Showing papers on "Lysis published in 1968"

Further characterization of particulate fractions from lysed cell envelopes of halobacterium halobium and isolation of gas vacuole membranes

Abstract: Lysates of cell envelopes from Halobacterium halobium have been separated into four fractions. A soluble, colorless fraction (I) containing protein, hexosamines, and no lipid is apparently derived from the cell wall. A red fraction (II), containing approximately 40 per cent lipid, 60 per cent protein, and a small amount of hexosamines consists of cell membrane disaggregated into fragments of small size. A third fraction (III) of purple color consists of large membrane sheets and has a very similar composition to II, containing the same classes of lipids but no hexosamines; its buoyant density is 1.18 g/ml. The fourth fraction (IV) has a buoyant density of 1.23 g/ml and contains the "intracytoplasmic membranes." These consist mainly of protein, and no lipid can be extracted with chloroform-methanol. Fractions I and II, which result from disaggregation of cell wall and cell membrane during lysis, contain a high proportion of dicarboxyl amino acids; this is in good agreement with the assumption that disruption of the cell envelope upon removal of salt is due to the high charge density. The intracytoplasmic membranes (IV) represent the gas vacuole membranes in the collapsed state. In a number of mutants that have lost the ability to form gas vacuoles, no vacuole membranes or any structure that could be related to them has been found.

Lysis of Yeast Cell Walls Induced by 2-Deoxyglucose at Their Sites of Glucan Synthesis

Abstract: Six sites of 2-deoxyglucose (2DG)–induced lysis on three yeasts (Schizosaccharomyces pombe, Pichia farinosa, and Saccharomyces cerevisiae) coincided with the regions of growth of their glucan layers. Identification of the glucan layer as the site of lysis suggests a mechanism of attack by 2DG or by its derivatives. It is proposed that the glucan layer grows by addition of glucose into internal breaks of polysaccharide molecules. 2DG inhibited resynthesis (insertion of glucose) of the broken glycosidic linkage.

Lytic agents, cell permeability, and monolayer penetrability.

Abstract: Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20–30 % lipid and 50–75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2–3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.

The Extraction of Intracisternal A-Particles from a Mouse Plasma-Cell Tumor

Abstract: Plasma-cell tumors in BALB/c mice contain numerous intracisternal A-particles which remain localized within microsomal vesicles when the tumor cells are disrupted by homogenization. Liberation of the particles has been achieved by subjecting microsome suspensions to mechanical shear in the presence of an optimal concentration of Triton X-100. The particles were concentrated by two cycles of sedimentation in sucrose-potassium citrate solutions, pH 7.2, and finally banded isopycnically in a sucrose density gradient containing dilute potassium citrate. Most of the particles were recovered in a density range of 1.20–1.24 gm/cu cm. A-particles extracted by this procedure retained their characteristic inner and outer shells. Electron microscopy of the gradient-isolated fractions revealed some residual contamination of the A-particles with microsomal membranes. The isolated material consisted of about 80% protein, 14% phospholipid (other lipids not studied) and 5 to 6% RNA. No DNA was detected. Deoxycholate treatment, which lysed the contaminating membranes and simultaneously stripped the A-particles of their outer shells, sharply reduced the phospholipid content of the fraction but did not remove RNA. Evidence is presented that 40 to 50% of the total RNA in the isolated fractions was contributed by other cytoplasmic components. The remainder may have represented RNA intrinsic to the A-particles themselves; however, further studies are required to establish this point.

