion of lysosomal enzymes from human neutrophils was studied. Contact of neutrophils with zymosan-treated serum or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) secretion and a concomitant accumulation of cyclic GMP without any loss of cell viability. Acetylcholine (0.1 muM), in the presence of calcium, enhanced the immunologic stimulation of cyclic GMP accumulation and lysosomal enzyme discharge. A marked and rapid association of 45CaCl2 with neutrophils occurred during cell surface contact with zymosan-treated serum, and this effect on calcium association was enhanced with 0.1 muM acetylcholine. The precise mechanism of the neutrophil-calcium interaction is presently not well understood. However, the finding that 0.5-1.0 muM A-23187 also provoked a rapid association of extracellular calcium with neutrophils suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between cells and calcium. The close temporal relationship between beta-glucuronidase secretion, cyclic GMP accumulation, and calcium mobilization during cell contact with membrane active agents such as immune reactants, acetylcholine, and ionophores suggests that these three cellular events bear a cause and effect relationship. On the basis of our findings to date, we propose that the immunologic secretion of lysosomal contents from human neutrophils is signaled by intracellular cyclic GMP and that extracellular calcium, by gaining access to the intracellular processes responsible for cyclic GMP accumulation, serves as the link to stimulus-secretion coupling.

Dose-related inhibition of chemical carcinogenesis in mouse skin by caffeine.

THERE is some evidence that caffeine protects against the effects of chemical carcinogens1,2. Leitner and Shear3 found a reduction in the effectiveness of benzo(a)pyrene as a carcinogen when it was administered by subcutaneous injection together with various purines, including caffeine. An enhancement of the antitumour effect of 1,3-bis(2-chloroethyl)-1-nitrosourea by caffeine has been reported4 but whether the caffeine acts as an anticarcinogen or as a membrane-active agent (a property of caffeine which has been demonstrated5) is not clear. Tumour production in vivo has been shown to be partially suppressed by theophylline6 and it is possible that caffeine which is chemically closely related to theophylline may similarly suppress tumour production.

The interactions between vitamin A, vinblastine, and cytochalasin B in parathyroid hormone secretion.

The role of, and interrelationships between, microtubules, microfilaments, and membrane active agents in the secretion of parathyroid hormone were determined utilizing VB, a microtubule disrupter, CB, a microfilament disrupter, and Vit A, an agent known to interact with membranes.

Studies on action mechanism of clotrimazole as compared with other membrane-active agents and their combinations effective in synergism or antagonism

Studies on action mechanism of clotrimazole as compared with other membrane-active agents and their combinations effective in synergism or antagonism

Effects of colicin E2 on DNA and the bacterial membrane in vivo

1. Colicin E2 labilises the association of pulse-labelled DNA and the bacterial membrane 5–10 min after it induces double-strand breaks in the chromosome in vivo. 2. Newly synthesised DNA was partially protected against colicin E2-induced exonucleolytic activity. 3. Under certain conditions membrane-active agents, β-phenethylalcohol and novobiocin, enhanced colicin E2-induced exonucleolytic activity. Inhibitors of DNA synthesis, mitomycin and phleomycin, had little effect whereas intercalating drugs inhibited colicin action.

Stimulation of cell division by membrane-active agents

Agents that decrease membrane stability (e.g. dimethyl sulfoxide, lysolecithin, sodium oleate, and short-chain alcohols) stimulate multinucleoid, serpentine filaments of Agmenellum quadruplicatum strains SN12 and SN29 to divide into cellular equivalents within approximately one generation time. Agents that increase membrane stability (e.g. long-chain alcohols) antagonize this timulation. Thus, the physical properties of the cell membrane appear to be involved in the regulation of cell division. These observations suggest that the invagination of the cell wall may be regulated by agents that interact with the plasma membrane and with enzymes involved in peptidoglycan synthesis.

The effect of membrane-active agents on the mitotic frequency in normal and carcinogen-treated epithelium.

Die Mitosehäufigkeit im Epithel der Backentasche des Goldhamsters nach 12 Wochen dauernder Behandlung mit Dimethylbenzanthracen (DMBA) Vitamin A-palmitao, Cortison-Acetat, Chlorpromazin sowie Kombinationen dieser Stoffe wurden untersucht und die beobachteten Wirkungen werden mit der Modifikation der carcinogenen Wirkung von DMBA durch die letzteren drei Substanzen verglichen.

