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.     

Distribution and effects of 2,4,5-trichlorophenoxyacetic acid in cells of Bacillus megaterium.

Cell death in a resting population of an asporogenous Bacillus megaterium was accelerated by ambient concentrations of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) equal to or greater than 10 mug/ml or 5 mug/mg of cells (dry weight), but only after prolonged exposure. Conversely, populations of growing cells were not markedly influenced even at 100 mug/ml. Effects on cell respiration were not manifest until the ambient concentration reached 1,000 mug of 2,4,5-T/ml, or 500 mug/mg. Cells of B. megaterium did, however, accumulate 2,4,5-T passively to a level approximately twofold above the ambient concentration. Most of the accumulated compound was easily washed from the cells, but, of the firmly bound herbicide, about 0.5 mug/mg of cells (dry weight), nearly 60% by weight, was localized in the protoplast membrane. The foregoing results, obtained with a purified preparation of 2,4,5-T were also elicited by 2,4,5-T analytical standards. The extracted contaminants did not produce the results alone nor did they influence the results when present in combination with 2,4,5-T.     

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.     

Influence of metal ions on the formation of mycobactin and salicylic acid in Mycobacterium smegmatis grown in static culture

Mycobacterium smegmatis was grown on trace-metal-free medium in static culture. Throughout the growth phase, the concentration of mycobactin increased continuously, reaching a maximum of about 30 to 40 mug of mycobactin/mg of cell dry weight after 6 days; the concentration of salicylic acid remained approximately constant at 1 to 2 mug of salicylic acid/mug of cell dry weight. Fe(2+) (or Fe(3+)), Zn(2+), Mn(2+), and Mg(2+) were all essential to a maximum formation of mycobactin. Optimum concentrations required were: Fe(2+), about 1.8 mum; Mn(2+) and Zn(2+), about 0.5 mum; and Mg(2+), at least 0.17 mm. Higher levels of Fe(2+) (9 to 90 mum) and Zn(2+) (2 to 7 mum) repressed mycobactin to about half the maximum value. No other cation or anion apparently is required for mycobactin biosynthesis. Salicylic acid concentration increased about fourfold when iron was omitted from the medium, but this is not as great as the increase reported previously for this strain of M. smegmatis. Mycobactin formation in another strain of M. smegmatis, NCIB 8548, showed similar dependencies on Fe(2+), Zn(2+), and Mn(2+). Maximum accumulation of mycobactin with this strain was 85 mug of mycobactin/mg of dry cell weight, under iron-deficient (1.8 mum Fe(2+)) conditions.     

植物叶片原生质体分离的可能机制

分析了植物叶片在分离液环境中形成原生质体的过程,文中提出,分离液配方中的酸性物质使植物叶片处于酸性环境中并导致植物正常细胞首先发生细胞壁酸性降解,随后出现原生质体脱离细胞壁进入分离液,继而又进一步发生质膜的酸性降解,使细胞核和细胞器进入分离液中,最终分离液中的细胞器以细胞核为中心进行细胞器重组,最后产生外貌形态一致的新的原生质体。植物细胞壁和质膜是植物细胞的包被系统。植物细胞包被系统的酸性降解使植物细胞器重组并产生新的原生质体成为可能。     

Trehalose degradation and glucose efflux precede cell ejection during germination of heat-resistant ascospores of Talaromyces macrosporus

Talaromyces macrosporus forms ascospores that survive pasteurization treatments. Ascospores were dense (1.3 g ml(-1)), relatively dry [0.6 g H(2)O (g dry weight)(-1)] and packed with trehalose (9-17% fresh weight). Trehalose was degraded to glucose monomers between 30 and 100 min after heat activation of the spores. The maximal activity of trehalase was calculated as 400-520 nmol glucose formed min(-1) (mg protein)(-1) as judged by measurements of the trehalose content of spores during germination. During early germination, glucose was released from the cell (10% of the cell weight or more). The intracellular concentration of glucose only peaked briefly. After 160-200 min, the protoplast encompassed by the inner cell wall was ejected through the outer cell wall in a very quick process. Subsequently, respiration of spores increased strongly. The data suggested that trehalose is primarily present for the protection of cell components as glucose is released from the cell. Then, an impenetrable outer cell wall is shed before metabolic activity increases.     