Immune Lytic Transformation: A State of Irreversible Damage Generated as a Result of the Reaction of the Eighth Component in the Guinea Pig Complement System

Abstract: 1. 1. Data presented here indicate that during the sequential complement reaction a state of irreparable cellular damage is reached after the reaction of the eighth component, but before the reaction of the ninth component, in the guinea pig complement system. 2. 2. Spontaneous lysis of the EAbC′—8 complex was shown to be similar to the lysis of the classical E* complex (EAbC′—9) in that it proceeded to a greater extent during a fixed incubation in buffer at 0.15 M rel NaCl than in buffer of lower ionic strength; and also in that it was completely inhibited in 0.086 M EDTA. 3. 3. The rate of lysis in an EAbC′—8 population remained almost constant during 180 min at 37°C, whereas, the rate of lysis in a population containing very small numbers of C′9 sites was very much higher at the beginning of the incubation period and decreased rapidly so that by 90 min it was indistinguishable from that of a population of EAbC′—8. This difference in lytic pattern indicates that the lysis of EAbC′—8 is not a reflection of contaminating C′9 sites. 4. 4. The EAbC′—8 complex did not manifest increased lytic susceptibility compared with other stable intermediate complexes in buffers of low osmotic pressure, on subjection to mechanical trauma, or in buffers at pH between 5.0 and 7.5. 5. 5. The EAbC′—8 complex was found to be specifically more susceptible to lysis in the presence of low concentrations of sodium desoxycholate. Further, the lysis of this intermediate in the presence of sodium desoxycholate was found to be similar to its lysis in the presence of C′9 in that it was temperature dependent, and inhibited in buffer of low ionic strength. 6. 6. Under carefully controlled conditions of sodium desoxycholate concentration and incubation time, the amount of lysis of EAbC′—8 was proportional to the average number of C′8 sites per cell in the population.

Comparison of the action of vantocil, cetrimide and chlorhexidine on Escherichia coli and its spheroplasts and the protoplasts of gram positive bacteria.

Abstract: SUMMARY The actions of chlorhexidine, cetrimide and Vantocil (the hydrochloride of a polymeric biguanide) on bacteria and spheroplasts or protoplasts derived therefrom have been compared. Lysis of Escherichia coli spheroplasts by chlorhexidine or cetrimide is due to rupture of the cytoplasmic membrane. Membranes ruptured with cetrimide can reform to give small, empty envelopes. With increasing concentrations of cetrimide or chlorhexidine more and more granules appear in the cytoplasm, increasing in size with increasing concentration, until the structure becomes transformed to a granular body similar in size to the original spheroplast. With chlorhexidine or Vantocil this now electron-dense body is rigid, but with cetrimide it collapses to a flat disc which does not lyse. The granules appear to consist of coagulated cytoplasm. This effect of excess of these antiseptics not only prevents osmotic lysis of the spheroplast or the collapsed disc but renders them immune to lysis by a variety of chemical agents. In isolated cytoplasm, chlorhexidine or cetrimide causes precipitation over a narrow range of concentrations but greater than those required for lysis. With Vantocil precipitation occurs gradually over a wide range of concentrations, starting at concentrations equal to those which cause a limited amount of lysis of the spheroplast or a relatively small increase in permeability of intact bacteria. Lanthanous and uranyl ions also precipitate cell contents but do not lyse spheroplasts; spheroplasts treated with these ions are protected from lysis by other agents.

Structures Containing Polyphosphate in Micrococcus lysodeikticus

Abstract: Granular structures containing inorganic polyphosphate were found in Micrococcus lysodeikticus. These structures were isolated by fractionation of the bacterial extract obtained by lysing the organisms with lysozyme. The composition of the fraction which was enriched with these structures was found to be: protein, 24%; lipids, 30%; and polyphosphate, 27%. This fraction also contained small amounts of ribonucleic acids, carbohydrate, and polyvalent cations. The effect of different reagents and enzymes on the integrity of the granules was examined. It was noticed that they accumulate in the bacteria during the logarithmic phase of growth but disappear gradually during the stationary phase.

Spontaneous transformation in Bacillus subtilis

Abstract: Cultures of Bacillus subtilis release transforming DNA during the early exponential and stationary phases of growth. The pattern of release of transforming DNA was followed by measuring transformation in a system consisting of a non-transformable DNA donor and a differently marked transformable recipient. Transformation in this system seems to be at least as efficient as that induced by purified DNA. Fluorescence microscopy revealed that released DNA remained bound extracellular to intact cells. The release of DNA during early exponential growth seemed to be correlated with the cells' proneness to lysis; both DNA release and cell lysis were inhibited by chloramphenicol. In stationary cells, the release of DNA was neither correlated with a similar proneness to lysis nor inhibited by chloramphenicol.

