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Showing papers on "Lysis published in 1982"


Journal ArticleDOI
01 May 1982-Cell
TL;DR: Direct comparison of the degree of ricin resistance conferred by transfer of antiricin antibodies revealed pinosome lysis to be equal, if not superior, to injection mediated by red blood cells.

323 citations


Journal Article
TL;DR: The mechanism of cytolysis by murine NK cells was analyzed using a variety of metabolic inhibitors that have proven informative in studying the lytic mechanism of CTL and the mechanism of histamine release by mast cells to lend support to the stimulus-secretion model.
Abstract: The mechanism of cytolysis by murine NK cells was analyzed using a variety of metabolic inhibitors that have proven informative in studying the lytic mechanism of CTL and the mechanism of histamine release by mast cells. Target cell binding occurred in the absence of calcium and was inhibited by only one of the agents studied, cytochalasin B. Lysis was initiated by addition of Ca2+ ions, as in the case of CTL. Subsequent to target cell binding, but prior to programming for lysis by Ca2+, NK cell lytic activity could be suppressed by inhibitors of chymotrypsin-like, but not trypsin-like proteases, in contrast to CTL. In addition, 3-deaza-SIBA, an inhibitor of transmethylation reactions and quinacrine, an inhibitor of phospholipase A2, appear to act before the Ca2+-dependent programming for lysis. Sr2+ ions blocked the lytic function, as did trifluoperazine (stelazine), the former presumably competing for ionic calcium, the latter known to block binding of Ca2+ to calmodulin. 8Br-cAMP and colchicine blocked later steps required for lysis. With the possible exception of trifluoperazine, all of the agents that blocked NK cell lysis are known to inhibit histamine release from mast cells. These results lend support to the stimulus-secretion model, originally proposed to explain the mechanism of CTL cytolysis, as relevant to the mechanism of lysis by NK cells.

134 citations


Journal ArticleDOI
TL;DR: The results suggest a possible role of β-1.3-glucanases in the mechanism of release of β -glucosidase from cell walls of T. pseudokoningii; this is discussed.
Abstract: The formation and excretion of beta-glucosidase from Trichoderma pseudokoningii was studied during growth on different carbon sources. The enzyme was present under all conditions examined, but increased activity was found during growth on carbon sources favouring slow growth. Two different patterns of beta-glucosidase excretion were observed: on carbon sources allowing fast growth a relatively high percentage of total activity was found in the culture fluid, which decreases as the culture grows older, but which increases again during the phase of cell lysis; on carbon sources favouring slow growth, excretion is initially low, but commences at later culture stages. Changes in cell wall composition and cell wall lytic enzyme activities associated with the cell walls were examined during phases of high and low ratios of extracellular to cell-wall bound beta-glucosidase activities. With no component of the cell wall (chitin, alpha-glucan, beta-glucan, galactosamine) could correlation with beta-glucosidase excretion be identified. Among a number of cell-wall lytic, cell-wall associated enzymes (alpha-glucanases, beta-glucanases, glucosaminidase, galactosaminidase), beta-1.3-glucanase activity correlated well with the excretion of beta-glucosidase. The results suggest a possible role of beta-1.3-glucanases in the mechanism of release of beta-glucosidase from cell walls of T. pseudokoningii; this is discussed.

130 citations


Journal ArticleDOI
TL;DR: Results suggest that Ca2+ transport of intraerythrocytic parasites is coupled to a proton-motive force across the Plasmodia plasma membrane, suggesting that a DCCD- and CCCP-sensitive membrane potential in P. chabaudi-infected cells is suggested.
Abstract: The calcium content and transport processes of Plasmodium chabaudi-infected rat erythrocytes were analyzed by atomic absorption spectroscopy and 45Ca2+ flux measurements. Infected erythrocytes, after fractionation on metrizamide gradients according to stage of parasite development, exhibited progressively increasing levels of Ca2+ with schizont and gametocytes containing 10- to 20-fold greater calcium levels than normal cells (0.54 +/- 0.25 nmol/10(8) cells). 45Ca2+ flux experiments showed both increased influx and decreased efflux in infected erythrocytes. Tris/NH4Cl lysis of normal erythrocytes preloaded with 45Ca2+ with the Ca2+ ionophore A23187 released less than 90% of cell calcium after incubation in ethyleneglycol bis(aminoethylether) N,N'-tetraacetic acid containing buffer, whereas lysis of the infected erythrocyte membrane resulted in release of 10-20% cell Ca2+, with the remaining portion associated with the isolated parasite fraction. This information together with the effects of various metabolic inhibitors indicates the presence of a parasite Ca2+ compartment in P. chabaudi-infected erythrocytes. Dicyclohexylcarbodiimide (DCCD) an inhibitor of proton ATPases of chloroplasts, bacteria, yeast, and mitochondria, and the proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), inhibited Ca2+ influx and stimulated efflux from infected cells. These results combined with evidence for a DCCD- and CCCP-sensitive membrane potential in P. chabaudi-infected cells (Mikkelsen et al., accompanying manuscript) suggest that Ca2+ transport of intraerythrocytic parasites is coupled to a proton-motive force across the Plasmodia plasma membrane.