An Ultrastructural Study on Pollen Protoplasts and Pollen Tubes Germinated From Them in Gladiolus gandavensis

Large quantities of protoplasts were isolated enzymatically from the mature pollen grains in Gladiolus gandavensis. Regeneration of cell wall and germination of pollen tubes were performed during culture of purified pollen protoplasts in Ks medium supplemented with 32% sucrose, 0.1 mg/1 2,4-D, 1 mg/1 NAA and 0.2 mg/1 6-BA, with a germination rate up to 47.7%. The materials were fixed gently with gradually increasing concentration of glutaraldehyde, followed by osmium, then preembedded in a thin layer of agar and surveyed under an inverted microscope so as to select desired specimens for subsequent procedure. Small agar blocks containing specimens were dehydrated through ethanal-propylene oxide series, embedded in Araldite and ultratomed. Electron microscopic observations show that the pollen protoplasts are surrounded by a smooth plasma membrane and with ultrastructurally intact cytoplasm, a vegetative nucleus and a generative cell. After 8h of culture, wall regeneration commences resulting in a multilayered, fibrillar wall structure which is different from the intine. No exine is formed. Numerous vesicles participate actively in the wall formation. The wall is uneven in thickness around its periphery; a thickened area somewhat resembling to germ furrow is formed, from which pollen tube emerges. The tubes contain abundant plastids, mitochondria and dictyosomes. Vesicles are released out of the plasma membrane and involved in tube wall formation. After 18h of culture, the vegetative nucleus and generative cell have migrated into the tube. Technical points of preparing pollen protoplast specimens for ultastructural studies and the fearnres of wall regeneration in pollen protoplast culture are discussed.     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

Isolation, Chemical Composition, and Ultrastructural Features of the Cell Membrane of the Mycoplasma-Like Organism Spiroplasma citri

Thin sections of Spiroplasma citri, a mycoplasma-like organism isolated from citrus infected with "Stubborn" disease, showed the organisms to be limited by a single trilaminar plasma membrane. An additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. The organisms were found to be extremely sensitive to lysis by osmotic shock. The cell membrane of S. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical composition. The isolated membranes showed the characteristic trilaminar shape in section and the typical particle-studded fracture faces in freeze-etched preparations. Protein and lipid formed over 80% of the total dry weight of the membrane, which had a density of ~1.180 g/cm(3). Cholesterol constituted over 20% of the total membrane lipid. Phosphatidyl-glycerol, synthesized by the organisms, was the major phospholipid. Significant amounts of hexosamine (15 to 35 mug/mg of membrane protein) could be found in the membrane preparations. Our results support the thesis that S. citri does not possess a cell wall, either of the gram-positive or the gram-negative type, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily.     

Changes in wall and internal structure of allomyces-resistant sporangia during germination

The electron microscope was used to examine changes which take place in wall, as well as in internal, structure during germination of mature resistant sporangia of Allomyces neo-moniliformis. When the resistant sporangia are first placed in water to initiate germination, nuclei, mitochondria, and endoplasmic reticulum are not evident, though after the sporangia have been in water for more than 30 min all of these structures become visible. At this time no cracks are evident in the resistant sporangial wall and the cell membrane appears highly convoluted. Within the next 30 min the outer wall splits and the inner wall expands considerably as the protoplast increases in volume. At the same time the cell membrane straightens out, apparently in response to the protoplasmic expansion. The “cementing substances” begin to dissolve about this time so that 1 1/2 hr after placement in water the outer wall is completely separated from the inner wall which now acts as the cell wall. Cleavage appears to be initiated by the invagination of the cell membrane and by the appearance of segments of endoplasmic reticulum with filled vesicles at one end. Between 2 1/2 and 3 hr after placement in water zoospores are released.     

Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

Mode of antibacterial action by gramicidin S

To elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experiment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 microgram/ml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6 micrograms/ml, and the cells were temporarily saturated with GS at 10 micrograms/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by adsorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. coli increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall.     

