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PubMedDate not supplied

Compaction agent protection of nucleic acids during mechanical lysis.

Mechanical lysis is an efficient and widely used method of liberating the contents of microbial cells, but the sensitivity of large nucleic acids to shear damage has prevented the application of mechanical lysis to DNA purification. It is demonstrated that polycationic compaction agents can protect DNA from shear damage and allow chromosomal and plasmid DNA purification by mechanical lysis. In addition to being substantially protected during mechanical lysis, the compacted DNA can be separated with the insoluble cell debris, washed, and selectively resolubilized, yielding a substantially purified DNA product. An additional benefit of this method is that lysate viscosity is greatly reduced, allowing the use of much smaller processing volumes when compared with traditional lysis methods used in nucleic acid purification.

Chromatography, Agarose↗
PubMed1992

Bacteriophage lysis: mechanism and regulation.

Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E.

Amino Acid Sequence↗
PubMedDate not supplied

[Mechanical lysis of human erythrocytes. Membrane stabilization by plasma proteins].

Mechanical properties of erythrocyte membranes play an important role in red cell functions. Stability of human erythrocytes under deforming mechanical tensions which occur in the rapidly moving fluid is studied. The activation energy of the mechanical hemolysis determined by the temperature dependence of the hemolysis rate is 55 + 7 kJ/mol. The fragility of erythrocytes rises sharply as the salt concentrations increase. Glutaric dialdehyde forms a certain number of interprotein bonds which increase the fragility of erythrocytes. The mechanical stability of the erythrocyte membrane falls at high (0.5 M) ethanol concentrations. Blood plasma proteins, particularly human serum albumin, have a pronounced stabilizing effect. The hemolysis occurring during the rapid mixing is not probably associated with an osmotic mechanism since high sucrose concentrations do not prevent this process. The mechanical hemolysis depends both on the deforming tension arising in the membrane and on the state of the erythrocyte membrane.

Biomechanical Phenomena↗
PubMed2003

Prophylaxis for acute exacerbations of chronic bronchitis using an antibacterial sublingual vaccine obtained through mechanical lysis: a clinical and pharmacoeconomic study.

Chronic obstructive bronchitis (COB) complicated by acute exacerbations, or exacerbations, triggered by episodes of infection is the most demanding respiratory disease in our country today, to the extent that the implementation of every possible prevention strategy, in terms of both behaviour and prophylactic vaccination, is fully justified. From this point of view, in addition to the classic anti-viral vaccines, increasing importance is also being placed on antibacterial vaccines obtained by mechanically crushing pathogen bacteria (without the use of chemicals to denature the antigenic structures), which can be sublingually administered. In this study, conducted on 57 patients aged over 75 suffering from COB and affected by at least one exacerbation over the past 12 months, we evaluated the incidence of these exacerbations during a follow-up period, subsequent to prophylaxis with polyvalent bacterial mechanical lysate. As well as the absolute number of episodes, we also evaluated the seriousness of the episodes, the length of any antibiotic therapy and the overall cost of this therapy and of the prophylactic treatment. We then compared this data with that of the same period for the previous year, during which time no antibacterial prophylaxis had been administered. Both the absolute number of exacerbations, their length and seriousness were shown to be significantly reduced during the period of treatment compared to the control period. Similarly, the need to use antibiotic treatments, as well as the overall cost of the treatment of these patients, were shown to be reduced during the period of treatment compared to the control period.

Acute Disease↗
PubMed2005

Suppression of adenoviral gene expression in the liver: role of innate vs adaptive immunity and their cell lysis mechanisms.

BACKGROUND: Injection of adenoviral constructs causes liver infection prompting immunity, which suppress viral gene expression. Innate and adaptive immunity mediate these processes raising the question which pathways are the most prominent. METHODS: Adenovirus expressing the beta-galactosidase (beta-gal) gene was injected into normal and immunodeficient mice. Elimination of beta-gal-expressing hepatocytes and increases in liver enzymes were assayed. Major histocompatibility complex (MHC) class I densities, perforin channel insertion and apoptosis by Fas and tumor necrosis factor (TNF)-alpha were assayed. RESULTS: At high virus doses, suppression of viral gene expression was as efficient in immunodeficient as in normal mice, while at low doses effects of cytotoxic T lymphocytes (CTL) were demonstrable. Despite CTL priming and elimination of infected hepatocytes no liver injury is detected. Hepatocyte MHC I densities were able to trigger CTL granule exocytosis and perforin lysis in vitro but not in vivo. This is we show is because of decreased sensitivity of hepatocytes from infected mice to perforin and increased sensitivity to Fas and TNF-alpha lysis. CONCLUSION: Effector cells of the innate immune system are exceedingly effective in suppressing adenoviral gene expression. Perforin-independent pathways, those mediated by TNF-alpha and Fas are very efficient in hepatocytes from virus-infected livers.

