Showing papers on "Lysis published in 1979"

Hydrolytic enzymes in the central vacuole of plant cells.

Abstract: The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. ( a ) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. ( b ) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. ( c ) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: α-mannosidase, β- N -acetylglucosaminidase, β-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. Our data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome. None of the vacuolar enzymes investigated was found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter (24% sucrose, w/w).

Mechanism of cell-mediated cytotoxicity at the single cell level. I. Estimation of cytotoxic T lymphocyte frequency and relative lytic efficiency.

Abstract: A single cell method has been developed for directly measuring the frequency of cytotoxic T lymphocytes (CTL). The method is based on the ability of CTL to bind corresponding target cells and to form CTL-target conjugates visible by microscopy. Conjugates are incubated at 37°C in agarose to prevent CTL recycling. Single target lysis is assessed by trypan blue uptake. Target cell lysis was evident as early as 15 min and reached a plateau at 2 hr with an average of 60 to 80% of the conjugated target cells lysed. In a population of nylonwool nonadherent BALB/c peritoneal exudate T lymphocytes sensitized to C57BL/6 alloantigens, the frequency of CTL was estimated to range from 12 to 24% of the total T lymphocytes. The frequency of CTL and the kinetics of lysis were a function of the state of immunity, the method of sensitization, and the target cell employed. The single cell method described here has several features, namely, its independence of CTL recycling, the elimination of the variability in 51 Cr release by target cells, and its prevision for a direct estimate of CTL frequency. Furthermore, the method is simple, reproducible, highly quantitative with very low numbers of CTL, and adaptable to non-T cell-mediated cytotoxic systems.

Bactericidal activity of the alternative complement pathway generated from 11 isolated plasma proteins.

Abstract: Exposure of E. coli K12 W1485 to the cytolytic alternative pathway assembled from the 11 isolated pathway proteins resulted in killing of the bacteria, as evidenced by loss of viability. Lysis of the bacteria required introduction of lysozyme into the reaction mixture. The time-course and dose dependency of bacteriolysis in the isolated system were identical to those in C4-depleted serum. The bacteriolytic activity of the pathway was highly dependent on the concentration of the pathway proteins and became insignificant at 1:16 physiological concentration. Electron microscopic visualization of killed and of lysed bacteria revealed numerous complement membrane lesions and partial disintegration of the outer phospholipid membrane. Scanning electron microscopy showed that killed bacteria were enlarged, partially collapsed and exhibited irregular surface protrusions. Lysed bacteria were fragmented and appeared polymorphic. This study demonstrates that the alternative pathway, in absence of immunoglobulins, has the potential or eradicating gram-negative bacteria.

From Penicillin-Binding Proteins to the Lysis and Death of Bacteria: A 1979 View

Abstract: The mechanism by which interference with the biosynthesis of bacterial cell wall causes death and lysis of bacteria appears more complex than originally thought. In an earlier model of the mode of action of beta-lactams, it was assumed that, in the presence of the antibiotics, bacteria synthesize a mechanically weak (poorly cross-linked) cell wall that ruptured under the osmotic-mechanical pressure of the normally growing cytoplasmic mass. However, recent findings suggest a much more complex picture. Lysis and, in at least some bacteria, loss of viability as well, seem to be catalyzed by autolytic enzymes (murein hydrolases), the destructive activity of which is triggered in the beta-lactam-treated bacterium via a poorly understood mechanism. Furthermore, different species of bacteria respond quite differently to treatment with the same beta-lactam: some bacteria are both killed and lysed, others only lose viability, whereas still other species respond mainly by a reversible inhibition of growth (beta-lactam-tolerant bacteria). In addition, structurally different beta-lactams may cause quite different biochemical, morphological, and antibacterial effects, even within the same bacterial species. It is conceivable, therefore, that there is more than one mechanism for loss of viability and/or lysis. Most of the bacteria examined so far contain a number (four to eight) of different penicillin-binding proteins. Genetic and physiological evidence obtained in E. coli indicate that these proteins play essential roles in a variety of physiological functions, such as maintenance of structural integrity, shape, and cell division. Pneumococci with a suppressed autolytic system are resistant to he lytic (and, partially at least, to the bactericidal) effect of beta-lactams. Interference with cell wall synthesis seems to trigger autolysin activity by upsetting the cellular control of autolytic enzyme. It is suggested that the irreversible antimicrobial effect of beta-lactams may have an indirect mechanism in other bacteria as well.

Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes.

Abstract: A study was carried out to determine the sequence of events of T-cell mediated target cell lysis in microcinematography and electron microscopy. Highly efficient cytotoxic T lymphocytes (CTL) were generated in vivo and in vitro using preimmunized spleen cells and purification procedures. Such CTL were highly specific. This specificity correlated well with the number of adhesions formed between CTL and targets and this criterion was used to study killer-target cell interaction. Microcinematography showed that target cell lysis at the single cell level, despite time variations, could be clearly separated into three phases: (a) a recognition phase, visible by random crawling of CTL over the target cell surface until firm contact was established; (b) a post-recognition phase, during which firm contact between CTL and target was maintained without gross modification of either cell; (c) a phase of target cell disintegration, mainly characterized by vigorous blebbing of the cell membrane resulting in a motionless carcass of the target cell but not in its total dissolution. Only later this carcass decayed and formed a necrotic ghost. Electron microscopic observations were put into sequence according to microcinematography. Post-recognition phase was characterized by a tight apposition of the membranes of CTL and target cell. No gap junctions could be observed. During target cell disintegration, profound cytoplasmic and nuclear changes occurred simultaneous with surface blebbing. Most noticeable were extensive internal vacuolization, mitochondrial swelling, nuclear pycnosis and dissolution of the nucleolus. These observations suggested that target cell lysis does not start with a surface phenomenon similar to complement lysis, but a process involving practically the whole cell simultaneously. It is conceivable, therefore, that the signal from the CTL is transmitted across the target cell, and that the switch to sudden cell death is manipulated deep inside the cell.

Mechanism of Cell-Mediated Cytotoxicity at the Single Cell Level: II. Evidence for First-Order Kinetics of T Cell-Mediated Cytolysis and for Heterogeneity of Lytic Rate

Abstract: The kinetics and rate of T cell-mediated cytolysis was assessed by measuring the times required for lysis of isolated target cells by single cytotoxic lymphocytes. Single target cell lysis was determined microscopically by observing trypan blue uptake as a function of time of incubation of effector-target conjugates in agarose. Lysis of EL-4 target cells by alloimmune peritoneal exudate lymphocytes was initiated without a lag and was essentially complete at 2 hr. Both zero-order and first-order kinetics equations were analyzed for fit to the 0 to 2 hr lysis values. Statistically, the zero-order kinetic function could be rejected (p greater than 0.05), but the first-order kinetics function (p less than 0.01) could not. This strong evidence for first-order kinetics of T cell-mediated cytolysis implies that within each CTL-target cell population, cytolysis occurs exponentially as a random decay process and that one event in the entire process of cytolysis is rate limiting. The first-order equation was then applied to measurements of the rate of cytolysis in many different individual effector-target cell combinations. Significant differences in the lytic rate were apparent when either the effector or target cell were varied, with the rate constants spanning a 5-fold range. The heterogeneity of lytic rates is consistent with the hypothesis that lytic efficiency is a function of both the effector and target cells used.

Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics.

Abstract: A biochemical method was developed to quantitatively compare the effectiveness of beta-lactams in triggering murein degradation (autolysin activity) in Escherichia coli. Bacteria prelabeled in their cell walls with radioactive diaminopimelic acid in growth medium were exposed for 10 min to the antibiotics at the appropriate minimal growth inhibitory concentrations and at multiples of these values, and the rate of cell wall degradation was followed during subsequent penicillin-binding protein (PBP)-1 were the most effective triggers of autolytic wall degradation; beta-lactams selective for PBP-2 were the poorest; and antibiotics preferentially binding to PBP-3 showed intermediate activities. The relative effectiveness of beta-lactams in autolysin triggering was found to parallel the effectiveness of the same drugs in causing rapid loss of viability, culture lysis, and spheroplast formation. Autolysin triggering was suppressed by inhibitors of protein and ribonucleic acid biosynthesis but not by inhibitors of deoxyribonucleic acid synthesis. The beta-lactam-induced cell wall degradation did not seem to involve a direct stimulation of enzyme activity or synthesis of new enzyme molecules, and murein sacculi isolated from cells that had been preexposed to a triggering dose of beta-lactam treatment exhibited the same sensitivity to crude, homologous autolysins as sacculi prepared from untreated control bacteria. On the basis of these observations, mechanisms are considered for the triggering of E. coli autolysins and for the role of autolytic activity in bacterial spheroplast formation, lysis, and death.

