Showing papers on "Lysis published in 1973"
TL;DR: It was demonstrated that the superoxide free radical per se was not the agent causing lysis, and evidence is presented that the free radical causing the lysis of the lysosomes is the hydroxyl free radical (OH·).
644 citations
TL;DR: The fatty acid composition and cholesterol content of the membrane lipids of Acholeplasma laidlawii B were systematically altered and the rates at which glycerol and erythritol passively diffuse into intact cells and into liposomes prepared from the total membrane lipid were measured.
179 citations
TL;DR: Carotenoids incorporated into the liposomal membrane protect against both lipid peroxidation and Liposomal lysis, and other 1O2 quenchers and free radical absorbers also protect liposomes from photodynamic damage.
Abstract: — When liposomes (a model membrane system) are subjected to a dye-sensitized photo-oxidation, lysis, as measured by glucose leakage or a change in light scattering, results. Before lysis occurs, the membrane lipids undergo peroxidative damage, as determined by the appearance of malondialdehyde. Carotenoids incorporated into the liposomal membrane protect against both lipid peroxidation and liposomal lysis. Other 1O2 quenchers and free radical absorbers also protect liposomes from photodynamic damage.
175 citations
TL;DR: The response of protoplasts of Bacillus megaterium relative to those of Micrococcus lysodeikticus reflected differences in bactericidal sensitivity though whole cells were much less sensitive to fatty acid-induced leakage effects than protoplast.
Abstract: Summary Fatty acids of chain length > C10 induced lysis of protoplasts at pH 7·4 when the concentration was nearly bactericidal At pH 6, lauric and linoleic acids produced lysis above bactericidal concentrations but, at pH 8, lysis was produced by the same acids below bactericidal concentrations The lysis was immediate at pH 8, but at pH 6 the effect was preceded by contraction of protoplasts At pH 7·4 the order of lytic activity between individual fatty acids was similar to that of bactericidal activity and the response of protoplasts of Bacillus megaterium relative to those of Micrococcus lysodeikticus reflected differences in bactericidal sensitivity though whole cells were much less sensitive to fatty acid-induced leakage effects than protoplasts Reversal agents antagonized the lysis of protoplasts by fatty acids A physicochemical basis for the action of fatty acids and reversal agents on protoplasts and whole cells is discussed
154 citations
TL;DR: The possibility of resolving the components in a natural enzyme mixture, yeast lysate, by counter-current distribution in this type of biphasic system has been investigated and several glycolytic enzymes are resolved.
Abstract: Proteins have been partitioned in dextran–poly(ethylene glycol)–water biphasic systems containing positively charged poly(ethylene glycol). The effect of pH, buffer concentration, polymer concentration, protein concentration and temperature on the partition has been investigated. Characteristic extraction profiles are obtained when the percentage of protein in the upper phase is plotted versus pH. By keeping the salt concentration low, very steep extraction profiles can be obtained. Thus, within an interval of one pH unit, a protein is transferred from one phase to the other. This pH-interval is located close to the isoelectric point of the protein.
The possibility of resolving the components in a natural enzyme mixture, yeast lysate, by counter-current distribution in this type of biphasic system has been investigated. According to activity measurements, several glycolytic enzymes are resolved by this procedure. Good separation is achieved. For some of the enzymes the activity is split into more than one peak in the counter-current distribution.
108 citations
TL;DR: A 14S RNA species was purified to a single peak by successive sucrose gradient centrifugations, followed by acrylamide gel electrophoresis, and it seems to code for a precursor protein slightly larger than the light chain.
Abstract: Polysomes released from microsomes of MOPC 41 mouse myeloma were used to prepare a poly(A)-containing fraction of RNA by chromatography on poly-(dT)-cellulose. From that fraction, a 14S RNA species was purified to a single peak by successive sucrose gradient centrifugations, followed by acrylamide gel electrophoresis. The RNA has an apparent molecular weight of 380,000 (1100 nucleotides), as estimated from the electrophoretic analyses. In a reticulocyte lysate this RNA directs the synthesis of a protein that migrates more slowly in sodium dodecylsulfate-acrylamide gels than does the light chain secreted by the same tumor. This difference in migration corresponds to a size difference appropriate for polypeptide chain about 20 amino acids longer than the light chain. The tryptic peptides of this protein correspond to those of the secreted light chain, except for the presence of two additional peptides from the product synthesized in vitro and for the absence of one light-chain peptide. The purified RNA is, therefore, the mRNA of the light chain, and it seems to code for a precursor protein slightly larger than the light chain. From the estimated size of the 14S mRNA, it appears that only 65% of the RNA is translated.
