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Showing papers on "Lysis published in 1997"


Journal ArticleDOI
TL;DR: Observations indicate that released dissolved organic matter supported bacterial growth and may be a pathway by which various elements are diverted in microbial food webs within marine plankton communities.
Abstract: The potential importance of the viral lysis of phytoplankton for nutrient and carbon cycling has been acknowledged, but no quantitative assessments of this phenomenon exist. Radiotracer experiments examined the release and bioavailability of C, N, P, Fe, and Se following viral lysis of the "brown tide" chrysophyte Aureococcus anophagefferens. Photochemical effects on the dissolved-particulate partitioning and biological uptake of virally released elements were also investigated. Viral lysis of A. anophagefferens released 50% mon C and Se than uninfected control cells to the dissolved phase, while N, P, and Fe remained in the particulate phase. There was a significant inverse correlation between A. anophagefferens and bacterial densities, as well as an increase in particulate organic nitrogen levels in cultures during viral lysis. These observations indicate that released dissolved organic matter supported bacterial growth and may be a pathway by which various elements are diverted in microbial food webs. Dissolved nutrients released by viral lysis were accumulated to varying degrees by natural assemblages of marine bacteria and cultured diatoms, and vitally regenerated N and P relieved diatom nutrient limitation. During a 4-wk incubation, 80% of C and P within cell lysis debris was released to the dissolved phase, likely due to bacterial activity. Photochemical degradation of cell lysis debris enhanced dissolved levels of Se (100%) and Fe (50%) and reduced total dissolved C by 15%. Photochemistry doubled the bioavailability of virally released Se to diatoms, while decreasing the bioavailability of C to bacteria threefold. The viral lysis of an A. anophagefferens bloom in the field could release 40 mu M dissolved organic carbon and rapidly transfer other released elements to bacteria. Such occurrences may significantly affect water column chemistry, species composition, and succession within marine plankton communities.

325 citations


Journal ArticleDOI
TL;DR: The results indicate that burst size is not limited by cell size or DNA composition, nor directly by the rate of metabolism, but rather by the rates of synthesis and assembly of phage components and by lysis time.
Abstract: Several parameters of phage T4 adsorption to and growth in Escherichia coli B/r were determined. All changed monotonously with the bacterial growth rate (μ), which was modified by nutritional conditions. Adsorption rate was faster at higher μ values, positively correlated to cell size, and increased by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface area, indicating a constant density of T4 receptors on cell envelopes irrespective of growth conditions. Parameters of phage development and cell lysis were μ-dependent. The rate of phage release and burst size increased, while the eclipse and latent periods decreased with increasing μ. Differentiation between the contribution of several physiological parameters to the development of T4 was performed by manipulating the host cells. A competitive inhibitor of glucose uptake, methyl α-D-glucoside, was exploited to reduce the growth rate in the same effective carbon source. Synchronous cells were obtained by the ‘baby-machine’ and large cells were obtained by pretreatment with low Pn concentrations. Lysis was delayed by superinfection, and DNA content and concentration were modified by growing a thy mutant in limiting thymine concentrations. The results indicate that burst size is not limited by cell size or DNA composition, nor directly by the rate of metabolism, but rather by the rates of synthesis and assembly of phage components and by lysis time. The rates of synthesis and assembly of phage components seem to depend on the content of the protein-synthesizing system and lysis time seems to depend on cellular dimensions.

313 citations


Journal ArticleDOI
TL;DR: Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187 and had antibacterial and cell lysis activities with many kinds of bacteria.
Abstract: Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187. The molecular weights of FI and FII were 30,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 60,000 and 30,000, respectively, by gel filtration. The pIs for FI and FII were 5.2 and 4.8, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FI were pH 8, 50 degrees C, pH 6 to 9, and 50 degrees C; those of FII were pH 7, 40 degrees C, pH 5 to 10, and 60 degrees C. The activities of both enzymes were activated by Cu2+; strongly inhibited by Mn2+, Mg2+, and Zn2+; and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol. Both chitinases showed lysozyme activity. The purified enzymes had antibacterial and cell lysis activities with many kinds of bacteria. This is the first report of a bifunctional chitinase/lysozyme from a prokaryote.