117 citations


Journal ArticleDOI
TL;DR: The largest of the fragments produced by AluI digestion of ϕX174 RFI DNA comprises genes E and J as well as parts of genes D and F, and it is shown that the expression of this gene alone is sufficient to trigger cell lysis.
Abstract: The largest of the fragments produced by AluI digestion of ϕX174 RFI DNA comprises genes E and J as well as parts of genes D and F. This DNA fragment (1007 bp) was cloned into the lac z′ gene of plasmid pUR222. In the recombinant plasmid pUH12, transcription of the ϕX174 genes is controlled by the lac p-o region. Induction of the cloned genes by addition of the lac inducer, IPTG, resulted in lysis of the bacteria. Cloning of the corresponding AluI-fragment from ϕX174am3 DNA, carrying an amber mutation in gene E, showed that the expression of this gene alone is sufficient to trigger cell lysis. The time interval between the addition of IPTG and the onset of lysis depended on the concentration of the inducer, however, the rate of lysis was similar at all IPTG concentrations used.

115 citations


Journal ArticleDOI
TL;DR: Chinese hamster ovary cells in exponential growth were incubated with various concentrations of hematoporphyrin derivative, and lysis of the cell membrane may be largely responsible for cellular inactivation following HpD photoirradiation.
Abstract: Chinese hamster ovary cells in exponential growth were incubated with various concentrations of hematoporphyrin derivative (HpD). Cellular porphyrin content was determined after 2 h incubation at 37°C using [3H]-hematoporphyrin derivative. Photoactivation of cell-bound HpD by red light resulted in a family of survival curves with terminal slopes proportional to cellular HpD concentration. The degree of cellular lysis, assayed 1 h after illumination using a chromium-51 labeling technique, was also found to be related to cellular HpD concentration. The amount of 51Cr released increased with post-irradiation incubation to a level parallel to cell lethality as measured by colony formation. These data suggest that lysis of the cell membrane may be largely responsible for cellular inactivation following HpD photoirradiation.

82 citations


Journal ArticleDOI
TL;DR: Density gradient centrifugation techniques, using iso-osmotic colloidal silica suspensions (Percoll), were developed for the isolation of insulin secretory granules from a transplantable rat islet cell tumour, finding that the isolated granules contained 150 or more proteins besides insulin-related peptides.
Abstract: Density gradient centrifugation techniques, using iso-osmotic colloidal silica suspensions (Percoll), were developed for the isolation of insulin secretory granules from a transplantable rat islet cell tumour. These procedures were readily completed within 7 h and from each animal yielded approximately 1 mg of granule protein. The isolated granules were essentially free of other subcellular organelles as evaluated by their contents of marker proteins, electron microscopy and by electrophoretic analyses. Their susceptibilities to lysis at low osmotic strength, at pH values above 7 or in media containing sodium ions were similar to those of granules partially purified from islets. Insulin comprised 50-60% of the total granule protein when determined by immunoassay or by densitometry of electrophoretic profiles. The proinsulin content was marginally higher than that of islets, as was the ratio of insulins I to II. Electrophoretic analyses revealed that the secretory granules contained 150 or more proteins besides insulin-related peptides. The majority of these had acidic isoelectric points and were located both within the granule interior and its enveloping membrane.