ADCC (K-cell) lysis of human erythrocytes sensitized with rhesus alloantibodies. II. Investigation into the mechanism of lysis.

Abstract: Summary. The mechanism of lysis of anti-D coated human erythrocytes by human mononuclear K-cells was investigated. Red cell lysis was measureable after 30 min incubation and reached a maximum by 18–20 h. Cell-to-cell contact was necessary for lysis, phagocytosis was not a prerequisite, and intact microfilament function was required. The divalent cations Ca2+ and Mg2+ were both required for lysis to occur. Studies with metabolic inhibitors indicate that some RNA and protein synthesis is required for maximum expression of ADCC and intact microtubule function is essential. In the present system lysis was mediated by IgG1 anti-D antibodies and was signficantly inhibited by IgG1 and IgG3 subclasses with some inhibition by IgG2 but not by IgG4, IgA or IgM. This suggests that the K-cell Fc receptor is specific for IgG but that there is major cross-reactivity between IgG1 and IgG3. The inhibiting effect of hydrocortisone suggests that ADCC inhibition may be one mode of action of corticosteroids in ameliorating autoimmune haemolytic anaemia.

Lysis of measles virus-infected cells by the purified cytolytic alternative complement pathway and antibody.

Abstract: The dependence of antibody-and-complement-mediated lysis of virus-infected cells on the alternative pathway was examined utilizing the isolated cytolytic alternative pathway--a system consisting of the six purified proteins of the alternative pathway of activation (C3, factors B and D, beta 1H, C3b inactivator and properdin), and the five proteins of the membrane attack pathway (C5--9) of complement. HeLa cells acutely infected with measles virus were lysed by anti-viral IgG and the isolated cytolytic alternative pathway with an efficiency comparable to whole human serum. IgG and its F(ab')2 fragment were equally effective in inducing lysis by the isolated cytolytic alternative pathway, binding of approximately equal to 5 X 10(7) molecules per cell being required for 50% lysis; in contrast, no lysis occurred when equivalen or greater amounts of Fab' were bound to the virus-infected cell. Properdin was required for lysis. No lysis occurred if properdin was deleted from the isolated cytolytic alternative pathway, and lysis was diminished by 80% in properdin-depleted serum. Uptake of [125I]C3b from the isolated alternative pathway onto measles virus-infected cells occurred in the absence of properdin, but was accelerated in the presence of properdin. The 11 proteins of the isolated cytolytic alternative pathway are thus sufficient for lysis of measles virus-infected cells bearing anti-viral IgG or F(ab')2 without any other serum protein.

Intracellular localization of the dermonecrotic toxin of Bordetella pertussis.

Abstract: Localization of the heat-labile dermonecrotic toxin of Bordetella pertussis strain 114 grown in chemically defined Stainer-Scholte medium was studied by using skin reaction in 4-day-old suckling mice as the assay for toxin. Through log phase and into stationary phase of growth the toxin was cell associated and not detected in the culture supernatant. Only about 4% of the activity present in a suspension of lysed cells was detected in a suspension of whole cells, and the dermonecrotic activity was not released by subjecting whole cells to osmotic shock, a procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After cell lysis and preparation of soluble and membrane fractions, 73 to 80% of the activity in the cell lysate was recovered in the soluble fraction, with only 3 to 6% present in a membrane fraction. Further evidence for the intracellular cytoplasmic localization of the dermonecrotic toxin was the insensitivity of the toxin to trypsin treatment of whole cells. Treatment of whole cells with trypsin (80 micrograms/ml) for 20 min at 37 degrees C did not decrease dermonecrotic or malate dehydrogenase activities, but did inhibit more than 95% of the extra-cytoplasmic adenylate cyclase activity. Identical trypsin treatment of a cell lysate decreased all the above activities by more than 90%.

