Antimicrobial Actions of Hexachlorophene: Lysis and Fixation of Bacterial Protoplasts

Hexachlorophene was found to be both a lytic and a fixative agent for protoplasts isolated from Bacillus megaterium. Concentrations of 50 to 100 mug of drug per mg of original cell dry weight were required to lyse 4.4 x 10(9) protoplasts (2 mg of original cell dry weight). At higher drug concentrations, protoplasts became fixed against osmotic stress and reduced in sensitivity to disruption by n-butanol. Lower drug concentrations caused proportionate lysis in the protoplast population. Intact cells lost the ability to become plasmolyzed at these same hexachlorophene concentrations. Nonplasmolyzed, drug-treated cells were resistant to the action of lysozyme, whereas plasmolyzed, drug-treated cells were sensitive. But the sensitivity of isolated cell walls to lysozyme digestion was not markedly altered by hexachlorophene treatment. These effects appeared to be secondary in the killing of cells by hexachlorophene because they occurred at concentrations higher than the minimum lethal concentration.     

Antimicrobial Actions of Hexachlorophene: Cytological Manifestations

Hexachlorophene is a soap-compatible bisphenol that has been widely used as an antiseptic, yet its mechanism of action is undefined. The relative threshold concentration for bactericidal effect on a susceptible test organism, Bacillus megaterium, was established to be about 10 mug/mg of cell dry weight. At this or at high (>/=100 mug/mg) concentration, adsorptive uptake by cells displayed saturation kinetics. At about 30 mug/mg, the time course of adsorption occurred in three distinct stages. The triphasic pattern was interpreted to represent successive penetration of and adsorption by the cell wall, the protoplast membrane, and the cytoplasm. This interpretation was substantiated by determinations of hexachlorophene adsorption by isolated cell components. Electron microscopy disclosed cytopathology, evidenced as gaps or discontinuities, in the protoplast membrane (but not in the cell wall or cytoplasm) at > 30 mug of hexachlorophene per mg of cell dry weight. Similarly, treatment with > 30 mug/mg allowed a fluorescigenic dye (tolyl-peri acid) to penetrate into the protoplast. However, no detectable cytological manifestations were discerned at the minimum lethal concentration of 10 mug/mg. Apparently, hexachlorophene is physically disruptive at intermediate or high relative concentrations but acts in a more subtle fashion at the minimal lethal concentration.     

Effects of inorganic salts on tissue permeability

Inorganic solutes are shown to alter the permeability of root and leaf tissues. Experiments with beet root tissues reveal that CaCl(2) decreases leakage of betacyanin from the tissue, that (NH(4))(2)SO(4) increases leakage, and that each salt can relieve the effects of the other. A comparison of cations and anions shows a range of effects with the various solutes. Experiments with Rumex obtusifolius L. leaf discs reveal that whereas CaCl(2) defers the development of senescence, (NH(4))(2)SO(4) hastens senescence and increases the leakage of materials out of the leaf discs. The solute effect on Rumex obtusifolius L. is prevented by gibberellin. CaCl(2) can relieve the (NH(4))(2)SO(4) effect. The results are interpreted as indicating that the inorganic solutes may serve to alter the permeability of membranes through alterations of interactions between water and macromolecules in the tissues; the interpretation is consistent with the evidence for opposite effects of Ca and NH(4), the effective concentrations being about 10(-3)m, and the reversibility of the effects of one solute by another of opposite stabilization-destabilization effect.     

Kinetics of toluene-induced leakage of low molecular weight solutes from excised sorghum tissues

The relationship between toluene concentration and the rate of leakage of solutes from toluene-treated roots and leaves of Sorghum bicolor, L. Moench, was studied to determine the effect of toluene on plant cell membranes. A threshold concentration of 0.2% toluene was needed to induce leakage. Maximal leakage rates were obtained with 0.5% toluene. Low molecular weight solutes, such as amino acids, sugars, and inorganic ions, leaked from treated tissue, while macromolecules, such as protein were retained. The rates at which the low molecular weight solutes diffused from treated cells decreased with increasing molecular weight. At 25°C, treatment of roots and leaves with 0.5% toluene resulted in the quasi-quantitative leakage of solutes within 180 minutes. At 1°C, roots and leaves differed in their response to toluene. The rates of leakage from roots at 1°C were much lower and the total amounts much smaller than at 25°C, while in leaves the difference between the two temperatures was very small.     

