Showing papers on "Lysis published in 1970"

Reactive lysis: the complement-mediated lysis of unsensitized cells : ii. the characterization of activated reactor as c56 and the participation of c8 and c9

Abstract: It has been shown that the "activated reactor" that is produced in certain human sera by complement activation is a stable complex of the fifth and sixth component of complement (C56). On interaction with C7, the indicator factor, a complex C567 is formed which for a short time (half-life less than 1 min) has an activated binding site and can attach itself to normal red cell membranes, conferring on them the hemolytic properties of the "heat stable" complement intermediate EC 1 ∼ 7, the capacity to be lysed by C8 and C9. These cells have neither antibody nor the complement components up to C3 bound on them. The binding site—activated C567c—can similarly bind to other hydrophobic surfaces, including agarose gel where it forms a "stainable line". If the complex is not bound to a surface, the binding site decays and the resulting complex will no longer give rise to lysis. However it will still inactivate C8 and C9 in solution. The sera that can generate activated reactor apparently do so because they have an excess of C5 and C6, compared to their content of C7. The phenomenon of reactive lysis thus represents complement-mediated lysis of unsensitized cells initiated at the C5 stage by a stable complex (C56) which was generated by complement activation at a distance. The immunochemistry of the phenomenon is described and some of its implications discussed.

Chemical Composition of Wild-type and Mutant Aspergillus nidulans Cell Walls. The Nature of Polysaccharide and Melanin Constituents

Abstract: SUMMARY Chitin and a β-linked glucan were the major chemical components of Aspergillus nidulans cell walls. Other monomeric residues identified in enzymic and acid hydrolyses of whole cell walls and cell-wall fractions included galactose, mannose, glucuronic acid and galactosamine. The β-glucan contained (1 → 3) and (1 → 6) linkages and was two-thirds digested by an exo-β-D-1,3 glucanase prepared from a cell-wall lysing Streptomyces species. An α-glucan was identified as a cell-wall component and it also contained (1 → 3) linkages. This latter polysaccharide was distinguishable from nigeran (an α-1,3; α-1,4 glucan present in other Aspergillus species) by infrared spectroscopy and by its low susceptibility to hydrolysis by an endo-α-1,3; α-1,4 glucan glucanohydrolase. Both glucans were alkali-soluble, but the β-glucan was completely solubilized only after acid extraction of the wall. The N-acetylglucosamine to galactosamine ratio in the A. nidulans cell wall was 1.32 and the two hexosamines were shown to be constituents of distinct polymers. The remaining cell wall was accounted for by protein, lipids, readily extractable and bound, and, in the wild-type, melanin. The melanin was distributed throughout the cell wall but was associated particularly with the chitin fraction. The pigment has been partially characterized chemically and contains indolic residues; this result does not substantiate earlier views that indolic melanins are peculiar to the animal kingdom. Melanin appears to be a finite heteropolymer both in terms of its molecular size and its chemistry.

Lysis of erythrocytes by complement in the absence of antibody

Abstract: A new pathway of complement-mediated hemolysis has been described. It is independent of antibody and does not require binding of the first four complement components to the target-cell surface. The actual attack of the target cell begins with the attachment of C5, C6, and C7. The binding reaction is catalyzed by C4, 2, 3, an enzyme which may be formed in cell-free solution. C4, 2, 3 may effect binding of C5, 6, 7 by acting from the fluid phase or from the surface of another cell to which it is specifically bound (EAC 4, 2, 3). In either case, the resulting product is EC5, 6, 7 which is susceptible to lysis by C8 and C9. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were particularly susceptible to lysis by the above described mechanism. PNH cells, but not normal human erythrocytes, could also be lysed through activation of complement by cobra factor. These observations allow the operational distinction of an activation and an attack mechanism of complement.

Structure and composition of resistant layers in bacterial spore coats.

Abstract: SUMMARY: Treatment with mercaptoethanol caused spores of various bacteria to become sensitive to lysis by lysozyme, a spore enzyme and hydrogen peroxide; further treatment with alkali caused greater sensitization to these lytic agents. Alkali removed a protein from the outer coats of mercaptoethanol-treated spores. The protein from Bacillus cereus, B. coagulans and Clostridium sporogenes contained high levels of acidic and basic amino acids; that from B. coagulans contained a high proportion (over 7% of its residues) of tyrosine. Electron microscopy of thin section and freeze-etch samples showed that the alkali-soluble protein contributed to a striking banding pattern containing 57 A spaced fibres on the outer surface of B. coagulans spores. This layer may function as a protection against the enzymes of potential predators.