Alternative Complement Pathway-Mediated Lysis of Measles Virus Infected Cells: Induction by IgG Antibody Bound to Individual Viral Glycoproteins and Comparative Efficacy of F(ab′)2 and Fab′ Fragments

Abstract: Lysis of measles virus-infected cells by human serum is dependent on IgG antibody and an intact alternative complement pathway The binding sites of IgG measles virus antibody, or its fragments, on the surface of measles virus-infected cells were determined by cell surface radioiodination and immunoprecipitation It was found that IgG bound to either of the two measles virus surface glycoproteins was effective in inducing cell lysis by the alternative pathway Thus, IgG bound only to the viral hemagglutinin (HA) or only to the fusion protein (F) could induced lysis in C4-depleted human serum; the same number of IgG molecules bound to either HA or F induced equivalent dose-related lysis Divalent antibody bound preferentially to HA on the surface of intact cells; this enabled antibody to F to be obtained free of antibody to HA by adsorption Quantitative studies of binding and lysis showed that lysis of measles virus-infected cells by whole or C4-depleted serum required binding of at least 10 times more Fab′ than F(ab′) 2 or IgG molecules per cell In these experiments, C4-depleted serum was quantitatively and kinetically equivalent to whole serum in mediating lysis of measles-infected cells Hence, the binding of IgG to either of the two measles viral polypeptides (HA, F) expressed on the surface of infected cells induces lysis by the alternative pathway, and there is a clear requirement for divalency for effective induction of lysis

Studies on the Mechanism of T Cell-Mediated Lysis at the Single Effector Cell Level I. Kinetic Analysis of Lethal Hits and Target Cell Lysis in Multicellular Conjugates

Abstract: The time required for lethal hit of each conjugated target cell was measured by adding EDTA to the incubation medium of individual multicellular conjugates at various time intervals. Evidence could be obtained that a CTL induced irreversible lesions in only one of the conjugated target cells at a time, i.e., lethal hits were given sequentially rather than simultaneously.

Isolation and partial characterization of vacuoles from tobacco protoplasts.

Abstract: Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5′-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.

Important variables in granulocyte chemiluminescence.

Abstract: SummaryA CL system consisting of canine granulocytes stimulated with opsonized zymosan was used to examine factors which lead to variability in light production. Variability of CL response was associated with granulocyte aggregation. Granulocyte aggregation was reduced by: (i) reducing centrifu-gation and mixing forces used to prepare cell suspensions, (ii) preparing cell suspensions at 4° in Ca2+- and Mg2+-free cell media containing divalant cation chelating agents, and (iii) lysing red blood cells by the ammonium chloride technique.The type of cell media used during the CL run was another important variable. Solutions containing complex mixtures of amino acids and vitamins gave markedly reduced levels of CL. Tryptophan, ascorbic acid, iron salts, and all proteins tested caused significant suppression of CL. Glucose, Ca2+, and Mg2+ in the final reaction mixture were necessary for maximal CL response. The optimal cell media for CL, therefore, was buffered salt solution containing glucose, Ca2+, and Mg2+, ...

Effects of lysozyme on Bacillus cereus 569: rupture of chains of bacteria and enhancement of sensitivity to autolysins.

Abstract: Summary: Bacillus cereus 569 is known to be resistant to lysis by lysozyme because of the presence of deacetylated glucosamine residues in its peptidoglycan, and cultures continued to grow even in the presence of lysozyme at 200 μg ml−1. However, lysozyme caused rupture of the chains of bacteria and promoted the rate of autolysis in a non-growing cell suspension, causing a doubling of the rate of release of radioactively labelled wall material. Heatinactivated cells did not autolyse and were not lysed by lysozyme unless they were supplemented by unheated cells or cell-free autolysate. Enhancement of autolysin activity could also be effected by pre-treatment of heated cells with lysozyme. The action of lysozyme on isolated cell walls released some free reducing groups, indicating limited breakage of the polysaccharide chains of peptidoglycan, and it was concluded that lysozyme modified the peptidoglycan and made it more susceptible to autolysin(s). Lysozyme also enhanced the rate of septum separation and the probable significance of the results in relation to the control of cell separation is discussed.