Permeability of the peroxisomal membrane to cofactors of beta-oxidation. Evidence for the presence of a pore-forming protein

Peroxisomes were purified from livers of clofibrate-treated rats. Permeability measurements on the isolated organelles revealed that peroxisomes are permeable to small solutes, including sucrose and the cofactors for fatty acid oxidation NAD+, CoA, ATP, and carnitine. The intraperoxisomal distribution volume was equal for all solutes. Peroxisomal solute uptake was rapid, not saturable and not visibly influenced by temperature. NAD+ and carnitine uptake in the solute accessible volume was not diminished by a variety of analogs and inhibitors. Subfractionation of peroxisomes and reconstitution of the subfractions into liposomes preloaded with solutes made the liposomes reconstituted with the integral membrane protein fraction, but not those reconstituted with the other subperoxisomal protein fractions, permeable to the same solutes that entered intact peroxisomes. Solute leakage from the preloaded liposomes was rapid and not visibly influenced by temperature. Leakage activity was destroyed by heat treatment of the integral membrane protein fraction and was not present in lipid extracts of the membrane. Separation of the integral membrane proteins on sucrose density gradients and reconstitution of the gradient fractions into liposomes indicated that the leakage activity was caused by a polypeptide of rather low molecular weight. The gradient distribution of leakage activity corresponded most closely to the presence of a 22- and a 28-kDa polypeptide. Our experiments indicate that the nonspecific permeability of the peroxisomal membrane to small solutes is based on the presence in the membrane of a nonselective pore-forming protein.     

Elution of low molecular weight solutes from viable cells of Saccharomyces bisporus

Previous work has shown that high molecular weight compounds were released from Saccharomyces bisporus by -mercaptoethanol, 2 M KCl, 0.5 M KCl and osmotic shock without affecting viability of the cells. In this current experiment, it was shown that low molecular weight compounds were also eluted when cells were treated in sequence with the same reagents. Alanine, glutamate, serine, an unidentified amino acid, glucose, glycerol, and arabitol were all eluted by each of the first three reagents. The osmotic shock eluate contained a larger number and quantity of amino acids than the first three eluates but, otherwise, the compounds in this eluate were the same. One hundred percent of the cellular glycerol and 65–70% of the total amounts of the other above mentioned solutes were released by the 4 eluting treatments. A hot water treatment was needed to extract the remainder of these solutes. The hot water extract also contained almost all the cellular proline. It was suggested that the elutable solutes are contained by cells in compartments (or vesicles) whose membranes are accessible to the eluting reagents without affecting the plasmalemma.     

In vivo synergistic effect of the immunomodulator AS101 and the PKC inducer bryostatin.

The immunomodulator AS101 has recently been found to have radioprotective properties when injected prior to sublethal and lethal doses of irradiation. In addition, this compound was found to protect mice from hemopoietic damage caused by sublethal doses of cyclophosphamide (CYP) and to increase the rate of survival of mice treated with lethal doses of CYP. AS101 was previously shown to exert a synergistic effect with the PKC-inducer bryostatin in cytokine secretion in vitro. The present studies were designed to evaluate the effects of in vivo combined treatment with AS101 and bryostatin on bone marrow and spleen cellularity and on the number of committed progenitors in the bone marrow at various points of time after their treatment with a sublethal dose of CYP or irradiation. In addition, the combined effect was tested on the survival of mice irradiated with a lethal dose of irradiation. Our data show the presence of synergism which greatly enhances the number of bone marrow and spleen cells 48 hr and 9 days after CYP treatment or irradiation. The combined effect was also demonstrated when bone marrow colony-forming units granulocyte-macrophage (CFU-GM) progenitor cells were evaluated. Moreover, AS101 and bryostatin synergized in their protective effects against lethal damages of irradiation. These results strongly suggest that bryostatin, which lacks tumor-promoting activity, is a particularly good candidate in combination with AS101 for treatment in vivo in counteracting chemotherapy- or radiation-induced hematopoietic suppression or in generally improving the restoration of immune response under conditions involving immune or hemopoietic damage.     

