Showing papers on "Lysis published in 1978"

Target-effector interaction in the natural killer (NK) cell system. II. The isolation of NK cells and studies on the mechanism of killing

Abstract: We have previously shown that a subpopulation of nylon wool nonadherent, lymphoid cells in normal mice binds selectively to tumor cell targets that are sensitive to killing by natural killer (NK) cells. In the present report these target-binding cells (TBC) were enriched or depleted by velocity sedimentation and the isolation of single target-effector conjugates indicated that the majority of TBC killed their targets. Furthermore, the frequency of TBC correlated well (r = 0.86) with lysis in a population during kinetics experiments. TBC resembled small, resting lymphocytes with membrane specializations in the area of target cell contact as indicated by electron microscopy and cytochemical techniques. Target cell binding occurred before lysis in kinetics studies and regenerated in parallel with the lytic potential of a trypsinized population devoid of surface Ig or θ-bearing cells. Cell contact was prevented in the presence of EDTA whereas metabolic inhibitors or inhibitors of serine proteases suppressed lysis with no effect on the frequency of TBC. Conversely, an interferon-inducing agent elevated NK-mediated cytolysis with no effect on the level of TBC. These results suggest that i) the majority of TBC that we detect represents the NK cell and ii) lysis and binding are independently controlled events. A tentative model of NK-mediated lysis is proposed.

Murein-lipoprotein of Escherichia coli: A protein involved in the stabilization of bacterial cell envelope

Abstract: Two independent mutants of Escherichia coli lacking murein-lipoprotein have been found One mutant whose mutation was named lpo was subjected to detailed analyses The absence of both found and unbound lipoproteins was shown by electrophoretic analysis of 14C-arginine labelled membrane proteins of the mutant Nor was serologically cross-reacting material detected in the mutant by the Ouchterlony-method Sequestering magnesium from mutant cell suspensions by ethylenediaminetetraacetic acid caused cell lysis, which was prevented in the presence of 05 M sucrose Incubation in culture media at a very low level of magnesium resulted in the formation of blebs in the mutant Examination of mutant cells by electron microscopy showed that the outer membrane of the mutant was uneven with small irregular protuberances, some of which pinched off forming vesicles of various sizes Phosphotungstate used for negative-staining penetrated into the periplasmic space of the mutant cells The mutants leaked a considerable fraction of their periplasmic enzymes These physiological and morphological alterations in the lipoproteinless mutant suggest that murein-lipoprotein helps to maintain the outer envelope structure by connecting the outer membrane with murein so that the outer membrane may fulfil its physiological functions as a barrier to the environment

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Abstract: Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.

Estimation of dna strand breaks in single mammalian cells

Abstract: Lysis experiments demonstrate that unirradiated cell nuclei are rather stable in alkaline solutions (pH≥12) in the sense that DNA from each cell keep together as a separate unit for more than 30 min. Irradiated cells lyse more quickly. Based on this, a preliminary method is devised involving the dye acridine orange and microfluorometry, whereby the relative amounts of single-stranded and double-stranded DNA can be estimated from each cell after alkali treatment. This extends the “DNA strand separation method” of estimating DNA strand breaks to the level of single cells.

H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins.

Abstract: Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 μg of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.

Cellular lysis of Streptococcus faecalis induced with triton X-100.

Abstract: Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100-induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed.

Quantitative Influence of Antibody and Complement Coating of Red Cells on Monocyte-Mediated Cell Lysis

Abstract: Monocyte-mediated lysis in vitro of human red cells coated with measured amounts of immunoglobulin G (IgG) or complement were studied. 1,000-1,500 molecules of IgG anti-D are necessary to effect measurable lysis, and lysis increases linearly with increasing levels of antibody sensitization. 100 microgram/ml of IgG1 abolished lysis even at maximal levels of anti-D sensitization (15,000 molecules/cell). Two isoimmune IgG anti-A or anti-B antisera were 5 to 10-fold less efficient in promoting phagocytosis or lysis per molecule of IgG bound; however, because of the greater antigen density of A or B, more than 100,000 molecules IgG/cell could be bound, producing equivalent lysis to anti-D-coated cells. Although inhibition by IgG1 was similar at equivalent levels of sensitization with anti-A, anti-B, or anti-D at high levels of coating with anti-A or anti-B (not attainable with anti-D), lysis was not inhibited by IgG1. Cells coated with human complement components alone were not lysed by monocytes; however, complement coating augmented IgG-mediated lysis and reduced the quantity of anti-D necessary to produce lysis to less than 1,000 molecules/cell. After thorough degradation of C3b by serum to C3d, complement augmentation persisted.