Biochemical indication of microbial mass changes using ATP and DNA measurement in biological treatment of thiocyanate

This study was designed to monitor changes in the levels of adenosine 5'-triphosphate (ATP) and deoxyribonucleic acid (DNA) per unit of microbial mass during the autotrophic biodegradation of thiocyanate (SCN(-)). An artificial medium containing trace minerals and 500 mg SCN(-)/L was used as a substrate for bacterial growth. An SCN(-)-degrading bioreactor with a working volume of 6 L, equipped with temperature, pH, and dissolved oxygen controls, was operated in batch mode. During the exponential phase of SCN(-) biodegradation, the ratios of ATP and DNA to microbial dry weight varied from 0.6 to 1.1 mug ATP/mg of volatile suspended solid (VSS), and from 3.5 to 8.8 mug DNA/mg of VSS, respectively. The ATP and DNA concentrations correlated linearly with microbial mass (r (2) > 0.9) within the exponential phase. The linear regression equations were as follows: (1) microbial mass concentration (mg/L) = 0.663 x ATP concentration (mug/L) + 11.1 and (2) microbial concentration (mg/L) = 0.081 x DNA concentration (mug/L) + 10.9. The applicable ranges were 6.8 to 47.4 mug/L for ATP concentration and 41.5 to 395 mug/L for DNA concentration, respectively.     

牛磺酸对小麦幼苗生长的生理效应

采用牛磺酸溶液培育小麦幼苗,测定10、100、500、1 000、5 000 mg/L的牛磺酸对小麦幼苗的光合作用PS Ⅱ光化学效率、细胞膜相对透性和膜脂过氧化以及生长的影响.结果表明,与对照组相比,适宜浓度的牛磺酸处理可促进小麦幼苗的生长,使其根长、株高、单株幼苗的干重和鲜重增加,并在一定程度上提高叶片的光化学效率,降低细胞膜相对透性和膜脂过氧化产物的含量;最适处理浓度约为500 mg/L.说明牛磺酸对小麦幼苗细胞膜有一定的保护作用.     

Uptake and binding of dodine acetate by fungal spores

Uptake of the dodine cation from acetate solutions by conidia of Alternaria tenuis and Neurospora crassa was characterized by a rapid rate of sorption, a ‘Langmuir type’ adsorption isotherm, and independence of temperature: all of which suggests an ionic bonding mechanism. Metal cations competed with dodine for the anionic binding sites of the cell—regarded as carboxyl and phosphate groups—and dodine uptake also decreased as ionization of the carboxyl group was suppressed. Cell walls of A. tenuis had a greater capacity to bind dodine than did those of N. crassa. Binding at the cell wall may detoxify some of the large amount of dodine that must be accumulated by the spores to achieve toxicity. The dodine retained by N. crassa cell walls could not be exchanged or desorbed by washing and is probably bound covalently rather than by weaker ionic bonds. At sub-lethal concentrations there was no evidence that dodine disorganized cell wall structure. Disruption of spores which had been incubated with ¹⁴C-labelled dodine showed the fungicide to be associated with intra-cytoplasmic organelles. It is suggested that dodine reacts with the protoplast membrane so as to alter its permeability and allow more dodine to penetrate into the cytoplasm where it may destroy intracellular membrane structure.     

Effect of aeration and carbon dioxide on cell morphology of Candida utilis

The effect of CO2 availability on cell size, shape, and aggregation in continuous cultures of Candida utilis was studied in minimal medium with glucose or ethanol as the sole carbon and energy source. Enrichment with CO2 was achieved (i) by using the substrate with more C atoms, (ii) by using pure oxygen and thus decreasing aeration intensity at the same dissolved-oxygen concentration, or (iii) by adding CO2 to the aeration gas. The cells were always of yeast shape, and no filaments were formed. In cultures with a biomass concentration above 6 g (dry weight) per liter, no cell aggregates were observed. In cultures with a lower biomass, the daughter cells failed to separate from the parent cells and formed aggregates with thickened walls. The average cell number per aggregate was found to be higher, and the average protoplast volume lower, under conditions of probable CO2 limitation. Simultaneously, the ratio of total dry weight to wet weight of protoplasts was considerably higher, indicating an increased share of wall or extracellular material. The possible effect of the observed morphological changes for maintaining a suitable concentration gradient of CO2 around the cell is discussed.     