Association of the Bacillus subtilis Chromosome with the Cell Membrane: Resolution of Free and Bound Deoxyribonucleic Acid on Renografin Gradients

Abstract: Linear density gradients of Renografin have resolved two components of bacterial deoxyribonucleic acid (DNA) in sheared lysates. Component 1, at equilibrium density after 5 hr of centrifugation, is enriched for newly synthesized DNA and markers near the origin and terminus of replication. It contains 5% of total cellular protein, 25% of the phospholipids, 30 to 50% of the DNA, 4 to 11% of unstable ribonucleic acid (RNA), RNA polymerase, and low amounts of DNA polymerase. The material is sensitive to Pronase and Sarkosyl. In unsheared lysates, all of the DNA forms a band at this position. Shearing the lysate generates a slow-sedimenting fraction of DNA (component 2) which contains more uniformly labeled than newly synthesized DNA. These observations suggest that replicating DNA and DNA at the origin and possibly the terminus of replication are associated with membrane. The amount of uniformly labeled DNA in component 1 and an estimate of the number of chromosomal fragments suggest that other parts of the chromosome are possibly associated with the membrane.

Lysis of grouped and ungrouped streptococci by lysozyme.

Abstract: Thirty strains of streptococci were tested for lysis with lysozyme, and 29 of these could be lysed by the following method: (i) suspension of the cells to a Klett reading of 200 units (no. 42 filter) in 0.01 m tris(hydroxymethyl)aminomethane buffer, pH 8.2, after washing twice with the buffer; (ii) addition of lysozyme to a final concentration of 250 μg/ml with incubation for 60 min at 37 C; (iii) addition of sodium lauryl sulfate (SLS) to a final concentration of 0.2% and incubation up to an additional 15 min at 37 C. Significant lysis was obtained only after the addition of SLS. (Strains of groups A, E, and G were treated with trypsin at a concentration of 200 μg/ml for 2 hr at 37 C before exposure to lysozyme.) These parameters for optimal lysis of streptococci by lysozyme were established by testing the group D Streptococcus faecalis strain 31 which lyses readily with lysozyme and the group H strain Challis which is less susceptible to the action of the enzyme. Viability of S. faecalis decreased 96% after 3 min of exposure to 250 μg of lysozyme per ml, whereas the more resistant strain Challis retained 27% of the initial viability after the same period. After 60 min, there was almost total loss of viability in each case. Variations of three methods of lysing streptococci with lysozyme were compared with respect to the decrease in turbidity and the release of protein and deoxyribonucleic acid (DNA) effected by each variation. The method presented in this paper allowed the greatest release of these cytoplasmic constituents from S. faecalis and strain Challis. Transformation experiments using DNA obtained from strain Challis (streptomycinresistant) by this method showed that the DNA released is biologically active.

The separation of β-glucanases produced by Cytophaga johnsonii and their role in the lysis of yeast cell walls

Abstract: 1. When Cytophaga johnsonii was grown in the presence of suitable inducers the culture fluid was capable of lysing thiol-treated yeast cell walls in vitro. 2. Autoclaved or alkali-extracted cells, isolated cell walls and glucan preparations made from them were effective inducers, but living yeast cells or cells killed by minimal heat treatment were not. 3. Chromatographic fractionation of lytic culture fluids showed the presence of two types of endo-beta-(1-->3)-glucanase and several beta-(1-->6)-glucanases; the latter may be induced separately by growing the myxo-bacterium in the presence of lutean. 4. Extensive solubilization of yeast cell walls was obtained only with preparations of one of these glucanases, an endo-beta-(1-->3)-glucanase producing as end products mainly oligosaccharides having five or more residues. Lysis by the other endo-beta-(1-->3)-glucanase was incomplete. 5. The beta-(1-->6)-glucanases produced a uniform thinning of the cell walls, and mannan-peptide was found in the solution. 6. These results, and the actions of the enzyme preparations on a variety of wall-derived preparations made from baker's yeast, are discussed in the light of present conceptions of yeast cell-wall structure.

The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture

Abstract: Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-(3)H]uridine and the cellular DNA with [2-(14)C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-(14)C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.

Effect of the thiol-oxidizing agent diamide on the growth of Escherichia coli.