Adenoviridae↗
PubMed1982

Inducible macrophage cytotoxins. II. Tumor lysis mechanism involving target cell-binding proteases.

Thioglycollate-elicited C57BL/6 peritoneal exudate macrophage monolayers (PEMM) stimulated with poly I . poly C or LPS released a macrophage cytotoxin (MCT) that rapidly bound to syngeneic (EL 4) or allogeneic (NS-1, YAC-1) tumor cells but did not bind to normal splenocytes. No binding to human (K562) tumor cells was observed. PEMM stimulated with poly I . poly C destroyed allogeneic tumor cells (NS-1) when separated by cell-impermeable Millipore filters in vitro; in contrast, PEMM not stimulated with poly I . poly C were incapable of lysing targets when separated by membranes. The reversible inhibitors N alpha-p-tosyl-L-arginine methyl ester and soybean trypsin inhibitor and the irreversible inhibitors N alpha-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride, of trypsin-like proteases, significantly or totally inhibited MCT cell-lytic activity for L-929 cells in vitro. Furthermore, modification of MCT-associated arginine residues by 1,2-cyclohexanedione completely blocked lytic activity. MCT was concluded to be an inducible nonspecific cell-lytic effector molecule elaborated by activated macrophages, which could bind to potential target cells, and was itself or was associated with a protease.

Animals↗
PubMed1979

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

The mechanism of lysis of anti-D coated human erythrocytes by human mononuclear K-cells was investigated. Red cell lysis was measurable 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 significantly inhibited by IgG1 and IgG3 subclasses with some inhibition by IgG2 but not by IgG4, IgA or IgM. This suggests that the K-cell 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.

Animals↗
PubMed1985

The isolation of natural killer (NK)-resistant variants of the K562 cell line by mutagenesis and selection with antibodies which inhibit NK cell-mediated lysis.

Mechanisms involved in the lysis of tumor cells by natural killer (NK) cells were investigated by using mutagenized K562 targets resistant to the effects of NK cells. K562 cells were treated with the mutagen methyl methanesulfonate (MMS) and, to select for resistant mutants, rabbit anti-idiotypic (anti-id) antibodies were used. This anti-id was raised to a monoclonal antibody 9.1C3 which itself blocked lysis by NK cells by binding to the effector cells; the anti-id inhibited killing by binding to the K562 targets, presumably to a cell surface protein relevant to a secondary event in the NK lytic pathway. MMS-derived mutants showed a heterogeneity of staining with the anti-id, allowing the antibody to be used with flow cytometry to select a population of K562 cells relatively negative in antigen expression. The degree of reactivity of K562 cultures with the anti-id antiserum and the resistance to lysis by NK cells were inversely related. Cultures of NK-resistant K562 cells with low expression of the anti-id structure were cloned by limiting dilution: 96 clones were analyzed and one subclone, C9/2, which was six-to sevenfold less sensitive to lysis than the parental K562 cell line, was used in further studies by cold target inhibition and single cell binding assays. The increased resistance to lysis of C9/2 was not due to a reduced expression of target recognition structures, and resistance could not be overcome by prolonging the time allowed for lysis to 18 hr nor by adding exogenous recombinant leukocyte interferon. Killing of the NK-resistant variant was inhibited by mannose-6-phosphate but not by the monoclonal antibody against which the anti-id antibody was raised. It is therefore suggested that the structure on the K562 cells recognized by the anti-id antibodies is a novel secondary receptor which is important in the later stages of the NK cell cytolytic cascade.

Antibodies, Monoclonal↗
PubMed1998

Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis.

Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2003

Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation.

A highly effective, reagentless, mechanical cell lysis device integrated in microfluidic channels is reported. Sample preparation, specifically cell lysis, is a critical element in 'lab-on-chip' applications. However, traditional methods of cell lysis require purification steps or complicated fabrication steps that a simple mechanical method of lysis may avoid. A simple and effective mechanical cell lysis system is designed, microfabricated, and characterized to quantify the efficiency of cell lysis and biomolecule accessibility. The device functionality is based on a microfluidic filter region with nanostructured barbs created using a modified deep reactive ion etching process. Mechanical lysis is characterized by using a membrane impermeable dye. Three main mechanisms of micro-mechanical lysis are described. Quantitative measurements of accessible protein as compared to a chemically lysed sample are acquired with optical absorption measurements at 280 and 414 nm. At a flow rate of 300 microL min(-1) within the filter region total protein and hemoglobin accessibilities of 4.8% and 7.5% are observed respectively as compared to 1.9% and 3.2% for a filter without nanostructured barbs.