105 citations
TL;DR: The hypothesis formulated previously, that activating factor and zymogen reside in different organelles, has been substantiated and yeast vacuoles were obtained in good yield and practically free from other organlles.
80 citations
TL;DR: The infection of Acholeplasma laidlawii cells by MVL 51 virus has been studied, MVL51 is a bullet-shaped virus, about 14 nm wide and 71 nm long, and progeny virus assembly and maturation occur at the cell membrane as the viral particles are extruded.
61 citations
TL;DR: The results support the thesis that S. citri does not possess a cell wall, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily, and the organisms were found to be extremely sensitive to lysis by osmotic shock.
Abstract: 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.
58 citations
Journal Article•
TL;DR: The effector-dependent action on the target cell is completed much earlier than evidenced by chromium-51 release, and subsequent, effectorindependent steps must occur before completion of target cell lysis.
Abstract: Heparin was previously shown to inhibit the lysis of target cells by sensitized thymus-derived mouse lymphocytes in vitro. However, heparin failed to inhibit target cell lysis when added to the cultures after only 10% specific release of chromium-51 had occurred. For further exploration of this phenomenon, effector cells have been specifically inactivated at various times during their action on target cells. This was accomplished with alloantiserum made in target strain mice (DBA/2) against effector strain cells (C57BL/6). Treatment of effector cells with this antiserum and complement rendered them unable to initiate new cytolytic events on target cells (and incidentally did not release soluble toxins). However, when untreated cultures were incubated for about 60 min, after which the effector cells had caused 10% specific release of chromium-51 from the target population, treatment of the cultures with anti-effector serum and complement did not interfere with the subsequent lysis of more than half of the target cell population. Hence, the effector-dependent action on the target cell is completed much earlier than evidenced by chromium-51 release, and subsequent, effectorindependent steps must occur before completion of target cell lysis. Data suggest that divalent cations are required for the effector-dependent phase, but not for the effector-independent phase.
57 citations
TL;DR: The yeast plasma membrane fraction, which accounted for approximately 25% of the total protein content of the membrane preparation, was found to consist of two major fractions with respective buoyant densities, and the ease of release into the soluble fraction precluded its use as a satisfactory marker enzyme.
TL;DR: Bacterium CP-I isolated from Scottish waters is an aerobic prokaryote with a high guanine: cyto-sine ratio (68%), which causes lysis of a variety of blue-green algae.
Abstract: Summary
Bacterium CP-I isolated from Scottish waters is an aerobic prokaryote with a high guanine: cyto-sine ratio (68%), which causes lysis of a variety of blue-green algae. When suspensions of algae and bacteria are mixed the bacteria can be seen to glide towards the algae where they become attached end-on round the algal cells. There is evidence that the bacteria produce substances at the point of contact with the algae which are necessary for algal lysis to occur. Lysis of vegetative cells may occur within 30 minutes of the bacteria becoming attached. Heterocysts do not lyse in this way although their intracellular contents may eventually degenerate. When vegetative cells rupture, the protoplasmic contents, including gas vacuoles when present, are released and empty-looking ‘ghosts’ which retain the original cell shape may remain for a time although these too eventually break down. There is some evidence that the mode of filament lysis may be random, intercalary or terminal.
At the ultrastructural level the initial effect of CP-I is to cause lysis of the L2 layer of the algal cell wall and this effect is similar to that obtained using lysozyme. This is followed by rupture or disintegration of the other cell wall layers, and ‘scroll’-like structures, either of coiled up cell wall material (Microcystis) or of the plasmalemma (Nostoc) may be found scattered throughout the preparations of lysed cells. In fully lysed cells there is a loss of polyphosphate bodies, structured granules, gas vacuoles, and most other cellular inclusions. Released gas vesicles can be collected from the surface of the solution of lysed cells, and there is evidence that these vesicles may fracture easily near the conical ends. In fully lysed cells, the thylakoid membranes remain, often with wide lumens between. This thylakoidal system is contorted and branched, ramifies through the cell and shows constrictions of the membrane system at intervals. Continuity between the plasmalemma and the thylakoidal system can be seen clearly, Lipid droplets which are resistant to CP-I treatment are found in the lumen of the thylakoidal system. Heterocysts are not as markedly affected by CP-I as are vegetative cells, but eventually heterocyst disorganization also occurs. The use of CP-I provides a technique for preparing material for the study of algal membranes and gas vesicles.