Reactive haemolysis--a distinctive form of red cell lysis.

Abstract: This paper describes a form of red cell lysis differentiated from classical complement haemolysis by its occurrence in the absence of antibody on the cells and in the presence of EDTA. This type of haemolysis has been called reactive haemolysis. It is the result of the interaction, in the presence of red cells, of at least two serum factors called `reactor' and `indicator', respectively. Reactor only becomes active after incubation at 37° of serum with certain agents, notably antibody-coated bacteria, zymosan and agarose, in conditions similar to those required for complement activation. The potential for the formation of activated reactor could be demonstrated infrequently in healthy subjects but more frequently in sera from hospital patients. Activated reactor behaved as a protein sedimenting between 7S and 19S, and of α2—β1, electrophoretic mobility in agar. Indicator factors were present in all human sera studied, as well as in the sera of a number of mammalian species. They were demonstrable at high dilutions of the serum and required no prior activation for their action. They occurred maximally in the 7S fractions of serum proteins and migrated in the β2 position on electrophoresis. Reactive haemolysis was first observed and can most conveniently be demonstrated in a red cell—agarose gel. It can also be demonstrated in the test tube following partial purification of activated reactor and indicator factors. Studies in the test tube indicated that a soluble labile lytic factor was responsible for this type of haemolysis.

DEGRADATION OF THE DNA OF ESCHERICHIA COLI K12 REC- (JC1569b) AFTER IRRADIATION WITH ULTRAVIOLET LIGHT*

Abstract: — Degradation of the DNA of a rec- mutant of Escherichia coli K12 (JC1569 b) induced by u.v. light was investigated. The rate of degradation was much larger by growing bacteria than by stationary cells. When growing bacteria were starved for amino acids, their DNA became resistant to irradiation. The mode of u.v.-induced degradation was investigated by comparing the time course of release from the acid-insoluble fraction of the label for two growing cultures; the one was pulse-labeled with 3H-thymidine and the other was pulse-labeled and chased thereafter for 12 min. It was found that the label incorporated into the former culture begins to be lost from the acid-insoluble fraction prior to the loss of the label incorporated into the latter culture. It was concluded that breakdown of the replicating point precedes degradation of the bulk of the DNA. This result suggested that the replicating point is a sensitive site to irradiation and the u.v.-induced degradation of DNA seemed to be influenced by the state of chromosome at the time of irradiation. Experiments of centrifugation of lysed spheroplasts of bacteria uniformly labeled with 3H-thymidine in alkaline sucrose demonstrated that DNA of low molecular weight appeared after irradiation with only 5 ergs/ mm2, and that the molecular weight could not be restored by post-irradiation incubation. Considering these results, an hypothesis is proposed concerning the initiation of induced degradation of the DNA of the rec- mutant.

The effects of osmotic lysis on the oxidative phosphorylation and compartmentation of rat liver mitochondria

Abstract: Rat liver mitochondria isolated in 0.25 M sucrose were osmotically lysed with distilled water. The effect of osmotic lysis on mitochondrial compartmentation was monitored by following the changes in the specific Mg(++)-ATPase and the stimulation of this activity by DNP. Each resuspension in distilled water caused a progressive increase in the specific Mg(++)-ATPase and a decrease in DNP-stimulation. Lysed mitochondria yielded P:O ratios of slightly less than 1.0 when each of the "site-specific" substrates, NADH, D-beta-hydroxybutyrate, succinate, and ascorbate, were oxidized. These data indicate that only site 3 phosphorylation remained undiminished. The crude, lysed mitochondria were subfractionated by centrifugation on linear sucrose density gradients. Assays for protein, malate dehydrogenase, D-beta-hydroxybutyrate dehydrogenase, and succinate dehydrogenase indicated that the inner compartment could be clearly separated from the outer membrane vesicles. The results also suggested that the small vesicle fraction contained a small proportion of vesiculated inner membranes. Inner mitochondrial compartments, "contracted" by preincubation in the presence of ATP, sedimented to a markedly lower density on the gradients than did the unincubated preparations and about 50% of the ghosts showed a highly condensed morphology. In the contracted preparations, relatively low malate dehydrogenase and D-beta-hydroxybutyrate dehydrogenase activities were found in the fractions comprised of the inner compartments. The specific activity and distribution of succinate dehydrogenase were about the same as were found with the unincubated, lysed mitochondria.