Assembly of the cytolytic alternative pathway of complement from 11 isolated plasma proteins.

Abstract: The known cytolytic function of the alternative pathway in serum was quantitatively reproduced by combining 11 isolated plasma proteins at their respective serum concentrations. These proteins are: C3, Factor B, Factor D, C3b inactivator, beta1H, native properdin, C5, C6, C7, C8, and C9. In absence of activators of the alternative pathway, this mixture was stable at 37 degrees C as evidenced by lack of consumption of Factor B, C3, and C5. Upon addition of either rabbit erythrocytes or neuraminidase-treated sheep erythrocytes, cell lysis ensued and the extent of lysis was dependent on dose of the component mixture. The dose-response curves obtained with the isolated component mixture and with C4-depleted serum were virtually indistinguishable. Nonactivator erythrocytes (untreated sheep erythrocytes) were not lysed by the component mixture. Deletion of properdin resulted only in a twofold diminution of the hemolytic activity of the component mixture. No immunoglobulin requirement was apparent. These results indicate that the cytolytic systems studied are internally sufficient and capable of coupling the initiation and amplification sequence with the cytolytic membrane attack sequence.

Corticosteroids Block Newly Induced but Not Constitutive Functions of Macrophage Cell Lines: Myeloid Colony-Stimulating Activity Production, Latex Phagocytosis, and Antibody-Dependent Lysis of RBC and Tumor Targets

Abstract: Hydrocortisone at 10 -4 M added to macrophage cell lines had no effect on the following activities: J774.1 antibody-dependent phagocytosis and lysis of sheep RBC, RAW 264 antibody-dependent lysis of tumor targets, latex bead phagocytosis by PU5-1.8 cells, and constitutive production of myeloid colony-stimulating activity (CSA) by WEHI-3 cells. In contrast, 10 -4 M hydrocortisone completely inhibited LPS or PPD activation of the following functions: induced production of myeloid CSA, stimulation of latex phagocytosis and stimulation of antibody-dependent lysis of tumor targets by PU5-1.8 cell line, and stimulation of antibody-dependent lysis of RBC targets by RAW 264 cell line. Lower concentrations of hydrocortisone or dexamethasone at 10 -6 M inhibited the LPS induction of CSA from 55 to 90%. Growth inhibition of PU5-1.8 cells caused by 1 µg/ml LPS was also partially reversed by corticosteroids. Thus, steroids block these newly induced but not constitutive functions of these macrophage cells in culture.

Blood cell typing and compatibility test procedure

Abstract: A solid-phase blood typing procedure is described based upon either aggluation or immune lysis In this invention, a monolayer of cells is irreversibly bound to a solid matrix, and thereafter a serum containing antibodies is brought into contact with the bound cell layer Immunoadsorption of antibodies by the bound cells occurs where the antigens of the cell membranes and the antibodies in the serum are complementary to each other This antibody-sensitized monolayer of blood cells can either bind a second layer of blood cells carrying complementary antigen (solid-phase agglutination) or undergo lysis in the presence of serum lytic complement (solid-phase immune lysis) Carrying out these reactions with a monolayer of blood cells bound to a solid matrix allows quantitative evaluation of results by such standard instrumentable procedures as densitometric scanning, radioisotope counting, etc

Evidence for the specific association of the chromosomal origin with outer membrane fractions isolated from Escherichia coli.

Abstract: DNA-envelope complexes isolated from osmotically lysed spheroplasts of Escherichia coli contained 0.2 to 1% of the total cellular DNA after labeling with [3H]thymidine. Molecular weight determinations indicated that the amount of bound DNA was equivalent in most cases to a maximum of three binding sites per chromosome. Bound DNA from E. coli B/r was distributed approximately equally between inner and outer membrane components when envelopes were fractionated on sucrose equilibrium gradients. Outer membrane-DNA complexes, in particular, fraction H1, with a density of 1.24 g/cm3, were quite stable against shearing and against Sarkosyl NL97. In the case of E. coli B/r, H1-DNA was also relatively resistant to deoxyribonuclease. Inner membrane-DNA complexes, in contrast, were quite labile and readily dissociated to release free DNA. The outer membrane fractions did not appear to contain replication fork DNA, but small amounts may have been present in the inner membrane complexes. A two- to eightfold enrichment for chromosomal origin DNA in the envelope was obtained when cultures of E. coli K-12, synchronized for DNA replication, were pulse labeled at different times in the replication cycle. This enrichment was found invariably in the outer membrane fractions. However, the data do not exclude the possibility that this DNA is bound to regions of adhesion between inner and outer membranes which sediment with a density indistinguishable from that of the outer membrane.

