Complete Cell Lysis Research Articles

Ultrastructural changes produced in Ehrlich ascites carcinoma cells by ultraviolet-visible radiation in the presence of melanins.

Irradiation of Ehrlich ascites carcinoma (EAC) cells in the presence of pheomelanin, i.e., red hair melanin (RHM), has been reported to produce extensive cell lysis. Irradiation in the presence of eumelanin, i.e., black hair melanin (BHM), or irradiation in the absence of either type of melanin did not produce this effect. We observed that RHM particles penetrated the cell membrane without apparent structural damage to the cell or the cell membrane. Irradiation of the cells in the absence of melanin did not produce any changes in the ultrastructure of the cells. Incubation of the cells in the dark in the presence of RHM produced only minor structural, mainly cytoplasmic changes. Irradiation of the cells in the presence of RHM produced extensive ultrastructural changes prior to complete cell lysis; these changes were more severe than the effects of incubation of the cells in the dark in the presence of RHM. When the cells incubated in the dark or irradiated in the presence of latex particles or either one of the eumelanins particles, viz. BHM or synthetic dopa melanin, these particles did not penetrate into the cells or produce any ultrastructural changes. These particles were in fact not even ingested by the cells.

4] Purification and crystallization of staphylococcal leukocidin

This chapter describes the purification and crystallization of Staphylococcal Leukocidin. Staphylococcal Leukocidin, known to be important in the pathogenicity of certain staphylococcal diseases, consists of two protein components (S and F) that act synergistically to induce cytotoxic changes in human and rabbit polymorphonuclear leukocytes. These components when tested individually are not cytotoxic. The F and S components crystallize in the form of plates and needles, respectively. The amino-terminal residues of both components are alanine. Leukocidin activity is determined also by assaying for 86 Rb release from 86 Rb-labeled polymorphonuclear leukocytes. Similar data are obtained by examining morphological changes of polymorphonuclear leukocytes. The terminal event in leukocidin action is cytoplasmic degranulation, rupture of nuclei, and complete cell lysis. The purified F and S components of leukocidin stored at –80° in 0.1 M phosphate-buffered saline (pH 7.2) containing 0.5% gelatin are stable for at least 3 months.

Glucocorticoid receptor-like antigen in lymphoma cell membranes: correlation to cell lysis.

S-49 mouse lymphoma cells undergo lysis when treated with glucocorticoids; the mechanism of this effect is not understood. A protein was detected in the plasma membrane of these cells by means of direct immunofluorescent labeling with a monoclonal antibody to the soluble glucocorticoid receptor. Cellular heterogeneity in the content of this glucocorticoid receptor-like molecule was evident. By immunoadsorption to antibody-coated tissue culture plates, the cells were separated into populations positive (100%) and depleted (38%) for this membrane antigen. Gel electrophoresis, specific immunoblot, and autoradiographic (binding of [3H]dexamethasone mesylate) analysis of the membrane proteins from the membrane antigen-positive group revealed multiple protein bands ranging in size from 85 to 145 kilodaltons. Furthermore, comparison of the glucocorticoid sensitivity of these groups of cells showed complete lysis of the membrane antigen-positive cells and only partial lysis of the antigen-deficient group, which suggests that the lysis response of cells to glucocorticoids is mediated by a glucocorticoid receptor-like molecule located in the plasma membrane.

Cytotoxicity of enantiomers of gossypol.