Induction of Solute Release from Nicotiana tabacum Tissue 10

For determination of the effects of polymyxin B, polymyxin E,or ethylenediamine tetra-acetic acid (EDTA) on plant cell membranes,the rates at which three solutes, K⁺, P₁, and sugar, leakedfrom treated tissue culture cell suspensions of Nicotiana tabacumwere measured. The kinetics of leakage from cells treated witheither of the polymyxins was biphasic, whereas kinetics forcells treated with EDTA was monophasic. Only K⁺ leaked frompolymyxin-treated cells during the first phase, and all threesolutes leaked during the second phase. The slower first phaseis interpreted as leakage of K⁺ from the Donnan free space andcytoplasm, and the faster second phase as the leakage of solutesfrom the vacuole. The monophasic kinetics of EDTA treatmentindicated that solutes were leaking simultaneously from cytoplasmand vacuole. Of the divalent cations tested, only Ca⁺⁺ and Mn⁺⁺counteracted the effects of polymyxin and EDTA. Ca⁺⁺ even restoredP¹ and sugar uptake. Addition of Mg⁺⁺ or Sr⁺⁺ to polymyxin-treatedcells did not stop solute leakage but actually enhanced theleakage rates. A model is presented that suggests that polymyxinor EDTA induces solute leakage by forming pores in plant cellmembranes. The effects of divalent cations on membranes oncethe pores are formed are also discussed. Key words: Polymyxin, EDTA, Nicotiana tabacum, Solute leakage     

Uptake and utilization of free fatty acids supplied by liposomes to mammary tumor cells in culture.

Unesterified oleic acid was co-dispersed with various phospholipids in Ca-Mg-free saline and sonicated. Most of the fatty acid co-eluted with phospholipid vesicles when the sonicate was chromatographed on a column of Sepharose 4B. Cells from mouse mammary tumors, maintained in primary monolayer culture, were incubated with these phospholipid-oleic acid vesicles and optimal conditions for the uptake of oleic acid were established. When phosphatidylcholine and phosphatidylserine were used to make the vesicles, the oleic acid was taken up by cells more efficiently than was oleic acid complexed with albumin. The liposome-supplied unesterified oleic acid was rapidly incorporated into other cellular compounds, especially triglycerides and phospholipids. Growth of cells was measured after a 2-h pulse with the liposomes. The amount of DNA in treated cultures was 50% greater than in control cultures by 3 days after treatment. The magnitude of the growth response was sensitive to liposome dosage and to cell density in the treated cultures. This survey suggests that liposomes can be employed to insert materials directly into epithelial cells via their apical surface, that oleic acid so supplied is readily metabolized, and that solutes from liposomes can participate in functioning of the cells.     

Release of biologically active peptides from Escherichia coli at subzero temperatures

Moss, C. Wayne (North Carolina State University, Raleigh), and M. L. Speck. Release of biologically active peptides from Escherichia coli at subzero temperatures. J. Bacteriol. 91:1105-1111. 1966.-Freezing and storage of Escherichia coli at -20 C in phosphate buffer resulted in loss of cell viability and a pronounced leakage of cellular material which had maximal absorption at 260 mmu. Greater loss in cell viability occurred when cells were frozen in distilled water, but only small amounts of 260 mmu absorbing material were detected. Unfrozen cells stored at 2 and 22 C in each menstruum showed little loss in viability, but cells in phosphate buffer released significant amounts of material during storage. Leakage material from cells in phosphate buffer contained greater amounts of ribonucleic acid and amino acids than did material from cells in distilled water. Leakage material from frozen cells contained protein in the form of peptides of relatively small molecular weight; this was not observed for unfrozen cells. These compounds protected a dilute cell suspension from the lethal effects of freezing, and also possessed biological activity for the recovery of cells which had been "injured" by freezing. Direct cell counts indicated that the material released was not a result of cell lysis.     