TL;DR: Thermoplasma acidophilum grows optimally under aeration at 59 C and pH 2, and both intact cells and membranes disaggregate below pH 1 and above pH 5, producing no sedimentable particles.
Abstract: Thermoplasma acidophilum grows optimally under aeration at 59 C and pH 2. Both intact cells and membranes disaggregate below pH 1 and above pH 5, producing no sedimentable particles. Increase in ionic strength at pH 5 or below results in cellular lysis and membrane disaggregation. Membranous components produced by lysis at alkaline pH reaggregate upon reduction of both pH and ionic strength. Osmotic environment plays little role in cellular stability. Membranes prepared by sonic lysis at pH 5 exhibit vesicular structures and are composed of multiple proteins. Although the amino acid composition of the membrane proteins is similar to other mycoplasmal membranes, the number of free amino and carboxyl groups is less than half of those in Acholeplasma. Reduction of the number of free carboxyl groups results in membrane stabilization over a wide range of pH. Increase in the number of free amino groups reverses the stability of membranes relative to pH. Acidophily in Thermoplasma can be related to a significant reduction in repulsing negative charges on the membrane proteins.
TL;DR: The results indicate that high-molecular-weight single-stranded DNA can be isolated when certain basic proteins are present at the time of lysis, in the absence of such protective agents as lysozyme or cytC.
Abstract: The deoxyribonucleic acid (DNA) extraction procedure of Piechowska and Fox was evaluated to determine which steps are required for the isolation of high-molecular-weight single-stranded material from transformed cultures of Bacillus subtilis. The results indicate that high-molecular-weight single-stranded DNA can be isolated when certain basic proteins are present at the time of lysis. In the absence of such protective agents as lysozyme or cytC the single-stranded DNA is degraded. The single-stranded DNA can also be protected by being treated with lysozyme at low temperature. The high molecular weight of this single-stranded material and its kinetics of appearance are consistent with its being an intermediate in the transformation process.
TL;DR: In this paper, a rapid method for the isolation of plasma membranes from normal lymphoid and acute lymphoblastic leukaemic cells of AKR mice was developed, which entailed lysis in a hypotonic medium containing borate and EDTA from which a fraction enriched in surface membrane ghosts was obtained by centrifugation.
TL;DR: It is concluded that antibody and complement produce surface rings, prelytic leakage of K+, colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.
Abstract: It is known that there are 100 A-wide circular structures associated with the erythrocyte membrane in immune lysis. To determine whether these structures were functional holes extending through the membrane, freeze-etch electron microscopy was carried out. Sheep erythrocytes incubated with either rabbit complement or rabbit antibody (anti-sheep erythrocyte antibody) did not hemolyze and did not reveal any abnormalities in freeze-etch or negative-stain electron microscopy. Erythrocytes incubated with both complement and antibody revealed rings on the extracellular surface (etch face) of the cell membrane. Allowing for the 30 A-thick Pt/C replica, the dimensions of the surface rings were similar to those seen by negative staining. The ring's central depression was level with the plane of the membrane; some rings were closed circles, others were crescent shaped. The cleavage face of the extracellular leaflet revealed globule aggregates, each aggregate appearing to be composed of about four fused globules. The cleavage face of the cytoplasmic leaflet was normal. When immune lysis was carried out in the presence of ferritin, ferritin was subsequently detected in all lysed erythrocytes. If ferritin was added after immune lysis was complete, only 15% of the cells were permeated by ferritin, indicating that transient openings exist in the cell membrane during immune lysis. No abnormal structures were detected when C6-deficient rabbit serum was used as a source of complement. It is concluded that antibody and complement produce surface rings, prelytic leakage of K+, colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.
TL;DR: The abundance and similarity in chemical and physical properties of protein bound to all forms of polyoma DNA suggest that part of the protein molecules may serve in a structural capacity in both r-py complexes and py complexes.