73 citations


Journal ArticleDOI
TL;DR: In this paper, the DNA-protein complex after lysis of macronuclei from the ciliated protozoan Oxytricha at 0.5-2 M NaCl was analyzed.
Abstract: On lysis of macronuclei from the ciliated protozoan Oxytricha at 0.5-2 M NaCl, the DNA, which is normally found as discrete molecules ranging from 0.5 to 20 kilobases, appears in high molecular weight aggregates. Various treatments of the macronuclear lysate (i.e., nucleases, proteases, variation of salt, pH, and temperature) indicate that preservation of the aggregate structure depends on both nucleic acid-nucleic acid and nucleic acid-protein interactions. Purification of the DNA-protein complex after lysing the nuclei in 2 M NaCl shows that one major nuclear protein copurifies with the DNA. As shown by DNA-protein binding experiments, this protein has a high affinity for DNA; however, no evidence for sequence specificity of the protein binding was obtained. Chromatin reconstitution experiments suggest that the protein in itself is not sufficient for DNA aggregation in nuclei, but other factors, possibly the native chromatin structure, are required. Electron microscopy of the purified DNA-protein complex showed structures similar to those observed previously with in vitro-aggregated purified macronuclear DNA (14). A model is presented in which the terminal inverted repeat sequences found on all macronuclear DNA molecules interact with each other forming multistranded DNA complexes. The formation of these structures may be accelerated and stabilized by a protein in vivo.

72 citations


Journal ArticleDOI
TL;DR: Induction of an Sts clone showed that the S gene product is stable and capable of inducing lysis long after the cessation of synthesis of S geneProduct, and a model for S action is proposed.
Abstract: The functions of the bacteriophage lambda lysis genes S, R, and Rz were investigated. Different combinations of wild-type and inactive alleles of all three lysis genes were cloned into the plasmid pBH20 and were expressed under the control of a lac operator-promoter. The involvement of the Rz gene in lysis was proposed in our previous work and was confirmed by the Mg2+-dependent lysis defect of clones in which part of the Rz gene is deleted. Membrane vesicles prepared from induced S+ cells were shown to have a severely reduced capacity for active transport of glucose; this defect was detectable at least 20 min before lysis. Cell viability was also shown to decrease very soon after induction, long before physiological death and lysis; this decrease in viability is absolutely dependent on S expression and independent of R and Rz. The nonviable fraction of cells at any time after induction was demonstrated to be equal to the fraction committed to eventual lysis. Induction of an Sts clone showed that the S gene product is stable and capable of inducing lysis long after the cessation of synthesis of S gene product. A model for S action is proposed.

72 citations


Journal ArticleDOI
TL;DR: Methanobacterium bryantii was found to undergo rapid lysis when grown in a prereduced chemically defined medium under H2-CO2 (4:1, vol/vol).
Abstract: Methanobacterium bryantii was found to undergo rapid lysis when grown in a prereduced chemically defined medium under H2-CO2 (4:1, vol/vol). The addition of 20 mM MgCl2 to the medium gave, rather than rapid lysis, a gradual formation of phase-dark spherical bodies which in thin section appeared as true protoplasts. In general, the protoplasts were stabilized by divalent but not monovalent cations and, unlike whole cells, were sensitive to lysis by Triton X-100. Electron microscopic examination revealed that protoplast formation was preceded by a general breakdown of the cell wall with an apparent squeezing out of the protoplast through the degraded wall. The growth of cells was greatly increased and not accompanied by detectable lysis in a medium modified by elevating the levels of nickel and ammonium.

66 citations


Journal ArticleDOI
T Kamiryo, M Abe, K Okazaki, S Kato, N Shimamoto 
TL;DR: The purification procedure includes the effective conversion of cells to spheroplasts with Zymolyase and sodium sulfite and the separation of the organelles at extremely low ionic strength.
Abstract: Yeast peroxisomes were purified to near homogeneity from cells of Candida tropicalis grown on oleic acid for the purpose of examining the possible presence of DNA in this organelle. The purification procedure includes the effective conversion of cells to spheroplasts with Zymolyase and sodium sulfite and the separation of the organelles at extremely low ionic strength. The mitochondrial contamination was less than 1%, based on several criteria, and the yield of peroxisomes was about 40%. The purified peroxisomal fraction contained a very small amount of DNA, which yielded restriction fragments indistinguishable from those of mitochondrial DNA. The absence of DNA in peroxisomes was also supported by cesium chloride density gradient centrifugation of the organelles lysed with a detergent, staining of the organelles with a fluorescent dye specific to DNA, and labeling of the DNA with [3H]adenine.

Journal ArticleDOI
TL;DR: Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.