The function of the suspending medium during the freeze-drying preservation of Escherichia coli

The role of added solutes in the freeze-drying preservation of bacteria is examined. Escherichia coli were washed, suspended in solutions of selected hydroxy-substituted compounds of various molecular weights, and frozen at rates of the order of one degree C per minute. The frozen materials were freeze-dried and rehydrated in several different ways. Freeze-drying survival was correlated with the development and persistence of an amorphous solute matrix and the desorption of residual water. Protection from potentially harmful effects of freeze-drying was attributed to the dispersion, by the aforementioned amorphous matrix, of metabolites released from the cells during the preparation, and freezing of the bacterial suspensions.     

Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury

Several studies demonstrated that treatment with mesenchymal stem cells (MSCs) reduces cisplatin mortality in mice. Microvesicles (MVs) released from MSCs were previously shown to favor renal repair in non lethal toxic and ischemic acute renal injury (AKI). In the present study we investigated the effects of MSC-derived MVs in SCID mice survival in lethal cisplatin-induced AKI. Moreover, we evaluated in vitro the effect of MVs on cisplatin-induced apoptosis of human renal tubular epithelial cells and the molecular mechanisms involved. Two different regimens of MV injection were used. The single administration of MVs ameliorated renal function and morphology, and improved survival but did not prevent chronic tubular injury and persistent increase in BUN and creatinine. Multiple injections of MVs further decreased mortality and at day 21 surviving mice showed normal histology and renal function. The mechanism of protection was mainly ascribed to an anti-apoptotic effect of MVs. In vitro studies demonstrated that MVs up-regulated in cisplatin-treated human tubular epithelial cells anti-apoptotic genes, such as Bcl-xL, Bcl2 and BIRC8 and down-regulated genes that have a central role in the execution-phase of cell apoptosis such as Casp1, Casp8 and LTA. In conclusion, MVs released from MSCs were found to exert a pro-survival effect on renal cells in vitro and in vivo, suggesting that MVs may contribute to renal protection conferred by MSCs.     

Isolation of Chinese hamster cells hyposensitive to ethyl methanesulfonate and their characterization in induction and antagonization of sister-chromatid exchanges

Dominant lethal tests were performed on female mice injected intraperitoneally with cyclophosphamide (200 mg/kg) or with mitomycin C (0.2 or 5 mg/kg) at the preovulatory stage of oogenesis. Complementary experiments were undertaken to clarify the results obtained. Embryo culture showed that sterility found after treatment with cyclophosphamide or with the high dose of mitomycin C was the reflection of true dominant lethal effects. Mortality after cyclophosphamide treatment occurred predominantly at the 2- and 3-cell stages, while it was reported in all preimplantation stages after treatment with the high dose of mitomycin C. Embryos treated with the low dose of mitomycin C developed normally to the blastocyst stage, confirming the absence of preimplantation effects found with this dose in the dominant lethal test. Cytogenetic analysis of female pronuclei at the first cleavage division were performed after mating treated females with males homozygous for one Robertsonian translocation. This method allowed one to distinguish easily the female pronuclei from the male ones, which exhibited one translocated 'marker' chromosome. After treatment with cyclophosphamide, most female pronuclei showed multiple chromatid exchanges or shattering of the entire genome. After treatment with the high dose of mitomycin C, various types of premature chromosome condensation were found, and they were often accompanied by important interchromosome associations. After treatment with the low dose of mitomycin C, no structural chromosome aberrations were found, and the number of numerical anomalies was not significantly different from that found in control embryos. These last results suggest that the increase in rate of postimplantation loss obtained in the dominant lethal test with the low dose of mitomycin C was not due to clastogenic effects of this compound in the female germ cells, but rather to indirect effects on the maternal organism.     