Abstract: Oxidation of glutathione within Escherichia coli cells by diamide, (CH3)2NCON=NCON(CH3)2, stops growth but does not cause cell death. Normal growth rates are resumed after periods which vary in length according to the diamide concentration. Consumption of excess reagent with added glutathione quickly reverses the inhibition. Another thiol-oxidizing agent, azoester, C6H5N=NCOOCH3, causes lysis.

Isolation, Characterization, and Immunogenicity of Mycoplasma pneumoniae Membranes

Abstract: Membrane and soluble fractions of Mycoplasma pneumoniae, M. pulmonis, and M. laidlawii B were prepared by hypotonic lysis of whole cells. The membranes of M. pneumoniae and M. laidlawii B contained, as percentage of dry weight: 34 to 37% protein, 59 to 61% lipid, 3 to 4% carbohydrate as hexose, and 0.2% ribonucleic acid as ribose. NADH2 and NADPH2 oxidase activities were localized in the soluble fractions of M. pneumoniae and in the membrane fraction of M. laidlawii B. NADH2 oxidase activity was localized in the soluble fraction of M. pulmonis. The lipids of M. pneumoniae were labeled when the organism was grown in the presence of either radioactive palmitic acid, oleic acid, cholesterol, or glycerol. The lipids were not labeled when grown in the presence of radioactive acetate. Palmitic acid radio-activity was found in neutral lipid, glycolipid, and phosphatide fractions. Immunodiffusion analyses of whole cells and membrane fractions demonstrated three reactive antigens. Two immunodiffusion antigens were localized in the membrane fraction. One of these apparently contains lipid. A third antigen, also considered lipoidal, was found in whole cells. Membrane and soluble fractions of M. pneumoniae were immunogenic. The immunogens eliciting metabolic-inhibiting antibodies were localized in the membrane. The membrane preparation also induced the formation of antibodies which fixed complement with an antigen extracted with lipid solvent. The soluble fraction contained a distinct immunogen which induces antibodies reactive in complement fixation with an antigen prepared by phenol extraction.

Early Intracellular Events in the Replication of Bacteriophage T4 Deoxyribonucleic Acid VI. Newly Synthesized Proteins in the T4 Protein-Deoxyribonucleic Acid Complex

Abstract: Shortly after infection of Escherichia coli B with T4 phage, the phage deoxyribonucleic acid (DNA) can be isolated as a fast-sedimenting, proteinaceous complex. Formation of the complex is inhibited by the addition of chloramphenicol between 3 and 4 min after infection, suggesting that phage-coded proteins are necessary to form the complex and may contribute to its structure. To determine whether the phage DNA is associated with a random collection of proteins after infection or whether the complex contained a specific set of proteins, total protein from phage-infected cell lysates was compared to complex protein isolated from similar lysates by gel acrylamide electrophoresis. The proteins obtained from complexes exhibited a distinctly different pattern of separation, indicating that the complex contained a specific set of those proteins newly synthesized after infection. The proteins of the complex appear to be associated directly with the DNA rather than with some other component which could impart the characteristic of fast sedimentation to the complex. Fast-sedimenting complexes were isolated from a 3H-leucine-labeled cell lysate. Part of this material was treated with pancreatic deoxyribonuclease. Deoxyribonuclease-treated and untreated complexes were resedimented in sucrose gradients. Virtually all the untreated complex remained fast-sedimenting, whereas most of the 3H-leucine label of the deoxyribonuclease-treated material was located toward the top of the gradient. These data suggest a direct association of DNA and protein in the complex.

Effect of Bactericidal Substance from Staphylococcus aureus on Group A Streptococci I. Biochemical Alterations

Abstract: A bactericidal substance isolated from phage type 71 staphylococci has been studied relative to its mechanism of action on streptococcal cells. The substance exerts its effect best at 37 C. No cell lysis occurs as evidenced by lack of alteration in optical density of susceptible cell suspensions. The bactericidal substance results in immediate cessation of protein and deoxyribonucleic acid synthesis as well as degradation of newly and previously formed ribonucleic acid (RNA). The action on RNA seems to be independent of protein synthesis.

Diffusion, permeation, or enzyme limitation: a probe for the kinetics of enzyme induction.