Abstract: Short-lived nucleoprotein complexes (r-py complex) containing replicating polyoma DNA were isolated from infected cells after lysis with Triton X-100. The Triton lysing procedure of Green, Miller, and Hendler (1971) releases most complexes containing supercoiled viral DNA (py complex) from nuclei, but liberates only a portion of r-py complexes. r-py Complexes are associated more strongly with nuclear sites but can be extracted by prolonged incubation of nuclei in lysing solution. Complexes containing replicating polyoma DNA appear to be precursors to stable complexes containing supercoiled DNA. Sedimentation and buoyant density studies indicate that protein is bound to both r-py complexes and py complexes at a ratio of protein to DNA of about 1 to 2/1. Both types of complexes sediment as if the viral DNA is more compact than free DNA and both undergo major reversible configurational changes with increased salt concentration. Changes resulting from enzymatic and chemical treatment indicate that there may be two or more protein components in both r-py complex and py complex. One component is digested by Pronase and trypsin while another is resistant to the enzymes but released by deoxycholate. The abundance and similarity in chemical and physical properties of protein bound to all forms of polyoma DNA suggest that part of the protein molecules may serve in a structural capacity.
Journal Article•
TL;DR: It has been found that, both on lecithin and on sphingomyelin-cholesterol liposomes, marker release and characteristic EM lesions can be found upon the sequential activity of C567 and C8 and C9 and that for both these parameters of lysis the action of C7 and C7 as well as of C9 are required.
Abstract: Experiments are described using the reactive lysis system on phospholipid dispersions to study the terminal phases of complement lysis. It has been found that, both on lecithin and on sphingomyelin-cholesterol liposomes, marker release and characteristic EM lesions can be found upon the sequential activity of C567 and C8 and C9 and that for both these parameters of lysis the action of C8 and C9 as well as of C567, are required.
Lysis of liposomes is accompanied by the appearance of traces of the products of phospholipase activity in the supernate but it is believed that these represent the activity of contaminating enzymes and are the consequence and not the cause of lysis. However the possibility that lysis could be due to lipolytic activity cannot be totally excluded because of the negligible hydrolysis theoretically needed to form lesions.
TL;DR: The specific catalytic activity of the lysozyme‐polyacrylamide system is found to decline as the density of derivatized surface groups is increased; a model of protein deactivation due to excess surface coupling is presented as a possible rationale for such specific activity variations.
Abstract: Lysozyme immobilized on polyacrylamide beads or cellulose fibers is found to retain activity for hydrolysis of the cell walls of Micrococcus lysodeikticus. The immobilization on cellulose is somewhat reversible; the polyacrylamide immobilized lysozyme does not release any enzyme upon washing as evidenced by UV and lytic activity tests. The specific catalytic activity of the lysozyme‐polyacrylamide system is found to decline as the density of derivatized surface groups is increased; a model of protein deactivation due to excess surface coupling is presented as a possible rationale for such specific activity variations.
TL;DR: The bulk DNA in bleomycin-sensitive ascites hepatoma, AH66, was more susceptible to single strand scission by this antibiotic than that in the resistant AH66F cells, which suggests induction of breakage by the antibiotic of an alkali-labile linkage in the chromosomal DNA.
Abstract: The bulk DNA in bleomycin-sensitive ascites hepatoma, AH66, was more susceptible to single strand scission by this antibiotic than that in the resistant AH66F cells. The break was repairable in the resistant cells, while it was incompletely repaired in the sensitive cells. Rejoining was inhibited by actinomycin D but not by cycloheximide. The sedimentation coefficient of bulk DNA was reduced with increasing lysis time of the cells in alkali-sodium dodecyl sulfate before sucrose gradient centrifugation, while that of damaged DNA in bleomycin-treated cells did not change further under the same conditions. This suggests induction of breakage by the antibiotic of an alkali-labile linkage in the chromosomal DNA.
TL;DR: The localization of alkaline phosphatase in the specific granules of rabbit polymorphonuclear leukocytes was investigated and the results obtained suggest very strongly that alkalineosphatase is a component of the granule membrane.