Journal ArticleDOI
TL;DR: Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.
Abstract: Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption. Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate. This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control. Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells. When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2. However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control. When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed. Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.

Book ChapterDOI
TL;DR: The hypothesis that the resulting cell lesion is identified with an alteration of the lipid bilayer membrane had been confirmed by single killer cell study and it is proposed that phospholipase enzymes are involved in the membrane alterations.
Abstract: T cell mediated lysis has been extensively studied using the standard chromium release test (1) which measures semi-quantitatively the activity of cytotoxic T lyphocyte (CTL) suspensions from in vivo (2, 3) and in vitro (4) experimental systems. As reviewed by Berke (5) and more recently by Henney (6, 7), biological conditions required for T cell mediated cytotoxicity (T CMC) were well defined and the lytic process was dissected into three succesive stages: A) Binding, which is dependent upon the specific recognition by effector cells (E) of sensitizing antigens carried by targets (T). E-T cell contact is a necessary step for target lysis. B) Lethal hit during which stage a lesion occurs on the target cell. This process is at once “the most interesting and most enigmatic,” (7) since we do not know yet the nature of the “hit.” One hypothesis indeed proposed that target cell destruction is caused by a soluble mediator secreted by the killer lymphocytes (8). C) Cytolysis which does not require the continuous presence of the effector cell. The target cell undergoes a series of membrane permeability alterations leading to cell destruction (5–7).

Journal Article
TL;DR: An exceptional cell type of Burkitt's lymphoma cell lines is described which appears to have a reduced tolerance to certain types of DNA damage, the reduced tolerance being expressed by early cell lysis or disruption.
Abstract: Three Burkitt9s lymphoma cell lines were studied for their response to cis-diamminedichloroplatinum(II) (cis-DDP) and l-phenylalanine mustard (L-PAM) with the objective of relating cytotoxicity to DNA cross-linking in human cells. Cytotoxicity was measured by cell proliferation and colony formation assays. DNA interstrand and DNA-protein cross-linking were measured by alkaline elution. Two of the cell lines showed quantitative agreement between cytotoxicity and DNA cross-linking assays following treatment with cis-DDP or L-PAM. The correlation with DNA-protein cross-linking was not as good as with interstrand cross-linking. With the third cell line, cytotoxicity and interstrand cross-linking were correlated in response to L-PAM but not in response to cis-DDP. When this line was treated with cis-DDP, cross-linking was low, but cytotoxicity was high. This line differed from the other two lines in that, following treatment with either cis-DDP or L-PAM, substantial cell lysis or disruption occurred 12 to 14 hr following treatment. The results are consistent with a general correlation between DNA cross-linking and cytotoxicity in human cells treated with bifunctional agents. An exceptional cell type, however, is described which appears to have a reduced tolerance to certain types of DNA damage, the reduced tolerance being expressed by early cell lysis or disruption.

Journal ArticleDOI
TL;DR: The results of these experiments indicate that the lambda S product is present in the inner membrane, that it increased the permeability of the membrane for all of the small molecules that were tested, and that its action is reversible.
Abstract: The S gene of bacteriophage lambda is a late gene required for cell lysis, but unlike the other two lysis genes, R and Rz, it does not code for an endolysin Earlier studies have shown that the S gene product inhibits respiration and macromolecular synthesis and makes the inner membrane permeable to sucrose In this study, the effect of the S gene product on a number of Escherichia coli membrane functions (active transport, permeability, respiration, and transhydrogenase and ATPase activity) were measured, and a product of the lambda S gene was identified in the inner membrane fraction by two-dimensional polyacrylamide gel electrophoresis The results of these experiments indicate that the lambda S product is present in the inner membrane, that it increased the permeability of the membrane for all of the small molecules that were tested, and that its action is reversible The simplest explanation of these results is that the S gene product forms a hydrophilic pore through the inner membrane, allowing small molecules and lambda lysozyme to pass through

Journal ArticleDOI
TL;DR: Several membrane properties of vitamin E-deficient and normal erythrocytes were studied after incubation of these cells with hydrogen peroxide to speculate that changes in membrane function, which follow peroxidant injury, contribute to the shortened red cell survival in the vitamin E deficient state.
Abstract: Summary: Several membrane properties of vitamin E-deficient and normal erythrocytes were studied after incubation of these cells with hydrogen peroxide. Measurements of mean corpuscular volume, cation permeability, membrane Na+, K+ ATPase activity, red cell filterability through 5 μ millipore filters, and membrane protein pattern on sodium dodecyle sulfate-gel electrophoresis revealed marked alterations before lysis. Vitamin E sufficient cells were unaffected by a similar incubation with hydrogen peroxide. We speculate that the changes in membrane function, which follow peroxidant injury, contribute to the shortened red cell survival in the vitamin E deficient state.