The effect of gossypol on cell proliferation was studied using 24-well multiwell dishes. Cells were added to each well and incubated for 24 hours before the addition of different concentration of gossypol, which was dissolved in dimethyl sulfoxide immediately prior to use. After a further 3-day incubation, cell growth was determined by measuring cellular protein content. Standards and controls were included and dose response curves constructed by plotting increase in protein content over the 3-day growth period against gossypol concentration. Gossypol inhibited proliferation in tumor-derived cells as well as normal fibroblasts. BCL-D1 cells, a human diploid strain of fibroblasts, were sensitive to gossypol and further studies were undertaken using these cells. In contrast of most of the tumor-derived cells that have been used, these cells maintained consistent patterns of growth over periods of many months. The enantiomers of gossypol were prepared and analyzed as described in Matlin & Zhou (1984). The (-) enantiomer of gossypol was more cytotoxic than the (+) form. In the case of all the tumor-derived cells, the concentration of the (-) enantiomer required to produce cytotoxicity was -10% of that required in the case of the (+) enantiomer. At lower concentration, gossypol inhibited cellular proliferation, but at higher concentrations the compound was responsible for complete cell lysis. Even with high doses of (-), gossypol cell lysis did not occur rapidly. When gossypol was added to cultures and then removed with thorough washing at time intervals, it was found that 30 minute exposure was adequate to bring about the cytolytic effect. Thus, the biochemical effects of gossypol occurred rapidly after addition even though cellular changes may not be obvious for some hours. Once gossypol has been added to tissue culture medium, there is a loss of cytotoxic potential. When gossypol was added to culture fluid and incubated at 37 degrees Centigrade before addition to cells, the chemical lost -10%/hour of its cytotoxic potential. The likely explanation is a binding of gossypol to plasma proteins that form the serum supplement to the growth medium. More than 10,000 men have participated in clinical trials relating to the anti-fertility effects of gossypol; a low incidence of side effects has been reported. The question arises as to why gossypol fails to have an effect on bone marrow cell proliferation. Most likely the answer lies in the protection afforded by higher protein concentrations.

A fast-acting cytotoxin derived from Con A-activated porcine leukocytes: I. Biochemical characteristics and target cell specificity

We here describe a new cytotoxin which was detected in serum-free culture supernatants of mitogen stimulated porcine leukocytes. The factor was precipitated at 35–45% (NH 4) 2 SO 4 concentration, sensitive to heat, low pH, and trypsin, thus indicating its protein nature. Column chromatography on hydrophilic or hydrophobic matrices revealed ampiphilic properties. The smallest unit which was sufficient to induce complete target cell lysis had a molecular weight of 33K. This porcine cytotoxin (PCT) could be distinguished functionally from lymphotoxin (LT) and tumor necrosis factor (TNF). It is a fast acting mediator which exerts strong cytostatic and cytotoxic anti-tumor activity using a panel of T lymphoma target cells.

Lysis of Vibrio cholerae cells: direct isolation of the outer membrane from whole cells by treatment with urea.

Cells of Vibrio cholerae underwent rapid autolysis when suspended in media of low osmolarity under non-growing conditions. Chaotropes like urea and guanidine. HCl which are potent protein denaturants caused complete and immediate lysis of whole cells. This unique sensitivity of V. cholerae to protein denaturants led to the development of a rapid method for the selective isolation of the outer membrane upon treatment of whole cells with urea. The composition of the outer membrane isolated from both whole cells and crude envelopes by treatment with urea was comparable with that of the outer membrane isolated by other conventional methods.

Hyperplasia of pancreatic duct epithelium produced by exposure to sodium deoxycholate

Severe epithelial hyperplasia was produced in a canine model by the perfusion of the main pancreatic duct with 15 mM of deoxycholate at rates as low as 1.5 ml/day in 6 to 14 days. At higher rates (5 ml/day) deoxycholate caused complete epithelial cell lysis in the duct closest to the tip of the cannula with hyperplastic changes downstream from this section. Perfusion with a buffer solution alone and cannulation alone produced none of these changes in similar duct segments. No hyperplasia was seen in the up-stream cannula obstructed duct, even in the presence of severe atrophy. Long-term (81 days) perfusion with 3 mM of deoxycholate at 3 ml/day resulted in more severe hyperplasia that still appeared benign. When glycine-conjugated deoxycholate was perfused through the duct, hyperplasia but no cell lysis was seen. In vitro, deoxycholate caused epithelial cell lysis in pancreatic duct fragments at concentrations of 0.5 mM and above. The results of this study suggest that secondary bile salts or other similar surface-active cytotoxic agents present in the biliary tree or duodenum may play a more important role in the pathogenesis of pancreatic ductal epithelial hyperplasia associated with pancreatic cancer than ductal obstruction.

The mechanism of synergistic complement-mediated lysis of rat red cells by monoclonal IgG antibodies.