The ion-specific increases in membrane permeability with a group of membrane-active antibacterial agents.

The ion-specific increases in membrane permeability with a group of membrane-active antibacterial agents.

Studies on negative chemotaxis and the survival value of motility in Pseudomonas fluorescens.

SUMMARY: Multiplication of aerobic piliated fla+ and fla− Pseudomonas fluorescens growing in aerated mixed culture gave fla+:fla− ratios approximately 1:1 over a 24 hr growth period. When fla+ and fla− were cultured simultaneously in non-aerated media, fla+ then outgrew the fla− mutant to a final (24 hr) ratio of 10:1 or greater, thereby lending supporting evidence to the view that motility is a survival factor in environments where ‘nutrients’ are limited or discontinuous. Gravity, magnetism, and light did not influence a negative chemotactic response of fla+ P. fluorescens to hydrogen ions. Temperature variations, pH, or viscosity alterations either inhibited or stimulated motility, thereby making impossible a determination of their specific effects. Various antibiotics, membrane-active agents, and protein or DNA synthesis inhibitors were employed in attempts to interfere with the chemotactic response, but none completely inhibited chemotaxis without also affecting motility. The cytoplasmic membrane may act as a transducer of environmental stimuli in chemotaxis.

ATPase activities of rat epididymal adipose tissue

1. 1. A Mg 2+-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.4) has been observed in rat epididymal adipose tissue. The activity was concentrated in the microsomal subcellular fraction. Evidence indicated that a large fraction of this microsomal Mg 2+-ATPase activity was located in the cytoplasmic membrane in the intact cell. 2. 2. The incubation of microsomes with [ γ- 32P]ATP resulted in incorporation of 32P into trichloroacetic acid-precipitable material. No appreciable fraction (larger than 5 μμmoles/mg protein) of this phosphorylated material possessed a rate of turnover compatible with its being a possible intermediate in the Mg 2+-ATPase-catalyzed reaction. 3. 3. The influence of several membrane-active agents (detergents, vitamin A, gramicidin, histones, phospholipase C and lysolecithin) suggested a very intimate relationship between the integrity of the membrane and the activity of the ATPase. 4. 4. Evidence is presented establishing the existence of a Mg 2+ requiring ( Na + + K +)-activated ATPase activity in adipose tissue.

The Action of Polyene Antibiotics on Phospholipid-Cholesterol Structures

Artificial spherules of phospholipid were prepared with and without cholesterol present, so as to sequester anions (H2PO4- or CrO4=) or glucose. The effect of several polyene antibiotics upon release of anions or glucose was determined, and it was found that, whereas filipin and etruscomycin released similar amounts of these markers whether or not cholesterol was preincorporated into the spherules, amphotericin B and nystatin were far more effective when cholesterol was preincorporated. Ergosterol could be substituted for cholesterol. Release of anions or glucose from spherules by other membrane-active agents, such as etiocholanolone, deoxycorticosterone, diethylstilbestrol, streptolysin S, or Triton X-100, was unaffected by the presence or the absence of cholesterol. It was found that an ovolecithin to cholesterol ratio of 15:1 was optimal for release of markers both by filipin and by amphotericin B. By electron microscopy, the lamellar substructure of polyene-disrupted spherules was seen to be altered. Whereas filipin or deoxycorticosterone induced such morphological changes in spherules prepared with and without cholesterol, amphotericin B produced morphological changes only in spherules formed in the presence of cholesterol. These studies support the hypotheses that polyene antibiotics interact with purified lipids present in biological membranes and that amphotericin B and nystatin preferentially disrupt membranous structures which contain cholesterol. They also show that filipin and etruscomycin disrupt spherules of pure phospholipid prepared in the absence of cholesterol, and thereby suggest qualitative differences in the mode of action of the several polyene antibiotics.

Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture.

Basic proteins and polyamino acids are taken up by mammalian cells at rates up to 3000 times greater than serum albumin. When given together with serum albumin they increase the albumin uptake by a factor that correlates with their own rate of uptake and can reach more than 50-fold. The lowest threshold of activity detected (10(-10)M)is comparable to the activities of the most potent membrane-active agents.