Journal Article
TL;DR: It is suggested that the glucocorticoid-induced lysis of human CLL cells is similar to the phenomenon observed in rat or murine lymphocytes and is mediated by interaction of the steroid molecule with the cytoplasmic receptor and appears to activate specific gene(s) the products of which eventually cause cytolysis.
Abstract: Human chronic lymphocytic leukemia (CLL) cells like prothymocytes and immunoactivated T-lymphocytes are readily lysed in vitro by pharmacological concentrations of glucocorticoids such as cortisol, whereas peripheral blood lymphocytes and thymocytes are unaffected by the hormone. In this study, metabolic and ultrastructural aspects of the cortisol-induced killing process of CLL cells are recorded. In vitro lysis was found to be temperature dependent and was detected only after 6 to 8 hr incubation with cortisol by means of the trypan blue exclusion test. However, 30 min of incubation with cortisol at either 37° or 4° followed by the removal of the hormone was still sufficient to induce the lytic process. Ultrastructural studies demonstrated sequential changes in the cytoplasm, including swelling of mitochondria and cytoplasmic decompartmentalization, followed by loss of surface microvilli with the appearance of “holes” in the cell membrane, and subsequent condensation of nuclear chromation. The large holes in the membrane appearing after 6 hr of incubation with the hormone may be the cause for the penetration of the viable stain into the dead cells, as seen by light microscopy. Addition of metabolic inhibitors including actinomycin D, puromycin, and cycloheximide following administration of cortisol resulted in inhibition of the cell lysis. An excess of an antagonist such as cortexolone was found to inhibit the cortisol-induced cytolysis of the CLL cells. It is suggested that the glucocorticoid-induced lysis of human CLL cells is similar to the phenomenon observed in rat or murine lymphocytes and is mediated by interaction of the steroid molecule with the cytoplasmic receptor. The resulting complex appears to activate specific gene(s) the products of which eventually cause cytolysis.

Journal ArticleDOI
TL;DR: The tBHP-treated cells provide a useful model for analysis of the sequence of events in oxidative hemolysis, and their effects are related to the formation of methemoglobin (MetHb), which catalyzes the homolytic cleavage of t BHP to form OH • radicals.

Patent
21 Jul 1982
TL;DR: In this paper, a target cell lysis factor (TCLF) is produced by exposing a human cell line capable of producing TCLF to an inducer, and collecting and purifying the accumulated TCLFs.
Abstract: Target Cell Lysis Factor (TCLF) is produced by exposing a human cell line capable of producing TCLF to a TCLF inducer to induce TCLF production, and collecting and purifying the accumulated TCLF. The TCLF which is produced includes lymphotoxin and human Tumor Necrosis Factor (hTNF). The hTNF may be separated and purified. The human cell lines are preferably human leucocyte and human lymphoblastoid lines, such as BALL-1, TALL-1, NALL-1 Namalwa, MOLT-3, Mono-1, M-7002, B-7101, JBL, EBV-Sa, EBV-Wa, EBV-HO, BALM 2, CCRF-CEM, DND-41 and CCRF-SB, as well as human cell lines which are obtainable by transforming normal human monocytes, or granulocytes. All of these human cell lines are multipliable by implanting them in a non-human warm-blooded animal, or alternatively, allowing them to multiply in a conventional-type diffusion chamber by which the nutrient body fluid of a non-human warm-blooded animal is supplied to them.