Induction of limited DNA damage by the antitumor agent Cain's acridine.

Abstract: The antitumor drug methanesulfonyl-m-anisidine, 4′-(9-acridinylamino) monohydrochloride (AMSA) (Cain9s acridine, NSC 141549), causes a limited, partially reversible decrease in size of the DNA of mouse L1210 leukemia cells as analyzed by centrifugation of cell lysates on alkaline sucrose gradients. Exposure of cells for 30 min to AMSA, 2.5 µg/ml, in tissue culture or to 150 µg/mouse in vivo results in a shift of long-term prelabeled DNA from >170S fractions to a broad band of about 30S. Higher doses or longer exposure times do not produce DNA much smaller than 30S. Labeled AMSA cosediments with heavy DNA in neutral sucrose gradients in which no degradation is observed, but AMSA is dissociated from both heavy and 30S DNA in alkaline sucrose gradients and appears only at the top of the gradient. On neutral gradients degradation to 30S DNA after AMSA exposure is detected only when cells are previously lysed in an alkaline medium but not when they are lysed in a neutral 70% formamide solution or when the neutral lysate is heated to 83° before centrifugation. Treatment of L1210 cells with other DNA intercalating agents (ethidium bromide, acridine orange, and phosphine orange) caused a similar degradation of the DNA on alkaline sucrose gradients. The results are interpreted to indicate that AMSA causes alkali-sensitive lesions of DNA at a limited number of sites; this effect correlates with the antitumor action of the drug and with other work showing long-term damage to chromosome structure.

Kinetic characterization of plasma membrane ATPase from Saccharomyces cerevisiae.

Abstract: 1. Plasma membrane preparations have been isolated from spheroplasts of Saccharomyces cerevisiae, strain R XII, via lysis and subsequent differential centrifugation. These preparations are almost devoid of mitochondria) contamination. 2. The plasma membrane ATPase is fairly stable when refrigerated, but loses activity at 8 °C and above. Below pH 5.6 the ATPase is irreversibly inactivated. The enzyme also splits GTP and ITP, although to a lesser extent. 3. Mg2+-ions are essential as part of the reactive substrate, MgATP, and furthermore they activate the ATPase. Optimal conditions depend on substrate concentration. When the concentration of free Mg2+ ions exceeds about 0.1 mm, competitive inhibition occurs. 4. In the range of pH 5.6–9.2 two functional groups dissociate. One, with pKb = 8.1 ± 0.1 participated in substrate binding and another one with pKb′ = 8.1 ± 0.1 is involved in substrate splitting. 5. The experiments with group-specific inhibitors suggest that an α-amino group and a sulfhydryl residue are involved in substrate binding and conversion. Furthermore, imidazole, tryptophan and carboxyl residues may be important for the catalytic process.

Effect of benzylpenicillin on the synthesis and structure of the cell envelope of Neisseria gonorrhoeae.

Abstract: The effect of benzylpenicillin on the synthesis and morphology of the cell envelope of Neisseria gonorrhoeae was examined. Penicillin immediately stopped murein synthesis; it also enhanced the rate of turnover of glucosamine, but not diaminopimelic acid, in the murein. In addition, penicillin greatly increased the shedding of lipid and lipopolysaccharide into the medium. In the electron microscope, protrusions of the cell membrane were evident, as well as apparent holes in the murein cell wall. All of these changes occurred while active synthesis was taking place, before the lysis of the cells. Lysis could be prevented by growing the cells at low pH and high concentrations of Mg2+; however, the effects of penicillin on murein synthesis and turnover and on the release of lipid were not affected. Images

Lysis of C1q-Coated Chicken Erythrocytes by Human Lymphoblastoid Cell Lines

Abstract: Human lymphoblastoid cells lysed chicken erythrocytes (E) that carried cell surface bound human C1q. Antibody to E (A) was not required for the C1q-dependent reaction. The effect of C1q was inhibited by Fab′ 2 anti-C1q and by the serum C1q inhibitor. The action of the lymphoblastoid cells was inhibited by anti-metabolites and by pretreatment of the cells with trypsin which is known to destroy their C1q receptor. Lymphoblastoid cell lysate was inactive. The time course of the C1q-dependent lysis was comparable to that of the antibody-dependent cellular cytotoxic reaction of human K-cells. Lysis of EA by human peripheral lymphocytes was enhanced up to 50% by human C1q.

Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA

Abstract: Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNAHL (hybrid DNA, density approximately equal to 1.75 g/cm3), DNAint (intermediate density DNA, density approximately equal to 1.71 g/cm3), and DNAHH (DNA with both strands containing analog, density approximately equal to 1.80 g/cm3). Preparations of DNAint yield DNAHH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (BrdUrd + 3HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNAHH.Cross-linking after lysis has little effect. A large fraction of DNAHH is obtained after incubation of cells with caffeine. Extraction of DNA at high salt concentration or cross-linking with trioxsalen and near UV light drastically reduced the amount of DNAHH obtained from caffeine-treated cells. We conclude that most DNAHH arises from in vitro branch migration in isolated DNA growing points.

Membrane proteins in human erythrocytes during cell fusion induced by oleoylglycerol.

Abstract: 1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca(2+) used, at least 80% of the cells fused after 30min at 37 degrees C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in ;ghosts' prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca(2+), although some cells fused when no exogenous Ca(2+) was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of ;ghosts' prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1-2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca(2+) into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed.

Isolation of an Fcγ-Binding Protein from the Cell Membrane of a Macrophage-Like Cell Line (P388D1) after Detergent Solubilization

Abstract: Lactoperoxidase-catalyzed iodination, NP-40 lysis, and subsequent affinity chromatography on IgG-Sepharose were used in an attempt to define some of the molecular properties of the Fc receptor of P388D 1 , a macrophage-like mouse tumor line. Radioiodinated material retained on columns of Sepharose coupled either to monomeric mouse IgG2a or monomeric human IgG1 appeared on SDS polyacrylamide gel electrophoresis to contain principally three labeled components, a major band of about 57,000 m.w., and two minor bands of 28,000 and 24,000 m.w. The mobilities of these components changed little on reduction, which suggested that they represented single polypeptide chains. An identical pattern was obtained with Sepharose-linked Fc fragments of human IgG1, but neither Fab fragments of IgG1 nor IgM appeared to bind these components. Since the specificity of binding to the immobilized proteins is the same as that observed in vivo , it is postulated that these proteins represent either all or some portion of the P388D 1 Fc receptor.

Cell yield and cell survival following chemotherapy of the b16 melanoma.

Abstract: We describe in this paper cell survival studies, using in vitro clonogenic assays, performed on the B16 melanoma treated in situ with various cytotoxic agents. In addition we have determined the effects of these agents on the yield of cells obtained by trypsinization. In untreated tumours the mean cell yield was approximately 10(8)/g, which is 20--30% of the cells actually present in the tissue. The plating efficiency was approximately 40%. Most agents rapidly affected both cell yield and cell survival. For example, within 20--30 h, gamma-radiation and several alkylating agents reduced cell yield by about 40%. The cell yield change was associated with an increase in mean cell size. Cell yield was reduced even more (approximately 70%) by Vinca alkaloids. This large reduction was associated with extensive cell lysis, observed as an increase in the necrotic fraction of tumours from approximately 35% to approximately 70%. Adriamycin, bleomycin and Ara-C also produced a moderate reduction in cell yield (approximately 40%), but actinomycin D did not reduce cell yield and FU increased it by about 30%. Only gamma-radiation, cyclophosphamide, CCNU, BCNU and melphalan produced more than a 90% reduction in cell survival, although there was a small but measurable reduction with all other agents except vinblastine, HN2 and actinomycin D.

The structural events associated with the attachment of complement components to cell membranes in reactive lysis.

Abstract: Electron microscopic study of the events occurring at the cell membrane during reactive lysis by complement, showed that a foliaceous particle was formed at the C5b-7 stage, that enlarged to a particle with a variable number of arms at the C5b-8 stage. Up to this point, no typical complement lesions were found. At the C5b-9 stages, the particles were completely converted to typical complement lesions, i.e. hollow cylinders projecting from the cell membrane and partly penetrating it. C5b-9 complexes assembled in the fluid phase did not show the typical structure of the lesions, but were amorphous masses of fibres.