Senescence of Pear Fruit Cells Cultured in a Continuously Renewed, Auxin-deprived Medium

Abstract: Auxins and cytokinins support cell division in tissue and cell cultures. In cytokinin-independent pear ( Pyrus communis ) cells, omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from the medium for two successive transfers leads to rapid cell lysis, unless the osmolarity is raised to 0.4 molar with mannitol. Use of this system (nutrients plus mannitol minus 2,4-D) for the study of cell senescence was explored both in batch culture and in a system designed to permit medium renewal without withdrawal of live cells. In both systems, an initial period (1-6 days) of limited increase in cell number is characterized by a continuous decrease in the respiratory activity and in protein and RNA synthesis to very low basal rates. In batch culture, cell death occurs after 13 to 15 days with little or no change in metabolic activity, or in protein and RNA synthesis. With renewal of cell medium, death is slightly delayed and is preceded by a burst in RNA synthesis followed by a notable increase in protein synthesis. Cycloheximide inhibition of protein synthesis is transient and its effect on cell longevity variable. Nonetheless, in all instances cell death is preceded by a burst in protein synthesis. Actinomycin D (1.6 micromolar) did not significantly affect protein synthesis but delayed RNA synthesis and cell death. The possible roles of auxin, osmoticum, and macromolecular synthesis in cellular senescence and death are discussed.

Isolation and purification of large quantities of DNA replication intermediates by pH step alkaline elution.

Abstract: The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of < 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8–12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20–30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40–50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated 3H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 106 cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation.

Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics

Abstract: Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli χ2452, which showed a tolerant response against beta-lactam antibiotics when grown at 42°C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli χ1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of χ1776 or in χ2452 grown at 32°C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of χ2452 grown at 42°C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecillinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of χ2452 cells grown at 42°C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of χ2452 grown either at 42 or 32°C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings.

Does Penicillin Kill Bacteria

Abstract: The thesis is presented that the bactericidal action of penicillin and of other inhibitors of cell wall peptidoglycan synthesis, such as vancomycin and cycloserine, is secondary or tertiary to their ability inhibit specific reactions in the assembly of an osmotically protective cell wall. Examples are given of the inhibition of these reactions, which results in inhibition of cell growth (bacteriostatic action) in the absence of either cellular lysis or rapid loss of viability. Thus, in some instances, inhibitory concentrations of these drugs are, in effect, sublethal; this is true, for example, for Streptococcus mutans, a species of bacteria that is part of the normal flora of the oropharynx and that can cause subacute bacterial endocarditis. On the other hand, the damaging effects of the subminimal inhibitory concentrations of penicillin G on Streptococcus faecalis, a species with an active autolytic enzyme system, can be uncovered and converted to a lytic (and lethal) response by partial inhibition of fatty acid synthesis with low concentrations of cerulenin. Some theoretical and practical implications of the occurrence and inhibition of these secondary lethal consequences are discussed.

Regulation of membrane peptides by the Pseudomonas plasmid alk regulon.

Abstract: Pseudomonas putida strains carrying the plasmid alk genes will grow on n-alkanes. Induced alk+ strains contain membrane activities for alkane hydroxylation and dehydrogenation of aliphatic primary alcohols. P. putida cytoplasmic and outer membranes can be separated by sucrose gradient centrifugation after disruption of cells by either mild detergent lysis or passage through a French press. Both the membrane component of alkane hydroxylase and membrane alcohol dehydrogenase fractionated with the cytoplasmic membrane. Induction of the alk regulon resulted in the appearance of at least three new plasmid-determined cytoplasmic membrane peptides of about 59,000 (59K), 47,000 (47K), and 40,000 (40K) daltons as well as the disappearance of a pair of chromosomally encoded outer membrane peptides of about 43,000 daltons. The 40K peptide is the membrane component of alkane hydroxylase and the product of the plasmid alkB gene because the alkB1029 mutation altered the properties of alkane hydroxylase in whole cells, reduced its thermal stability in cell extracts, and led to increased electrophoretic mobility of the inducible 40K peptide. These results are consistent with a model for vectorial oxidation of n-alkanes in the cytoplasmic membrane of P. putida.