The mechanism of synergistic complement-mediated lysis of rat red cells was investigated using rat monoclonal antibodies against class I RT1Aa antigens. The increased lytic activity when using two antibodies simultaneously is due to the increase in the number of activated C1 molecules on the cell surface and this results from (a) an increase in the number of binding sites for C1q, (b) an increase in the functional affinity constant for C1q binding and (c) an increase in the rate of activation of C1. Complete lysis of red cells was only achieved if one member of the synergistic pair was of the gamma 2b isotype, and this isotype was the only one to which binding of 125I-labeled C1q could be detected. A partial synergistic effect was seen using an F(ab')2 fragment of antibody. Increased uptake and activation of C1 probably results both from the presence of two antibodies attached to each antigen molecule and from the formation of antigen-antibody catenars.

Effect of Hard Detergents on Algae in a High-Rate-Oxidation Pond

Regular concentrations of hard detergents in domestic wastewater do not affect algal growth in a high-rate-oxidation pond. The addition of nonionic hard detergent at concentrations above 60 mg/liter decreased the algal concentration in the batch culture, and complete lysis of algal cells was observed within a few days at a detergent concentration of 100 mg/liter.

Growth and Maturation of a Vesicular Stomatitis Virus Temperature-Sensitive Mutant and Its Central Nervous System Isolate

A temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), tsG31, produces a prolonged central nervous system disease in mice with pathological features similar to those of slow viral diseases. tsG31 and the subsequent virus recovered from the central nervous system (tsG31BP) of mice infected with tsG31 were compared with the parental wild-type (WT) VSV for plaque morphology, growth kinetics, thermal sensitivity of the virions, and viral protein synthesis and maturation. Several properties of the central nervous system isolate distinguished this virus from the original tsG31 and the WT VSV. The WT VSV produced clear plaques with complete cell lysis, and the tsG31 produced diffuse plaques and incomplete cell lysis, whereas the tsG31BP had clear plaques similar to those of the WT VSV. Although plaque morphology suggested that tsG31BP virus was a revertant to the WT, growth kinetics in either BHK-21 or neuroblastoma (N-18) cells indicated that this virus was similar to tsG31, with a productive cycle at 31 degrees C and no infectious virus at 39 degrees C. At 37 degrees C, however, the tsG31BP matured much slower than did the original tsG31 (and produced only 1% of the yield measured at 31 degrees C). WT VSV produced similar quantities of infectious virions at 31, 37, and 39 degrees C. The lack of infectious virions at 39 degrees C for the ts mutants was presumably not due to a greater rate of inactivation at 39 degrees C. Unlike WT VSV, which synthesized viral proteins equally well at all three temperatures, tsG31 had a reduced synthesis of all the structural proteins at 37 and 39 degrees C, compared with that at 31 degrees C; the formation of the M protein was most temperature sensitive. In addition, fractionation of the infected cells indicated that the incorporation of the M and N proteins into the cellular membranes was also disrupted at the higher, nonpermissive temperatures. Several characteristics of protein synthesis during tsG31BP infection at 39 degrees C distinguished this virus from tsG31: (i) no mature viral proteins were detected at 39 degrees C; (ii) several host proteins were [ill], suggesting that the virus was incapable of completely depressing host macromolecular synthesis; and (iii) a great proportion of the incorporated radioactivity was found in unusually high-molecular-weight proteins. In addition, at 37 degrees C, the tsG31BP virus showed a decreased synthesis of viral proteins and reduced assembly of the viral structural proteins.

EXTRACELLULAR LYSIS OF THE BLUEGREEN ALGA PHORMIDIUM LURIDUM BY BDELLOVIBRIO BACTERIOVORUS1

SUMMARYWhen either cells of the bacterium, Bdellovibrio bacteriovorus (Stolp & Starr), strain 15143, or a heat‐resistant lytic factor derived from these cells is added to viable cultures of Phormidium luridum var. olivacea Boresch all the algal cells underwent gradual lysis. This effect was obtained with a mean initial bdellovibrio:algal cell ratio of 7.5:1. When P. luridum was mixed with the bdellovibrio cultures the algal chlorophyll content showed an 8‐fold decrease. Concomitantly, this interspecies interaction caused, a 75% inhibition of algal photosynthesis after‐4 h. Heat, treatment of the B. bacteriovorus culture supernatant fluid increased its ability to inhibit photosynthesis approximately 14%. Light, microscopy showed pale granules and intracellular spaces to form in the P. luridum within 16 h after adding the bdellovibrio lytic factor. Subsequent morphological changes included the development of large intracellular spaces, intercellular spaces, spheroplast formation and finally Complete lysis of the algal cells.