Pedigree Analyses of Yeast Cells Recovering from DNA Damage Allow Assignment of Lethal Events to Individual Post-Treatment Generations

Haploid cells of Saccharomyces cerevisiae were treated with different DNA damaging agents at various doses. A study of the progeny of individual such cells (by pedigree analyses up to the third generation) allowed the assignment of lethal events to distinct post treatment generations. By microscopically inspecting those cells which were not able to form visible colonies we could discriminate between cells dying from immediately effective lethal hits and those generating microcolonies (three to several hundred cells) probably as a consequence of lethal mutation(s). The experimentally obtained numbers of lethal events (which we call apparent lethal fixations) were mathematically transformed into mean probabilities of lethal fixations as taking place in cells of certain post treatment generations. Such analyses give detailed insight into the kinetics of lethality as a consequence of different kinds of DNA damage. For example, X-irradiated cells lost viability mainly by lethal hits (which we call 00-fixations); only at a higher dose also lethal mutations fixed in the cells that were in direct contact with the mutagen (which we call 0-fixations), but not in later generations, occurred. Ethyl methanesulfonate (EMS)-treated cells were hit by 00-fixations in a dose dependent manner; 0-fixations were not detected for any dose of EMS applied; the probability for fixation of lethal mutations was found equally high for cells of the first and second post treatment generation and, unexpectedly, was well above control in the third post-treatment generation. The distribution of all sorts of lethal fixations taken together, which occurred in the EMS-damaged cell families, was not random.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location and Consequences of 1,1,1-Trichloro-2,2-bis(p-Chlorophenyl) Ethane Uptake by Bacillus megaterium

No detrimental effects of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) were observed when cells of Bacillus megaterium were grown from small inocula in nutrient media containing up to 100 mug of DDT/ml. However, when the ratio of DDT to biomass of resting cells was held constant, levels of DDT as low as 1 mug/ml (0.5 mug/mg of cell dry weight) enhanced the rate of death in the population. The lethal action of DDT was both time- and dose-dependent so that higher doses required less time to effect the same killing than did lower doses. Intact cells bound a maximum of about 1.7 mug of DDT/mg of cell dry weight, of which about 75% was localized in the protoplast membrane. Much of the bound DDT was subsequently lost to the suspending medium and the aqueous stability of the returned DDT was enhanced, possibly by association with solubilized cell materials. A small quantity of bound DDT was converted to 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane, which was released from cells somewhat faster than DDT. Apparently the lethal action of DDT was related to its binding in the membrane, but respiration was not inhibited. The atypical macroscopic appearance of membranes isolated from treated cells suggested that cell death may result from altered membrane chemistry.     

Early cell envelope alterations by tobramycin associated with its lethal action on Pseudomonas aeruginosa

The immediate activities of the aminoglycoside antibiotic tobramycin were investigated in Pseudomonas aeruginosa PAO1. The lethal action of a low concentration of tobramycin (8 micrograms ml-1) occurred rapidly (1-3 min) and was associated with leakage of certain cellular components into the supernatant. The presence of magnesium at the time of initial exposure protected cells by preventing uptake of tobramycin; however, magnesium addition following a brief exposure did not restore viability. Analyses of supernatant material revealed a rapid 2-fold increase in protein released following tobramycin treatment. A prominent 29 kDa protein, observed by SDS-PAGE in the released material was identified as the periplasmic beta-lactamase. Brief exposure to tobramycin did not result in major morphological damage or cell lysis as observed by transmission electron microscopy, and release of LPS was not a primary event. Although activity at the ribosomal level was observed by 2-3 min, leakage was detected after only 1 min. These data indicate that leakage of cellular components, particularly beta-lactamase, occurs simultaneously, if not prior to inhibition of protein synthesis by tobramycin.     

Use of the PFGE assay for studies of DNA breakage induced by toxic chemicals

The relevance of the pulsed field gel electrophoresis (PFGE) assay for the estimation of the DNA damaging effects of chemicals was studied. Four chemicals were randomly chosen from the list of 50 Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) reference chemicals with known human acute systemic toxicity: acetylsalicylic acid, paracetamol, ethylene glycol and sodium chloride. Human fibroblasts (VH-10) were used as a model system. For the estimation of cytotoxic effect, cell monolayers were treated with chemicals for 24 hours. Cloning efficiency (colony-forming ability) at different concentrations of the test chemicals was estimated, and the 50% inhibitory concentration (IC50) was determined. The IC50 values obtained demonstrated a correlation with human lethal blood concentrations. The induction of DNA double-strand breaks, measured by PFGE as the fraction of activity released, was detected after treatment with paracetamol. However, the other three chemicals tested mainly induced DNA degradation.     