Abstract: The utilization of an exogenous substrate by enzyme inside a bacterial cell can be limited by diffusion up to the cell, penetration of the cell, diffusion within the cell, and/or attack by internal enzyme. For small molecular weight substrates such as galactosides, and for bacteria such as Escherichia coli the diffusion steps are not rate limiting even with the permeases fully induced and the external concentration of substrate low. In permeaseless organisms with more than about 20 enzyme molecules per cell, permeation of O-nitrophenyl-β-D-galactoside through the membrane is limiting. Thus, a single initiation of transcription of a lactose message suffices to yield enough enzyme molecules to switch an uninduced cell from enzyme limitation to permeability limitation. Subsequent initiations change the cellular activity very little. This transition can be followed by assaying enzyme activity of both intact and lysed cell suspensions. In this way the induction response amongst cells in growing populations at high inducer concentrations has been found to be uniform. It was found that nearly all of the cells from balanced growing culture are immediately inducible even with doubling times as short as 7.6 hrs. At 24 hrs about 1/3 of the cells are inert at any time, but all cells synthesize enzyme within a 3-hour period. At low inducer concentration or in the present of catabolite repressor the rate of initiation is greatly decreased; this leads to a non uniform distribution of enzyme within the cells, which is readily detected by the experimental technique. In addition to developing the kinetics for enzyme contained in cells, the distribution of the enzyme among uninduced bacteria is presented.

Localization in the Cell and Extraction of Alkaline Phosphatase from Bacillus subtilis.

Abstract: Study of protoplasts, lysed protoplasts, and cells treated with lysozyme in the absence of osmotic stabilizer suggested that the alkaline phosphatase (EC 3.1.3.1.) of Bacillus subtilis is located in the protoplasmic membrane. Cytochemical evidence in support of this view is presented. The enzyme protein was strongly bound to the membrane structure and could not be solubilized by a number of treatments known to release enzymes from membranes and other lipoprotein structures. Alkaline phosphatase was, however, solubilized by treatment of intact B. subtilis cells or isolated protoplasmic membranes with strong salt solutions at pH 7.2, suggesting that electrostatic forces are responsible for the association between membrane and enzyme protein. Dialysis of alkaline phosphatase solutions against buffer of low ionic strength resulted in precipitation of the enzyme.

Osmotic Properties of Spheroplasts from Saccharomyces cerevisiae Grown at Different Temperatures

Abstract: Spheroplasts were prepared from cells of Saccharomyces cerevisiae NCYC 366, grown at 30 or 15 C, by incubating cells with snail-gut juice after pretreatment with 2-mercaptoethanol. Walls of cells grown batchwise or in continuous culture at 15 C were more resistant to digestion with snail juice than walls on cells grown under the same conditions as 30 C. Spheroplasts lysed when suspended in hypotonic solutions of mannitol. The resistance of spheroplasts to osmotic lysis tended to increase when the test temperature was lowered below 30 C. The increased resistance was greater with spheroplasts from cells grown at 15 C. Cations, especially Ca2+, protected spheroplasts against osmotic lysis. In general, the protective effects, measured at 30 C, were smaller with spheroplasts from cells grown at 15 C compared with 30 C. Citrate and ethylenediaminetetraacetate (EDTA) decreased the resistance of spheroplasts to osmotic lysis. On the whole, the decrease was greater with spheroplasts from cells grown at 30 C rather than 15 C. In the presence of EDTA, spheroplasts from cells grown at 30 C were less resistant to osmotic lysis at 5 C than at 30 C; when spheroplasts from cells grown at 15 C were similarly examined, they were more resistant to lysis at 5 C than at 30 C. Spheroplast membranes from cells grown at 15 C had slightly but significantly greater contents of Mg2+, Ca2+, K+, and Na+ compared with spheroplast membranes from cells grown at 15 C. Mg2+ and Ca2+ were more easily extracted with EDTA from membranes of 30 C-grown cells than from 15 C-grown cells.

Cell walls of pirigularia oryzae

Abstract: Cell walls were prepared from Piricularia oryzae P2. Lytic enzymes were produced when Bacillus circulans WL 12 was grown with P. oryzae cell wall as a sole source of carbon. Mode of lysis of P. oryzae cell walls was compared with the lysis of walls of Aspergillus oryzae and Neurospora crassa. Activities of β-1→3-glucanase, chitinase, and β-1→6-glucanase were demonstrated in the culture filtrate. Modes of lysis of cell walls of P. oryzae P2 by a single and combined action of lytic enzymes were compared. Combination of β-1→3-glucanase and chitinase gave the most rapid and extensive lysis.

Effects of antibodies on L5178Y mouse leukemia cells cultured in vitro.