Effect of aeration and carbon dioxide on cell morphology of Candida utilis.

The effect of CO2 availability on cell size, shape, and aggregation in continuous cultures of Candida utilis was studied in minimal medium with glucose or ethanol as the sole carbon and energy source. Enrichment with CO2 was achieved (i) by using the substrate with more C atoms, (ii) by using pure oxygen and thus decreasing aeration intensity at the same dissolved-oxygen concentration, or (iii) by adding CO2 to the aeration gas. The cells were always of yeast shape, and no filaments were formed. In cultures with a biomass concentration above 6 g (dry weight) per liter, no cell aggregates were observed. In cultures with a lower biomass, the daughter cells failed to separate from the parent cells and formed aggregates with thickened walls. The average cell number per aggregate was found to be higher, and the average protoplast volume lower, under conditions of probable CO2 limitation. Simultaneously, the ratio of total dry weight to wet weight of protoplasts was considerably higher, indicating an increased share of wall or extracellular material. The possible effect of the observed morphological changes for maintaining a suitable concentration gradient of CO2 around the cell is discussed.     

Cytochemical localization of mercury in Saccharomyces cerevisiae treated with mercuric chloride

We describe a cytochemical method for localizing mercury at the electron microscopic level in the yeast Saccharomyces cerevisiae. After addition of a lethal concentration of mercuric chloride to growing yeast cells, mercury was associated with the cell wall and cytoplasmic membrane. Little or no mercury was present in the cytoplasm. Electron probe X-ray microanalysis (EPMA) confirmed that the cytochemical reaction, visualized as mercury-silver complexes, was localized in dense bodies consisting of a core of mercury sulfide polymers surrounded by a shell of silver atoms.     

Laser microsurgery: a versatile tool in plant (electro) physiology

Summary In plant cells the cell wall is a formidable obstacle in many physiological studies such as patch-clamp measurements and cell labelling with antibodies. Enzymatic digestion of the cell wall, in order to release a protoplast, has a number of disadvantages; therefore we worked out an alternative method to gain access to the plasma membrane. The wall of specialized cells from three higher plant species and one unicellular alga were perforated using the focussed UV light of a nitrogen laser. In order to enhance the absorption of the UV light by the walls, a dye was used that binds specifically to cell wall components. Extrusion of the protoplast or parts thereof was controlled by a regulated gradual decrease of the osmolarity of the solution surrounding the cells. Cytoplasmic streaming and chloroplast circulation were maintained in the protoplasts, demonstrating their viability after the wall perforation with the laser. Continuous deposition of new cell wall material by the polar tip of pollen tubes after surgical removal of the wall at the tip is another demonstration of the viability of the cells. Formation of high resistance seals between the plasma membrane and a patch pipet was surprisingly difficult. The role of Hechtian strands and continuing synthesis of cell wall material in seal formation is further investigated. Other applications for the surgical laser are: fusion of two cells or vacuoles, analysis of the composition of specific parts of the cell wall, and release of the vacuole from an identified cell type for patchclamp studies.Abbreviations CFW calcofluor white - PM plasma membrane     

Kinetic and Morphological Observations on the Yeast Phase of Histoplasma capsulatum During Protoplast Formation

Protoplast formation by Histoplasma capsulatum yeasts using high concentrations of MgSO(4) occurs either by lysis of the bud or lysis of the entire cell wall. Both mechanisms may also occur simultaneously. Neither the protoplast emerging through a hole in the cell wall nor the freshly released protoplast has a recognizable cell wall or the remnant of such. The protoplast contains all the organelles of the normal cell except for mesosomes. During protoplast formation the nucleus increases in size and produces several nuclear masses by the invaginations of the internal layer of the nuclear membrane. All these nuclear masses are surrounded by the external layer of the nuclear membrane. Several nuclei with a normal nuclear membrane are formed later.