Prevention of penicillin-induced lysis of Staphylococcus aureus by cellular lipoteichoic acid.

Abstract: The lysis of Staphylococcus aureus FDA 209P induced by benzylpenicillin was completely inhibited by cellular lipoteichoic acid isolated from an homologous organism. The cells prevented from penicillin-induced lysis were in static state and did not lose viability. The lipoteichoic acid inhibited either extracellular autolysin activity in culture supernatant or autolysis of whole cells in exponential phase of S. aureus. These results indicate that the prevention of penicillin-induced lysis of S. aureus by the lipoteichoic acid was brought about by the inhibition of autolytic activity of the organism.

Enzymatic lysis of yeast cell walls

Abstract: Lysis of yeast cell walls using zymolase and lysozyme was studied. During coupled zymolase–lysozyme treatment, nearly three times more reducing sugars were released from the yeast cells compared to controls. Enzyme treatment followed by extraction at pH 9 resulted in a yield of more than 80% of the total nitrogen of the yeast cell. Protein degradation occurred during enzyme treatment. The precipitation of proteins was significantly increased by succinylation after enzyme treatment. This also reduced the nucleic acid content of the yeast proteins to less than 2% and enhanced the extractability of nitrogenous material.

ADCC (K-cell)lysis of human erythrocytes sensitized with rhesus alloantibodies. I. Investigation of in vitro culture variables.

Abstract: An ADCC system has been developed using anti-D and papainized group O rhesus (D) positive red cells as the targets. Monocyte depleted mononuclear cell suspensions were effective in lysing appropriately sensitized red cells and papainization considerably enhanced the degree of specific lysis. Variation in culture volume and incubation in tubes or microplates were not critical to the degree of specific lysis obtained provided that the number of effector cells and target cells per culture was constant and the anti-D not diluted below the optimal concentration. Cytolytic activity was seen down to levels of 3 ng anti-D per culture. Specificity for lysis resided with the anti-D and not the effector cells. Several sources of anti-D were effective in inducing lysis of D positive red cells although individual variation was noted. Anti-c and anti-E were also shown to be effective in inducing specific lysis of red cells with the appropriate antigens.

Target cell recognition by cytolytic T cells: different requirements for the formation of strong conjugates or for proceeding to lysis.

Abstract: Conjugates between nonadherent peritoneal exudate lymphocytes from alloimmune mice, and neutral redlabeled or unlabeled tumor cell targets, were assayed by a procedure involving co-sedimentation of lymphocytes and target cells, vigorous resuspension, then visualization under phase-contrast or normal optics. The requirements for obtaining such conjugates, and for maintaining them in a state resistant to shear force, were compared to those for cytolysis in the usual 51 Cr release procedure. The following discrepancies were noted between the requirements for formation and visualization of such conjugates, and the requirements for the recognition stage of the cytolytic assay: 1) although in both cases divalent cations were required, more Mg ++ was necessary to obtain suspended conjugates than to obtain lysis; 2) although cytochalasin A added before cell contact was inhibitory in both cases, the post-recognition steps leading to lysis were not affected, whereas preformed conjugates were affected; 3) glucose was required to obtain suspended conjugates, whereas the hexose requirement in the 51 Cr release assay was restricted to a post-recognition state leading to lysis. The discrepancies between the two assays were explained by the observation that under all the inhibitory conditions tested (addition of EDTA, deoxyglucose, or cytochalasin A) preformed conjugates, although not immediately dissociated, were rendered labile to shear force. The resuspension step, necessary to visualize conjugates, imposed more stringent requirements on the T cell-target cell interaction than did the lytic assay using pelleted cells. The results suggest that the establishment of a tenacious linke between a cytolytic T lymphocyte and a target cell requires secondary, nonimmunologic interactions that follow the initial specific recognition event, and that these strengthening interactions are not necessary for cytolysis. The T cell receptor itself might only have a weak affinity, or be readily detached from the membrane, so that it has more of a “reading” than a “binding” role. A practical conclusion is that the metabolic requirements for T cell “recognition” are not fixed, but are imposed by the experimental conditions under which recognition is tested.