Abstract: Summary An in vitro system with the use of a rabbit serum medium-adapted subline of L5178Y cells and rabbit antisera was used in the present study. Heat-inactivated antisera inhibited both uptake and incorporation of glycine-2- 14 C into cell protein and killed the cells. These effects became demonstrable only after 6 hr and reached a maximum after 24 to 48 hr. Total cell counts decreased even more slowly and the initial cause of cell death did not seem to result from cell lysis. Sodium succinate partially protected the cells against killing or inhibition by antisera. Viable cell counts, uptake, and incorporation of glycine-2- 14 C increased greatly. Malonate reversed completely the action of succinate. Apparently, the energy derived from the oxidation of succinate is sufficient for many cells to grow and incorporate the amino acid into cell protein. Glucose and nicotinamide also showed similar protective effects. Antiserum was observed to have a transient stimulatory effect on viable cell counts, uptake, and incorporation of glycine-2- 14 C at about 2 hr after the addition of antisera. Whether this is due to stimulation of noncycling G2 cells by antiserum is currently under study. During the entire experimental period, i.e. , up to 4 days, a small portion of the cell population remained viable. Many of these looked much larger and the incorporation rate, but not uptake rate, of glycine-2- 14 C/10 6 viable cells was much higher than that of the controls. Addition of succinate increased the rate still further. Cultures treated with antiserum recovered if fresh medium without antisera was added at 24 to 48 hr. Possible modes of action of the antisera were discussed.

The distribution of polysomes, ribosomes and ribosomal subunits in exponential-phase cells of Bacillus licheniformis.

Abstract: 1 The distribution of ribosomal particles between the soluble and membrane fractions of exponential-phase cells of Bacillus licheniformis was studied. The cells were very gently lysed by a combined treatment with lysozyme and Brij 58 in 10 mM Tris-buffer, pH 7.6, with 60 mM K+, 10 mM Mg2+, 0.5 mM spermine and 0.75 mM spermidine. The proportion of the ribosomal material present in the membrane fraction was estimated after solubilization of the membranes by the combined action of Brij 58, deoxyribonuclease and lipase. 2 Under these conditions of lysis 96% of the total ribosomal material, consisting of subunits (12%) monosomes (20%) and polyribosomes (68%) and most of the cellular DNA, is found to be membrane-bound. 3 In the soluble fraction, containing only 4% of the total cellular amount of the ribosomal material, only subunits and 70S particles and no polyribosomes are found. 4 DNA does not seem to be involved in the binding of the ribosomal material to the membranes. 5 The results will be discussed considering the question of whether the distribution described in this paper represents the true distribution in vivo of ribosomes.

The NAD kinases of Saccharomyces cerevisiae.

Abstract: Subcellular fractions of a strain of Saccharomyces cerevisiae have been prepared by enzymic digestion of the cell walls, osmotic lysis of the protoplasts and sorbitol density gradient centrifugation of the lysate. The mitochondrial fraction has both NAD- and NADH-phosphorylating enzymes, whilst the cytoplasm contains a single NAD-specific kinase. These three enzymes differ in their kinetic properties and in their stability to heat and to an alkylating agent, 3-(bromoacetyl) pyridine. The properties of the cytoplasmic NAD kinase resemble those of NAD kinase partially purified from autolysates of pressed bakers' yeast.

Rapid lysis of cell walls of Micrococcus radiodurans with lysozyme: effects of butanol pretreatment on DNA

Abstract: A method is presented by which the walls of Micrococcus radiodurans are rendered sensitive to rapid decomposition by lysozyme after a preliminary extraction with an n-butanol-saturated buffer. The ...

Growth of bacteria within lysing fungal conidia in soil

Abstract: Examination of ultrathin sections of conidia of Cochliobolus sativus recovered from soil revealed bacterium-like organisms associated with lysed areas in the matrix of the fungal cell wall. The outer electron-dense layer of the cell wall was more resistant to lysis than inner fibrillar layers.

The inhibitory action of a mammalian viral RNA on the initiation of protein synthesis in a reticulocyte cell-free system.

Abstract: RNA from mouse encephalomyocarditis virus inhibited endogenous protein synthesis in reticulocyte cell-free systems and was not, itself, translated. The inhibition was caused by interference with globin chain initiation. The inhibitory effect was observed under a variety of experimental conditions, including those where the Krebs II ascites cell system translates the viral RNA. Preincubation of the reticulocyte lysate increased the severity of the inhibition because it increased the dependence of endogenous protein synthesis upon reinitiation. Reticulocyte and ascites cell ribosomes bound encephalomyoearditis [3H]RNA to cellulose nitrate filters with equal efficiency. RNA from bacteriophage f2 and tobacco mosaic virus also appear to inhibit globin chain initiation.