Journal ArticleDOI
TL;DR: Results from experiments in saliva versus buffer concluded that saliva had no major effect on the extent of lysozyme adsorption by S. sanguis 903 other than providing a source of ionic strength.
Abstract: Several strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii plus fresh isolates of Streptococcus salivarius were surveyed for their abilities to deplete lysozyme from human-whole-saliva supernatant. Bacteria were incubated in saliva for 60 min at 37 degrees C and then removed by centrifugation, and the recovered supernatant solutions were assayed for lysozyme activity by using whole cells of Micrococcus lysodeikticus as the substrate. Mean lysozyme depletions by bacterial strains varied over a wide (eightfold) range. The greatest mean depletion of lysozyme (60 to 70%) was observed with S. sanguis (biotype I), serotype b of S. mutans, and the fresh S. salivarius isolates. The lowest mean depletion was noted with S. mitis (15%) and biotype II S. sanguis (ca. 30%). The remaining species and strains exhibited an intermediate degree of depletion. In studies with S. sanguis 903, lysozyme was depleted by normal or heated (90 degrees C, 30 min) bacteria and could be recovered from the organism. Furthermore, under appropriate conditions, lysozyme depletion by cells at 0 and 37 degrees C was very similar. On the basis of these observations, we concluded that depletion was due to the adsorption of lysozyme by the organism. With S. sanguis 903, lysozyme adsorption depended on the concentration of bacteria, time of incubation, and the ionic strength of the medium. The extent of adsorption, however, was independent of pH's of 3.9 to 8.3. When a low concentration of S. sanguis 903 was used, lysozyme adsorption reached saturation (4 mug of adsorbed lysozyme per 10(7) cells) at 20 mug of lysozyme added per ml. Salivary lysozyme adsorption by several other oral microorganisms (A. viscosus WVU 626 and WVU 627, S. sanguis 73x11, S. mutans BHT, and S. salivarius NG) was similar to that of S. sanguis 903 in sensitivity to ionic strength. Lysozyme adsorption by S. sanguis 903 from either a buffer solution or a saliva supernatant was more sensitive to ionic strength at 0 than at 37 degrees C. On the basis of results from experiments in saliva versus buffer, we concluded that saliva had no major effect on the extent of lysozyme adsorption by S. sanguis 903 other than providing a source of ionic strength. A comparison of pH and ionic strength effects on lysozyme adsorption by S. sanguis 903 with literature reports of lysozyme lysis of whole cells and hydrolysis of cell walls, peptidoglycan, and (GlcNAc)(4) suggested that adsorption by S. sanguis 903 was more dependent on electrostatic interactions than was lysozyme catalysis. The possibility is discussed that anionic bacterial surface components mediate lysozyme adsorption and temper the potential effects of lysozyme on the microorganisms.

Journal ArticleDOI
TL;DR: In all the fungi studied, there is lysis of the cell walls during autolysis, confirmed by the change of thecell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.
Abstract: A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes. Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present. In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.

Journal ArticleDOI
TL;DR: Both light micrographic examination and the marker enzyme assays have shown that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible.
Abstract: Protein bodies were prepared from the cotyledons of pumpkin ( Cucurbita sp.) seeds by employing a nonaqueous isolation method. Both light micrographic examination and the marker enzyme assays have shown that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible. A proteolytic enzyme catalyzing the limited hydrolysis of carboxymethylated γ′ chain of globulin was found to be present in the protein bodies. The specific activity in the protein body (18 units per milligram protein) was higher than that in the whole cell extract (13 units per milligram protein), indicating that the limited proteolytic enzyme was localized in the protein body. After lysis of the protein bodies using hypotonic buffer solution, the suborganellar components (matrix, membranes, and crystalloids) were separated by sucrose density gradient centrifugation. The crystalloid was composed of only globulin, a major seed protein. The major proteins of matrix and membrane fractions were shown to have mol wt of approximately 10,000. About 90% of the limited proteolytic activity was found in the matrix region.