Abstract: The localization of alkaline phosphatase in the specific granules of rabbit polymorphonuclear leukocytes was investigated. The results obtained suggest very strongly that alkaline phosphatase is a component of the granule membrane. The enzyme remains attached to the membrane upon disruption of the granules by the use of detergents or by hypotonic shock and subsequent extraction with sodium sulfate, and can be isolated together with fragments of the granule membrane by isopycnic equilibration. Treatment of the granules with high amounts of Triton-X-100, sodium deoxycholate, or hexadecyltrimethylammonium bromide releases the enzyme in soluble form. In polymorphonuclear leukocyte homogenates, lysis of the granules is needed in order to render alkaline phosphatase fully accessible to substrates. This suggests that the catalytic site of the enzyme is exposed at the inner face of the granule membrane.
TL;DR: The retarded dissociation of the 125 I-labeled insulin bound to the plasma membranes of these vesicles suggests that at 24 °C “flip-flop” of the insulin-receptor complex to the cytoplasmic surface of the membrane occurs at a very slow or negligible rate.
TL;DR: The endonucleolytic activity from an extract of Micrococcus luteus is used to detect small amounts of damage in the DNA of bacteria exposed to ultraviolet light or γ rays.
TL;DR: Results indicate the presence of at least one RNA region in closed circular double stranded mitochondrial DNA in whole rat ascites hepatoma cells.
TL;DR: The autolytic N-acetylmuramidase present in Lactobacillus acidophilus strain 63 AM Gasser has an optimal pH between 5 and 6 when lysing intact cells or isolated cell walls, and it seems that sulfhydryl groups are required for both cell and wall autolysis.
Abstract: The autolytic N-acetylmuramidase present in Lactobacillus acidophilus strain 63 AM Gasser has an optimal pH between 5 and 6 when lysing intact cells or isolated cell walls. Cellular lysis at pH 5 is two to four times more rapid in citrate buffer of 0.01 M and 0.5 M or higher than in 0.1 M acetate buffer. It seems that sulfhydryl groups are required for both cell and wall autolysis. Heavy metal ions and p-chloro-mercuribenzoate, at low concentrations, are powerful inhibitors. Ethylenediaminetetraacetic acid stimulates cellular but not wall autolysis in acetate buffer to the level obtained in citrate buffer. The possible involvement of sulfhydryl groups in a mechanism of control of cellular autolytic activity is discussed. The autolytic enzyme, although unstable in solution at 37 C, can be extracted from walls by the use of solutions of bovine serum albumin (100 μg/ml) in 0.01 N NaOH. Soluble enzyme extracted from walls rebinds on to sodium decylsulfate-treated walls, but three times as much of the wall material is required to completely re-adsorb the activity.
TL;DR: A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells.
Abstract: The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.
TL;DR: The results have been interpreted as indicating that Na+ prevented the cell lysis by providing the sufficient mechanical strength of the envelope against mechanical stress and that K+ prev...
Abstract: In preventing the lysis of a slightly halophilic marine Vibrio alginolyticus, Na+ acted cooperatively with Mg2+, while K+ counteracted the effect of Na+ and Mg2+. When the cells were washed with a ...
Journal Article•
TL;DR: It is concluded that lysis of EAC1–8 results from a single membrane lesion caused by 1 molecule of C9, and the reaction is not enzymatic.
Abstract: The generation of EAC1–9 from EAC1–8 by reaction with C9 proceeds without time lag and the initial reaction velocity is directly proportional to the concentrations of C9 and EAC1–8. These characteristics, as well as the low temperature sensitivity, indicate that the reaction is not enzymatic. The subsequent lysis of EAC1–9 is highly sensitive to temperature, the Q 10 being approximately 2. Rabbit anti-guinea pig C9 blocks the reaction of C9 with EAC1–8, but does not interfere with lysis of EAC1–9. Input of 1 to 2 molecules of C9 suffices for lysis of 1 EAC1–8. The rate of lysis of EAC1–9 is proportional to the concentration of C9 per cell. The dose-response curve in the titration of C9 follows a monotonic course concave to the abscissa representing the concentration of C9. It is concluded from these results that lysis of EAC1–8 results from a single membrane lesion caused by 1 molecule of C9.
TL;DR: Plasma membranes were isolated from murine plasmocytoma cells in culture by a procedure involving lysis in hypoosmotic medium leaving the nuclei intact, and separation of surface membranes from the lysate constituents on a discontinuous sucrose gradient.
TL;DR: The exchange of chloride versus hydroxyl ions coupled to the buffering action of hemoglobin proved to deliver the most important contribution to the differences in the osmotic resistance in sucrose and in the lysis rate in glycerol solutions.