The adsorption of phage to group N streptococci. The specificity of adsorption and the location of phage receptor substances in cell-wall and plasma-membrane fractions.

Abstract: Summary The specificities of adsorption and multiplication of phages attacking some strains of lactic streptococci (serological group N) were investigated. At multiplicities of infection (m.o.i.) of 0.1 to 1 p.f.u./coccus, the specificity of adsorption was similar to that of multiplication, but some strains also adsorbed one or more heterologous phages. At m.o.i. ≈ 100 both homologous and heterologous strains of streptococci were lysed from without. The specificity of adsorption to cell walls was the same as to whole cocci and, with the exception of 2 phages, was irreversible at 30°. Three types of phage receptors, with different specificities, were recognized in the cell wall. Extraction of streptococci with lipid-solvents did not affect the adsorption of those phages which were irreversibly adsorbed to cell walls but reduced the adsorption of other phages. The plasma membrane of Streptococcus lactis strain ml 3, but not the cell wall, inactivated phage ml 3 but not heterologous phages in the presence of electrolytes. The plasma membrane of a phage-resistant mutant of this strain did not inactivate phage although it was similar in chemical composition to that of the parent strain.

Loss of sensitivity to EDTA by Pseudomonas aeruginosa grown under conditions of Mg-limitation.

Abstract: SUMMARY: Pseudomonas aeruginosa was grown in batch culture in simple salts medium under conditions of Mg-limitation and varying degrees of Mg-excess. Sensitivity to EDTA was measured in terms of lysis and decrease in colony count. The greater the degree of Mg-limitation the greater was the resistance to loss of viability and lysis. Loss of viability of sensitive bacteria occurred more rapidly than lysis. This suggests that bacterial death preceded cell lysis.

Isolation and characterization of three autolytic enzymes associated with sporulation of Bacillus thuringiensis var. thuringiensis

Abstract: Cells of Bacillus thuringiensis containing refractile spores autolyzed readily when suspended in buffer. The autolysate contained enzymes which lysed vegetative cell walls of the organism. Three enzymes were isolated from the autolysate, and each was purified approximately 30-fold. One enzyme, most active near pH 4.0, was found to be an N-acetylmuramidase. The other two enzymes exhibited pH optima at 8.5. One was stimulated by cobalt ions and the other was not. The cobalt-stimulated enzyme was shown to be an N-acetylmuramyl-l-alanine amidase. The cobalt insensitive enzyme exhibited both N-acetylmuramyl-l-alanine amidase and endopeptidase activity. The amidase activity may reflect incomplete separation of the cobalt-stimulated enzyme. The endopeptidase cleaved the peptide bond between l-alanine d-glutamic acid. A cell wall lytic endopeptidase with this specificity has not been previously reported. All three enzymes were extremely limited in the range of bacterial cell walls which they attacked. Except for cell walls of Micrococcus lysodeikticus, which were lysed by the muramidase, only cell walls of members of the genus Bacillus were attacked.

The isolation and properties of the yeast cell vacuole.

Abstract: SUMMARY: When glucose and a chelating agent such as EDTA were added to yeast protoplasts suspended in 8.5% (w/v) mannitol solution at pH 6.4 almost all the protoplasts lysed and the main vacuole was released as a discrete structure from about 75% of the protoplasts. The isolated vacuoles were themselves sensitive to osmotic shock and exhibited vital-staining reactions. They are therefore considered to be relatively undamaged. Some of the properties of the isolated vacuoles are described.