Journal ArticleDOI
TL;DR: From the different selectivity patterns of apMPh and NK cells, it is concluded that lysis of target cells is not based solely on inherent sensitivity to cytolysis, and is probably due to variations in expression of target‐cell structure recognized by each type of effector cell, and/or in susceptibility to the lytic mechanism of the various effector populations.
Abstract: Several murine tumor-cell lines were tested by isotope release assays for their susceptibility to lysis by either activated peritoneal macrophages (apMPh), macrophage-like (MPh-like) cell lines, or natural killer (NK) cells. The qualitative selectivity of tumor-cell lysis by these different effector cells was quite disparate. The rank order of target cell susceptibility to lysis by apMPh in 24 h assay was L5178Y greater than P815 approximately equal to RL male greater than YAC-1 approximately equal to MBL-2. This was seen regardless of whether peritoneal MPh (pMPh) were activated by LPS or poly I:C. Two MPh-like cell lines, PU-5R and FC-1, had a pattern of cytotoxic activity against these target cells that closely paralleled that associated with apMPh, although levels of reactivity differed quantitatively among the effector cells. In contrast, the MPh-like cell line RAW-264 expressed a qualitatively different pattern of target-cell selectivity, preferentially lysing MBL-2, which was relatively refractory to lysis by other MPh-like cell lines or apMPh in the 24 h cytolytic assay. When spontaneous or interferon (IFN)-augmented NK activity was measured against the same panel of target cells, the pattern of selectivity was qualitatively different from that observed for apMPh. The consistent rank order of susceptibility to lysis by NK cells was YAC-1 greater than RL male 1 greater than P815 approximately equal to L5178Y approximately equal to MBL-2. The characteristic patterns of target-cell selectivity for apMPh or NK cells were the same for all of the strains of mice tested. From the different selectivity patterns of apMPh and NK cells, it is concluded that lysis of target cells is not based solely on inherent sensitivity to cytolysis. Instead, selectivity of lysis is probably due to variations in expression of target-cell structures recognized by each type of effector cell, and/or in susceptibility to the lytic mechanism(s) of the various effector populations.

Journal ArticleDOI
TL;DR: The mechanisms involved in the activation of autolytic enzymes in Staphylococcus aureus, by leukocyte extracts, cationic proteins, phospholipase A2, amines, and membrane-damaging agents was studied, and it is suggested that the prolonged survival of intact bacterial cells in such a milieu may be due to the inactivation of autoytic enzymes.
Abstract: The mechanisms involved in the activation of autolytic enzymes in Staphylococcus aureus, by leukocyte extracts, cationic proteins, phospholipase A2, amines, and membrane-damaging agents was studied in a resting cell system as well as by growing staphylococci. The bacteria were labeled with [14C]N-acetylglucosamine and were subjected to a variety of agents either in 0.1 M acetate buffer, pH 5.0, or in phosphate buffer, pH 7.4. While intact log-phase cultures were found to undergo partial autolysis at pH 5.0 and almost complete lysis at pH 7.4, both heat-killed bacteria and bacterial cell walls were completely resistant to autolysis in buffers. Autolysis at pH 5.0 can be further activated by leukocyte extracts, nuclear histone, crystalline ribonuclease, egg-white and human lysozyme, phospholipase A2, as well as by spermine, spermidine, and polymyxins B and E. The addition of viable log-phase bacteria to radiolabeled heat-killed staphylococci or to radiolabeled cell walls which had been cleaned off autolytic enzymes resulted in degradation of the radiolabeled targets. The data suggest that the various inducers of autolysin activation caused leakage of autolytic enzymes from the intact bacteria which attacked the depolymerized the bacterial cell walls. Anionic polyelectrolytes like heparin, dextran sulfate, suramine, polyglutamic acid, and liquid (polyanethole sulfonic acid) markedly inhibited both spontaneous and induced lysis. Staphylococci which had grown in the presence of anionic polyelectrolytes became highly resistant to lysis triggered by any of the inducers of autolysis. Since inflammatory exudates are known to be rich in anionic polyelectrolytes, it is suggested that the prolonged survival of intact bacterial cells in such a milieu may be due to the inactivation of autolytic enzymes. It is also postulated that the degradation of certain bacterial species following phagocytosis or extracellular degradation may not be the result of the action of hydrolytic enzymes but rather the result of activation by leukocyte factors of autolytic enzymes which lead to bacteriolysis.

Journal ArticleDOI
TL;DR: Modification of tryptophan residues of Zymolyase B resulted in decreased sensitivity to yeast mannan and a decrease in the activity of the enzyme on yeast cell walls as well as heat-treatment.
Abstract: Zymolyase B decreased the turbidity of a yeast cell wall suspension by about 50% and caused release of peptide-mannan from the cell walls. However cell walls treated with the enzyme still maintained the cell shape. The effect of the enzyme on the cell walls was inhibited by yeast mannan and completely counteracted by treatment of the enzyme with DFP. The activity was not affected by pH, but was considerably reduced by incubation of the enzyme at 55°C for 15 min, a treatment that did not affect the proteolytic activity. Heat-treatment decreased the molecular weight of the enzyme from 29,000 to 22,500 and its sensitivity to yeast mannan. Yeast mannan caused noncompetitive inhibition of the proteolytic activity of the native enzyme and competitive inhibition of that of the heat-treated enzyme. Modification of tryptophan residues of Zymolyase B resulted in decreased sensitivity to yeast mannan and a decrease in the activity of the enzyme on yeast cell walls as well as heat-treatment. On the basis of these res...