The Effect of a Non-ionic Detergent on Some Plant Cells

The effects of a non-ionic detergent. Triton × 100, on protoplasmic streaming in some plant cells, on plasmolysis, and on leakage of solutes from beet cells were investigated. The results of the different tests showed some features in common: (1) There is a critical range between 0.007 and 0.01 % v/v. (2) Above this concentration of the surfactant, that is at a concentration close to 0.01 %, the effect is manifested in the following ways. Plasmolysis with sucrose is anomalous or impossible. Protoplasmic streaming ceases within a short time. Definite leakage of ions and sugars starts from beet tissues. (3) At concentrations lower than 0.01 to 0.007 % there is enhanced retention of solutes in beet disks. — It is thought that the critical micelle concentration is of paramount importance. The micelle effect may consist in solubilizing or in forming mixed micelles or a complex with the globular lipoprotein units of the outermost plasma layer, the plasmalemma. The effect of the lower Triton concentrations is discussed.     

Respiratory changes with chilling injury of soybeans

The leakage of solutes from cotyledons of soybeans (cv. Chippewa 64) was markedly stimulated by a chilling treatment (1 to 4 C) during the 1st minute of imbibition, but chilling after even 1 minute of water uptake resulted in little or no leakage increase. The respiratory rate of soybean particles was reduced more than 60% if a chilling treatment (15 minutes at 1 to 4 C) was given during the first minutes of imbibition, and little or no reduction was obtained if the chilling treatment was begun at 5 to 15 minutes after the start of imbibition. Using KCN as an inhibitor of cytochrome oxidase pathway of respiration and salicylhydroxamic acid as an inhibitor of the alternative pathway, it was found that the chilling injury involved a major reduction in the cytochrome pathway in whole axes and cotyledons and an engagement of the alternative pathway of respiration in cotyledon tissue. The suggestion is made that the chilling injury involves lesions resulting from temperature stress during the reorganization of membranes with water entry, and that both the leakage and the respiratory effects are consequences of these membrane lesions.     

Arachidonic acid inhibits luteinizing hormone-stimulated progesterone production in hen granulosa cells

Arachidonic acid has been proposed to function as a hormone-induced second messenger in a variety of mammalian endocrine tissues. The present studies were conducted to evaluate whether arachidonic acid, either added exogenously or released endogenously following treatment with physiologic (phospholipase A2) or pharmacologic (melittin) agents, influences basal and/or luteinizing hormone (LH)-induced cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone production in granulosa cells from domestic hens. Phospholipase A2 (PLA2) and melittin treatments failed to alter basal concentrations of progesterone, whereas arachidonic acid had a slight stimulatory effect (only at the 50-microM dose) on progesterone levels, and no effect on cAMP. By contrast, arachidonic acid, PLA2, and melittin each inhibited LH-promoted progesterone production in a dose-dependent fashion. The inhibitory effects of arachidonic acid on the progesterone response were determined to occur both prior and subsequent to cAMP formation since cAMP levels in arachidonic acid-treated cells were attenuated after treatment with 10 ng LH or 100 microM forskolin (at 10- to 100-microM doses of arachidonic acid), and progesterone production was decreased in the presence of 1 mM 8-bromo-cAMP (with 50 and 100 microM arachidonic acid). The post-cAMP mechanism of action is characterized by the inability of cells to convert 25-hydroxy-cholesterol, but not pregnenolone, to progesterone. The effects of arachidonic acid are probably direct, since pharmacologic inhibitors of the lipoxygenase (nordihydroguaiaretic acid) and cyclooxygenase (indomethacin) pathways of arachidonic acid metabolism failed to alter the suppression of