240 citations


Journal ArticleDOI
TL;DR: By destabilizing the membranes of P. aeruginosa, gentamicin increases the release of membrane vesicles three- to five-fold, which may help account for some of the bacterium-mediated toxicity encountered during patient treatment with aminoglycoside antibiotics.
Abstract: Pseudomonas aeruginosa (and various other gram-negative pathogens) liberate membrane vesicles during normal growth. These bilayered vesicles consist of endotoxin (lipopolysaccharide), outer membrane proteins and several potent hydrolytic enzymes including protease, alkaline phosphatase, phospholipase C and peptidoglycan hydrolase. The vesicles contain pro-elastase and alkaline phosphatase (which are periplasmic constituents) and so are important for packaging periplasmic components as they are liberated to the outside of the cell. Once liberated, the vesicles are capable of fusing with the membranes of epithelial cells and liberating their virulence factors into host cells where they degrade cellular components, thereby aiding infection by the pathogen. The aminoglycoside antibiotic, gentamicin, is thought to kill bacteria by inhibiting protein synthesis, yet this cationic antibiotic can also perturb the packing order of lipids, thereby destabilizing bilayered membranes. For pathogens with highly anionic lipopolysaccharide on their surface, such as P. aeruginosa, this membrane destabilization can be so serious that it can cause cell lysis; these cells are therefore killed by a combination of protein synthesis inhibition and surface perturbation. By destabilizing the membranes of P. aeruginosa, gentamicin increases the release of membrane vesicles three- to five-fold. This may help account for some of the bacterium-mediated toxicity encountered during patient treatment with aminoglycoside antibiotics.

211 citations


Journal ArticleDOI
TL;DR: Cholesterol that was assimilated by Lactobacillus acidophilus ATCC 43121 was not metabolically degraded; most of it was recovered with the cells, and the type of fatty acid in the phospholipid did not influence the assimilation.

176 citations


Journal ArticleDOI
TL;DR: Evidence is presented that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex and efflux of intracellular K+ without entry of the toxin into the cytosol.
Abstract: Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in animals, which is characterized by edema in several organs due to an increase in blood vessel permeability. The Madin-Darby canine kidney (MDCK) cell line has been found to be susceptible to epsilon-toxin (D. W. Payne, E. D. Williamson, H. Havard, N. Modi, and J. Brown, FEMS Microbiol. Lett. 116:161-168, 1994). Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K+ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37 labeled C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37 degrees C was more stable than that formed at 4 degrees C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.

147 citations


Journal ArticleDOI
TL;DR: Model cheese experiments showed a fourfold increase in release of L-Lactate dehydrogenase activity into the curd relative to the control strain and the holin-overproducing strain, demonstrating the suitability of the inducible nisin-inducible expression system for cheese applications.
Abstract: An attractive approach to accelerate cheese ripening is to induce lysis of Lactococcus lactis starter strains for facilitated release of intracellular enzymes involved in flavor formation. Controlled expression of the lytic genes lytA and lytH, which encode the lysin and the holin proteins of the lactococcal bacteriophage ΦUS3, respectively, was accomplished by application of a food-grade nisin-inducible expression system. Simultaneous production of lysin and holin is essential to obtain efficient lysis and concomitant release of intracellular enzymes as exemplified by complete release of the debittering intracellular aminopeptidase N. Production of holin alone leads to partial lysis of the host cells, whereas production of lysin alone does not cause significant lysis. Model cheese experiments in which the inducible holin-lysin overproducing strain was used showed a fourfold increase in release of L-Lactate dehydrogenase activity into the curd relative to the control strain and the holin-overproducing strain, demonstrating the suitability of the system for cheese applications.

142 citations


Journal ArticleDOI
TL;DR: A magnetic bead-based system for DNA isolation utilizing monodisperse beads was tested with the aim of producing a general approach for PCR-ready DNA, and DNA suitable for PCR was prepared in less than 30 min.
Abstract: A magnetic bead-based system for DNA isolation utilizing monodisperse beads was tested with the aim of producing a general approach for PCR-ready DNA This commercially available system was originally designed for isolating PCR-ready DNA from human whole blood We tested diverse organisms belonging to the major groups: bacteria, fungi, algae, vascular plants and vertebrates Optimization of sample amounts and lysis conditions was done using several types of tissue (fish epithelium, plant leaves, mammalian liver and muscle tissues, fungal fruit-bodies and mycelium) The standard lysis conditions used for blood could be applied with good results for most bacteria, algae and vertebrates, while plant leaves and fungal fruit-bodies had to be mechanically broken to obtain proper lysis For vascular plants and some cyanobacteria, lysis by heating to 65 degrees C gave better DNA yields than standard lysis at room temperature In all cases, DNA suitable for PCR was prepared in less than 30 min The PCR products yielded 350 to 500 bases of DNA sequence (99% accurate) by direct manual or automated sequencing