Journal ArticleDOI
TL;DR: The significance of the lytic reaction described here is not known but may lie in antibody mediated release of microbicidal substances from platelets.
Abstract: Antibody-coated erythrocytes are lysed by murine C5- whole blood but not by plasma separated from such blood. The lytic activity has been shown to derive from platelets that attach to sensitized cells probably through membrane receptors for C3b. Whole blood or platelet-rich plasma (prp) obtained from mice that have been treated with purified cobra venom factor has little or no activity unless it is fortified with fresh C5- plasma. Lysis is observed only if the reactants are incubated at 37 degrees C and mechanical shaking is practiced, at least intermittently, throughout the period of incubation. Adherence of platelets and subsequent lysis are mediated by antibodies of a variety of immunoglobulin classes, including those that fail to mediate complement-dependent lysis. Platelet-mediated lysis is limited to cells to which the platelets adhere; 51Cr labeled, unsensitized cells that are mixed with prp and sensitized, unlabeled cells do not release 51Cr. Normal murine lymphoid cells and ascites tumor cells of mice, rats, and guinea pigs were apparently unaffected by sensitization and incubation with prp. However, because adherence of platelets to these sensitized cells was not observed, it is not clear whether the cells are resistant to the lytic action of platelets or whether the conditions of incubation were unfavorable for the attachment of platelets to the surfaces of nucleated cells. The significance of the lytic reaction described here is not known but may lie in antibody mediated release of microbicidal substances from platelets.

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TL;DR: Biochemical and spectral analyses of the chromatin preparations show protein: DNA ratios similar to that found in other mammalian tissues and cells, but the RNA content is lower, andamination of the Chromatin protein by the cytoplasmic protein and membrane phospholipid is acceptably low.

Journal ArticleDOI
TL;DR: The pattern of relative abundanccs of the sequences in transported poly(A)-rich RNA was intermediate between that of polysomal and nuclear poly( A)-rich RNAs, and indicated that some degree of sequence-specific selection of processing, or transport, or both, can be maintained in isolated nuclei.
Abstract: 1 A variety of procedures for obtaining metabolically active nuclei from rat hepatoma tissue-culture (HTC) cells has been explored. All gave preparations which were, to a greater or lesser extent, contaminated with cytoplasmic RNA, which appeared to be absorbed non-specifically during the lysis of the cells. 2 A medium containing spermidine, monovalent and divalent cations, and cytosol has been defined in which the integrity of the isolated nuclei was maintained during incubation in virro. 3 The release of steady-state-labelled and pulse-labelled RNA from HTC cell nuclei during incubation in this medium has been studied. The transport of both was dependent on ATP and cytosol, but was distinguishable by the kinetics and by the fact that only the transport of pulse-labelled RNA appeared to involve hydrolysis of ATP. Transported RNA included ribosomal RNA and messenger-likc RNA as judged by criteria of size, oligo(dT)-cellulose binding, and organisation into ribonucleoproteins. A large proportion of the cytoplasmic RNA which adhered to the nuclei was also released during incubation in vitro. 4 Although the poly(A)-rich RNA transported in vitro was contaminated with poly(A)-rich RNA sequences arising from non-specific leakage of nuclear RNA and from the release of nucleus-adherent cytoplasmic RNA, the extent of this contamination was comparatively small (10% and 10–15% by weight respcctively). The transportcd poly(A)-rich RNA has been shown to consist of a complex mixture of sequences at heterogeneous abundances, resembling polysomal poly(A)-rich mRNA from HTC cells. 5 The pattern of relative abundanccs of the sequences in transported poly(A)-rich RNA was intermediate between that of polysomal and nuclear poly(A)-rich RNAs, and indicated that some degree of sequence-specific selection of processing, or transport, or both, can be maintained in isolated nuclei. Sequence-specific selection in vitro at the level of individual poly(A)-rich RNA sequences was detected using cloned cDNAs for poly(A)-rich polysomal RNA sequences.

Journal ArticleDOI
TL;DR: It is suggested that target cell susceptibility to NK-mediated lysis may in part be dependent on the stage of differentiation of the tumor cell target.