A kinetic model for the quantitative analysis of complement

A simple model has been developed which accurately predicts the time course of complement mediated lysis of sensitized red cells. The model assumes that the one hit theory of immune hemolysis is applicable and that the rate of lysis is directly proportional to the concentration of a complement component present in rate limiting amounts. It also assumes that the rate of lysis is dependent on the fraction of cells lysed. The model can be related to the classical von Krogh equation for end point complement analyses and can be used to estimate the rate constant for the critical step in hemolysis, as well as the efficiency of the critical complement component in the rate limiting step. Parameters derived from the model can be quantitatively related to complement concentration and can be used as the basis for a quantitative assay of complement activity. The model can also be used to calculate, for a particular sample, the concentration at which complement activity becomes undectable, the complement activity of the pure, undiluted sample, and the time required for the sample to produce complete lysis of the available cells.

TWO LYTIC FACTORS INVOLVED IN INDUCED SYNERGISTIC HEMOLYSIS IN NORMALLY NONHEMOLYTIC STAPHYLOCOCCI.

Smith, Doyle C. (Kansas State University, Manhattan), V. D. Foltz, and T. H. Lord. Two lytic factors involved in induced synergistic hemolysis in normally nonhemolytic staphylococci. J. Bacteriol. 88:1700-1704. 1964.-Synergistic hemolysis of "normally nonhemolytic" staphylococci results from the combination of two factors: Z, produced by hemolytic staphylococci that elaborate a secondary hemolytic zone on blood-agar, and T, produced by nonhemolytic staphylococci. Factors Z and T are produced in supernatant fluids of broth cultures and are detected by the synergistic hemolysis (SH) test. Complete lysis of sheep red cells is observed in the SH test only when both factors Z and T are present. Production of Z and T is greatly enhanced by the presence of 10 or 20% CO(2) during incubation. Both Z and T are heat-stable, withstanding 100 C for 15 min. Neither factor is precipitated by saturated Na(2)SO(4) or (NH(4))(2)SO(4). Hydrogen ion concentrations between pH 5.0 and 8.5 do not exert any effect on the two factors. Factor Z is produced in greater quantity than factor T. It is suggested that factors Z and T are not proteinaceous, enzymatic, or lipid in nature.

Observations on abortive infection of Micrococcus lysodeikticus with bacteriophage

Screening of ultraviolet irradiated Micrococcus lysodeikticus cultures for phage-resistant mutants yielded strains capable of producing plaques with virulent phage N6 but not with serologically related virulent phage N1. Adsorption tests conducted with a representative strain revealed that the culture was not truly resistant to N1 since this phage was efficiently adsorbed. Adsorption initiated an abortive infection which terminated with complete lysis of the host cells without liberation of infectious phage. An apparent reversal of the abortive infection was observed when the mutant cells were infected simultaneously with both phages. Under such conditions, 50–60% of the infected cells produced infectious phage, and single cell burst experiments showed that nearly every productive cell yielded both parental phage types and two new stable types in which the plaque morphology and host range characteristics were recombined. It was found that the percentage recovery of infectious centers from mixed-infected cells was influenced by the order in which the two phages infected the cells, and by the time interval between infection with the first phage and subsequent infection with the second phage. When cells were infected first with N6, delay of infection with N1 resulted in increased recoveries. When cells were infected first with N1, recoveries fell off at a rate which was exponential with respect to the time of delay in adding N6. During the course of the abortive infection, there was no increase in rate of oxygen uptake and no net synthesis of ribonucleic acid. Deoxyribonucleic acid was synthesized at a considerably higher rate, and the amount produced was two to three times greater than that found with any of the productive infections studied.

THE RATE OF ESCAPE OF HEMOGLOBIN FROM THE HEMOLYZED RED CORPUSCLE

A theoretical treatment is given of the rate of escape of hemoglobin from the hemolyzed red corpuscle. For complete permeability of the surface, as may perhaps be produced by strong lysins, the time taken for the hemoglobin to decrease to 10 per cent of its original concentration is calculated to be 0.16 seconds (for the human cell). For dilute saponin, giving complete lysis of human cells in 3 minutes, Ponder found a time of escape of 4 seconds, from which the permeability of the membrane to the pigment is calculated to be micro(H) = 5 x 10(-5) cm./sec.