Secretion of Cell Wall Polymers into the Growth Medium of Lysis-Defective Pneumococci During Treatment with Penicillin and Other Inhibitors of Cell Wall Synthesis

Abstract: Autolysin-defective pneumococci secrete into the growth medium choline-containing macromolecules during treatment with any one of a large number of inhibitors of cell wall biosynthesis, including beta-lactams, beta-halogeno-d-alanines, cephalosporins, and d-cycloserine. Secretion is closely related to the dose response of the bacteria to the various drugs: (i) secretion can already be detected at the minimum inhibitory concentration; (ii) the rate and extent of secretion is dependent upon the drug concentration; and (iii) secretion commences within minutes after the addition of the antibiotics to the cultures. Reversal of the growth-inhibitory effect of benzylpenicillin (by penicillinase addition) is accompanied by a halt in secretion just at the time when the bacteria resume normal growth. Secretion of the choline-containing macromolecules seems to be a specific consequence of the inhibition of peptidoglycan biosynthesis, since inhibition of growth by drugs affecting protein, ribonucleic acid, or deoxyribonucleic acid synthesis does not cause secretion. The choline-containing macromolecules include both the pneumococcal lipid-containing teichoic acid (Forssman antigen) and wall teichoic acids made after the addition of antibiotics. The appearance of these macromolecules in the growth medium is not due to the hydrolytic activity of an autolysin, since penicillin-induced secretion could be demonstrated in autolysin-defective mutants, in pneumococci grown on ethanolamine-containing medium (such cells are known to have defective autolytic systems), and in wildtype pneumococci grown under conditions nonpermissive for lysis. Images

Identification of lysis protein E of bacteriophage phiX174.

Abstract: The product of gene E, the lysis gene of phiX174, has been identified as a distinct band in a sodium dodecyl sulfate-gel electropherogram. The position of the band is consistent with the molecular weight of 10,589 calculated from the nucleotide sequence of the gene. The band is eliminated by a nonsense mutation in gene E. It is estimated that roughly 100 to 300 molecules of E protein are made in an infected cell; this appears to be less than one-tenth the amount of protein made by gene D, in which gene E is wholly contained.

Folded chromosomes of vegetative Bacillus subtilis : composition and properties

Abstract: The isolation of folded DNA from Bacillus subtilis, a Gram positive bacterium is described. When the lysis was achieved with 1 M NaCl a slow sedimenting nucleoid was obtained (1600-2000 S). Conversely, when the lysis was achieved with 0.2 M NaCl a fast sedimenting nucleoid was obtained (3500-4000 S). The yield of folded DNA was between 80 to 90 % of the total lysate DNA. Both nucleoids contained the same amount of RNA, but the relative proportions of lipids and proteins were different. Folded chromosomes were prepared in the presence of spermidine: artifactual protein binding is shown to be unlikely. Electrophoresis of nucleoid proteins showed a dominant polypeptide (MW = 36,000), which remained associated with DNA after sarcosyl treatment and could be partially removed by heat mediated DNA unfolding. In vitro transcription by endogenous RNA polymerase bound to the fast sedimenting-nucleoid was rifamycin resistant; the template capacity of the fast sedimenting-nucleoid was compared with that of the completely unfolded chromosomes.

Influence of R-plasmid RP1 of Pseudomonas aeruginosa on cell wall composition, drug resistance, and sensitivity to cold shock.

Abstract: R-plasmid RP1 was transferred to Pseudomonas aeruginosa cells, as indicated by their resistance to carbenicillin, ampicillin, cephaloridine, kanamycin, and tetracycline, and by the presence of a periplasmic beta-lactamase The wild-type cells (RP1-) were lysed by ethylenediaminetetraacetic acid but not by ethylene-glycol-bis(2-aminoethyl ether)-N,N-tetraacetic acid, whereas cells carrying the plasmid (RP1+) were resistant to both these chelating agents RP1+ and RP1- strains were both sensitive to the lytic action of polymyxin B and the lethal action of cold shock, but the effect was less marked in the RP1+ cultures A proportion of the RP1+ cells surviving cold shock lost resistance to carbenicillin, tetracycline, and kanamycin The chemical composition of whole cells and cell walls of RP1+ differed from that RP1- in the content of cation, phospholipid, and markers for lipopolysaccharide and peptidoglycan Differences in cell wall composition, response to ethylenediaminetetraacetic acid and polymyxin B, and the effects of cold shock are all compatible with the hypothesis that RP1 confers changes in the cell envelope, probably in the outer membrane, of P aeruginosa