Role of Galactose or Glucose-1-Phosphate in Preventing the Lysis of Streptococcus diacetilactis

Abstract: Cells of Streptococcus diacetilactis DRCI grown at 32 C in media containing glucose as the energy source were osmotically fragile and began to lyse immediately after growth was stopped (by the action of chloramphenicol or the exhaustion of glucose), unless they were then stabilized by hypertonic medium or spermine or by storage at low pH or low temperature, or both. In media containing excess glucose, with growth limited by exhaustion of some nutrient other than the energy source, the appearance of lysis was masked by the occurrence of a balance between lysis and synthesis. The osmotic fragility apparently resulted from inability of the organism to use glucose as an adequate precursor of galactosamine, and conditions of temperature and pH that promoted rapid growth on glucose were particularly conducive to the formation of cells that lysed readily. Growing the organism in media containing galactose, lactose, maltose, or glucose (at 17 C) as energy source resulted in the formation of cells that were resistant to lysis and richer in galactosamine than unstable cells formed on glucose at 32 C. The results indicate that the organism phosphorolyzes maltose to glucose plus β-glucose-1-phosphate, and suggest that it can use the β-glucose-1-phosphate in place of α-glucose-1-phosphate in the formation of cell materials.

Metabolic lysis of yeast protoplasts

Abstract: SUMMARY: Yeast protoplasts which were in osmotic equilibrium with a 0.55 M-mannitol solution underwent lysis when glucose was added to the suspension. Lysis was dependent on glucose metabolism and was accelerated when chelating agents were added to the medium. Lysis in the presence of glucose and chelating agents was inhibitied by alkali metal ions, magnesium, spermidine and by increasing the tonicity of the test medium. The inhibition of lysis by K+ and Na+ when chelating agents were present conformed to a Michaelis-Menten type relationship with apparent dissociation constants of 2–5 × 10–4 M and 3–6 × 10-3 M for K+ and Na+, respectively.

Autolytic Formation of Spheroplasts and Autolysis of Cell Walls in Clostridium botulinum Type A

Abstract: Cells of Clostridium botulinum type A strain 190 harvested at logarithmic growth phase rapidly autolysed in phosphate buffer and most of the cells were converted autolytically into spheroplasts in 0.5 M sucrose-phosphate buffer within 2–3 hr at 37 C. Electron microscope observations on the process of autolysis and spheroplast formation revealed that lysis of the cell wall commenced at one end of the cell and the cytoplasmic contents were released through such lesion. The rod cell was thusly transformed into a fragile spherical form in the hypertonic sucrose-buffer. The lysis of the cell wall proceeded centripetally and finally morphological integrity of the cell wall was completely lost. From these findings it is suggested that the autolysis of the organism is preceded by autodigestion of the cell wall at one end of the cell. A crude cell wall fraction isolated from log-phase cultures by sonication and fractionation rapidly autolysed in phosphate buffer. Reducing sugars and amino sugars of the wall were released from the autolysing wall fraction. Electron microscopy of the residues obtained from wall-autolysates demonstrated that the rigid structure of the wall completely disappeared and only fragile membranous or amorphous fragments remained after autolysis of the crude wall fraction. Heated wall preparations digested with trypsin and nagarse were dissolved by a soluble wall-autolysate, but not by a soluble cytoplasmic fraction. It seems likely that autolytic enzyme(s) may exist at or near the cell wall.

Activation of Isolated Lysosomes by Poliovirus-infected Cell Extracts

Abstract: SEVERAL reports have presented evidence that lysosomes may be involved in the “rapid degenerative type” of cytopathic effect (CPE) caused by certain viruses1–3. Although lysosomal enzymes are apparently able to cause cell damage4, neither the mechanism by which they are released nor their action on the cell during virus infection is understood. Amako and Dales5,6, working with the mengovirus-L cell system, speculated that a cytotoxic protein is synthesized under the regulation of the virus genome, which alters the permeability of the lysosomal membrane and allows the enzymes to leak out. We present here evidence that cell extracts from poliovirus-infected HEp-2 cells release twice the amount of (β-glucuronidase (a lysosomal enzyme) than the non-infected cell extract when exposed to isolated lysosomes at 37° C.