Animals↗
PubMed1991

Mechanisms of lysis by cytotoxic T lymphocyte clones. Lytic activity and gene expression in cloned antigen-specific CD4+ and CD8+ T lymphocytes.

Cloned murine Th having properties of either Th1 or Th2 cells as well as CD8+ CTL were tested for the capacity to lyse: 1) nucleated target cells bearing Ag or coated with anti-CD3 mAb, or 2) SRBC target cells coated with anti-CD3 mAb in a short term 51Cr-release assay. The lysis of SRBC occurs by a mechanism that does not involve nuclear degradation but presumably does involve membrane damage. Three patterns were observed: CTL and some Th2 cells lysed efficiently nucleated target cells and SRBC coated with anti-CD3 mAb. Th1 and some Th2 T cells lysed nucleated target cells but did not lyse efficiently the SRBC coated with anti-CD3 mAb. Finally, some Th2 cells failed to lyse efficiently either nucleated or SRBC targets. We also examined these clones for their expression of N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity, and for the expression of perforin or CTLA-1 (granzyme B) mRNA. Total N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity expressed by CTL and Th2 clones tended to be higher than that of Th1 cells. Perforin mRNA and CTLA-1 mRNA were readily detectable in CTL and some Th2 clones. Expression of perforin and CLTA-1 mRNA correlated well with the capacity of these clones to lyse SRBC coated with anti-CD3 mAb. Our results show that some but not all Th2 clones have lytic characteristics similar to those of CD8+ CTL. Two mechanisms appear to contribute to their lytic process, one mechanism of lysis involves membrane damage that correlates with the expression of perforin mRNA; a second mechanism involves the induction of DNA degradation in the target cells. In contrast, some CD4+ effector cells appear to lack the capacity to lyse efficiently via the mechanism involving membrane damage and may only have the lytic activity associated with the capacity to induce DNA degradation.

Animals↗
PubMed1965

OXYGEN-STABLE HEMOLYSINS OF GROUP A STREPTOCCI. IV. STUDIES ON THE MECHANISM OF LYSIS BY CELL-BOUND HEMOLYSIN OF RED BLOOD CELLS AND EHRLICH ASCITES TUMOR CELLS.

In studies of the mechanism of lysis of red blood cells by washed streptococci with hemolytic activity (cell-bound hemolysin, CBH) no components released spontaneously by RBC or streptococci, or by interaction between these cells, could be found to induce the formation of soluble hemolysin by the streptococci. It was also found that separation of RBC from streptococci even by Millipore filter or a very thin layer of agar could prevent their hemolysis. By means of cellulose columns it was possible to separate RBC from streptococci after a short incubation. RBC thus separated from streptococci with which they had been incubated underwent hemolysis on subsequent incubation at 37 degrees C. By varying the period of incubation prior to separation it was possible to demonstrate the transfer of increasing amounts of hemolysin from streptococci to RBC with increasing periods of incubation. A considerable part of this appeared to be at a constant rate. A theory is presented on the relationship between the streptococcal cell-bound hemolysin and the group of oxygen-stable streptococcal hemolysins, in terms of a transferable hemolytic moiety and binding sites for this moiety on the streptococcal cell, on various molecular species which can act as inducers of the oxygen-stable hemolysins, and on the RBC, with the affinity of the respective binding sites for the hemolytic moiety increasing in that order.

Animals↗
PubMed1982

Studies on the mechanism of NK cell lysis.

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.

Animals↗
PubMed1995

Mechanisms of lysis by cytotoxic T cells.

When a cytotoxic lymphocyte binds to an antigenic cell, there is a reorientation of the Golgi apparatus and a directed exocytosis of cytoplasmic granules toward the offending cell. In the model originally proposed by Henkart, these granules were hypothesized to contain lytic molecules that contribute to the demise of the target cell. Initially, perforin/cytolysin was believed to be the major player in the mechanism of lysis. Recent work, however, using a variety of biochemical, molecular biological, and genetic approaches, has provided convincing evidence that other granule-associated molecules contribute to the lytic pathway. Of particular note are the granzymes, whose proteolytic activity is intimately associated with the induction of DNA fragmentation and apoptosis. In this review we summarize these recent experiments and present an updated view of the granule-mediated killing mechanism. For some years the universality of the granule-killing mechanism has been challenged. Experiments reported in the last few years on the characteristics of target cell death, detailed studies with cytolytic T cell lines, and, ultimately, with lymphocytes from mice that have been genetically altered using homologous recombination, have proven the existence of a second pathway. We discuss whether the existence of this alternate, Fas antigen/Fas ligand, mechanism really deals a knock-out blow to the granule exocytosis followers. Most likely it represents an important immunoregulatory system rather than a defense against pathogenesis. The granule-mediated and Fas-dependent mechanisms of cytolysis represent, at first sight, completely different pathways. However, it is becoming increasingly apparent that once the kiss of death has been received, through either granules or Fas, the events within the target cell are remarkably similar. Considerable attention is now being focussed on a family of interleukin-1 beta-converting enzymes (ICEs) that become activated within the target cell after attack by a cytotoxic lymphocyte. These enzymes are particularly interesting as they have now been shown to be directly involved in developmentally controlled, programmed cell death.

Animals↗
PubMed1996

Mechanisms of lysis by activated cytotoxic cells expressing perforin and granzyme-B genes and the protein TIA-1 in muscle biopsies of myositis.

OBJECTIVE: Polymyositis (PM) and cermatomyositis (DM) are inflammatory muscle diseases of autoimmune origin. A chronic mononuclear cell infiltrate is always present around PM and DM muscle damage. In PM, the predominant cells are activated cytotoxic cells, natural killer, and CD8+ T lymphocytes. Cytotoxic cells can kill the target cells via 2 mechanisms. Both pathways induce target cell death by releasing the molecules (granule exocytosis) perforin (PF), which attacks the target cell membrane and causes cell death by necrosis, and TIA-1 protein and granzyme-B (GZB), possibly responsible for apoptosis. We studied the mechanisms of lysis in muscle biopsies of myositis. METHODS: We used a panel of monoclonal antibodies to determine the phenotypes of reactive lymphocyte subsets, in situ hybridization to study the expression of PF and GZB genes, and immunohistochemistry to evaluate TIA-1 protein production in muscle biopsies from 14 patients with myositis (11 PM/3 DM). Results were compared to those obtained from muscle biopsies of 12 control patients with muscle weakness, including patients with muscle dystrophy and vasculitis, but without myositis. RESULTS: Abundant CD8+ cells, especially in endomysial sites in PM, formed the predominant phenotype. The predominant mononuclear cells observed in DM were CD4+ T cells and CD22+ B lymphocytes, in perivascular sites. The GZB and PF genes and the TIA-1 protein were expressed simultaneously in muscle samples from patients with myositis. GZB, PF, and TIA-1 positive cells were predominantly located in endomysial sites of PM, in the nonnecrotic muscle fibers. In DM, these positive cells were rare. CONCLUSION: In myositis, especially PM, cytotoxic cells may cause muscle damage and muscle cell necrosis and/or apoptosis by releasing several proteins (PF, GZB, and TIA-1 proteins) responsible for the lysis of these stimulating target cells. Some drugs (prednisone and cyclosporine) inhibit the release of GZB and PF. Their efficacy may be due in part to this inhibitory effect.

Adult↗
PubMed2004

Granule-dependent mechanisms of lysis are defective in CD8 T cells of HIV-infected, antiretroviral therapy-treated individuals.

BACKGROUND: HIV-specific cytotoxic T-cell (CTL) responses are defective in HIV-infected patients undergoing antiretroviral therapy (ART). This defect has been attributed to the decreased antigenic burden secondary to ART-associated suppression of HIV-replication, and is responsible for the rebounds of viraemia that occur when patients interrupt therapy. CTL are stimulated by type 1 cytokines and can kill targets via granule-dependent (perforin and granzymes) and -independent (tumour necrosis factor-alpha, CD95) mechanisms. METHODS: Granule-dependent and granule-independent mechanisms of CTL killing, as well as type 1 cytokine production by CD4 T cells, were analysed in 57 chronically HIV-infected ART-treated or ART-untreated individuals. RESULTS: The results can be summarized as follows: the frequency of gp160 (env)-specific interferon-gamma-secreting CD8 T lymphocytes correlates positively with HIV viraemia in ART-treated and -untreated patients; Env-specific perforin- and granzymes-expressing CD8 T lymphocytes, and Env-stimulated perforin and granzymes mRNA, are reduced in ART-treated patients independently of HIV viral load and of type 1 cytokine production; tumour necrosis factor-alpha production is increased in ART-treated individuals; and Env-specific immature CD8+28+27+ cells are only marginally augmented in ART-treated patients, Similar results are observed in cytomegalovirus-specific CD8 T cells and peripheral blood mononuclear cells. CONCLUSIONS: A defect of CTL function that selectively affects the granule-dependent mechanisms of lysis is observed in ART-treated individuals. Because interferon-gamma production is higher in these patients, this could be a defect primarily involving CTL. These data suggest an independence of CD8 T-cell numbers and their lytic ability in HIV-infected, ART-receiving patients. Immunomodulants are needed to successfully treat HIV infection.

Adult↗