140 citations


Journal ArticleDOI
TL;DR: The isolated DNA is essentially free of polyphenols and other major contaminants based upon its lack of coloration, A260/A280 ratio, digestibility with restriction enzymes, melting profile, and reassociation properties.
Abstract: We have developed a protocol for isolating milligram quantities of highly purified DNA from tomato nuclei. The protocol utilizes fresh seedlings or leaves without freezing. Tissues are treated with ethyl ether, thoroughly washed, and placed in a buffer containing the nuclear-stabilizing agent 2-methyl-1,4-pentanediol. Nuclei are liberated from tomato cells by homogenization in a Waring blender. The interaction of nuclear DNA with oxidized polyphenols is inhibited by compounds that adsorb polyphenols or prevent oxidation reactions. Chloroplasts and mitochondria are preferentially eliminated with Triton X-100. Nuclei are concentrated using a Percoll gradient and lysed with SDS. DNA is subsequently purified by RNase and protease digestions and phenol/chloroform extractions. The isolated DNA is essentially free of polyphenols and other major contaminants based upon its lack of coloration, A260/A280 ratio, digestibility with restriction enzymes, melting profile, and reassociation properties.

136 citations


Journal ArticleDOI
TL;DR: Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent.
Abstract: Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times After 45 min, lysis efficiency was about 90% or more in all cases Total DNA yields varied between soils, from 2 to 35 μg g–1 The purification steps needed to obtain amplifiable DNA were different per soil To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches Both the direct and indirect DNA extraction methods yielded similar MPN estimates The dynamics of M chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils

125 citations


Journal ArticleDOI
TL;DR: A spectrophotometric hemolysis assay system is developed and a basic model for the interactions between HBL components and for the paradoxical zone phenomenon in blood agar is constructed, suggesting that lysis was caused by formation of a membrane attack complex on the cell surface.

Journal ArticleDOI
TL;DR: A rapid, inexpensive, large‐scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types and could be used in PCR directed at high‐copy number (bacterialsmall subunit rRNA) and single‐copy genes.
Abstract: A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types. DNA was extracted from 100 g of soil using direct lysis with glass beads and sodium dodecyl sulphate (SDS) followed by polyethylene glycol precipitation, potassium acetate precipitation, phenol extraction and isopropanol precipitation. The crude extract could be used in PCR directed at high-copy number (bacterial small subunit rRNA) and single-copy (fungal beta-tubulin) genes.

Journal ArticleDOI
TL;DR: The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen.
Abstract: A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L-1 of dry cell weight (OD600 = 120) within 24 h. A plasmid DNA yield of 100 mg L-1 (1.7 mg g-1 cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L-1) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L-1 was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10-12 g L-1 (OD600 = 25-30) and plasmid yields of 5-8 mg L-1 (approximately 0.7 mg g-1 cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (mu) from 0.69 h-1 to 0.13 h-1 and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales.

Journal ArticleDOI
TL;DR: The results show that polyamines are necessary for maintaining cell wall characteristics by strengthening the links between cell-wall components.
Abstract: summary Polyamines (PAs) and the cell wall have important roles in plant morphogenesis. An interaction between cell wall components (pectic substances) and polyamines is known and inhibition of polyamine biosynthesis induces inhibition of some morphogenic processes. This is the case of rhizogenesis in tobacco (Nicotiana tabacum L.) thin layers. This paper investigates whether inhibition of polyamine biosynthesis induces modifications in the structure, shape and size of the primary cell walls in tobacco thin layers cultured on a rhizogenic medium, and whether polyamines (putrescine and spermidine) administered in combination with their corresponding inhibitors dl-α-difluoromethylomithine +dl-α-difluoromethylarginine (DFMO + DFMA), cyclohexylamine (CHA), methylglyoxal-bis(guanylhydrazone) (MGBG) restore cell wall characteristics. In treatments with polyamine inhibitors, cell hypertrophy occurred in the lowermost layer of the explant in contact with the medium. Many unusual features were observed in the walls of the hypertrophic cells: an amorphous structure, loosening of the fibrillar component; detachment between contiguous cells, lysis of wall components and an uneven thickness often giving a wavy appearance (in transverse walls). The inhibitors reduced cell wall thickness, and caused a weak positive PATAg reaction for polysaccharides. Polyamines restored cell wall thickness and, in general, the other wall features. Our results show that polyamines are necessary for maintaining cell wall characteristics by strengthening the links between cell-wall components.

Journal ArticleDOI
TL;DR: During the massive mucllage event in the northern Adriatic Sea in July 1991 samples of macroaggregate were fixed in different ways: with formaldehyde, deep frozen and freeze-dried material, conventional microscopy revealed different autotrophic species embedded in gelatinous matl-ix.
Abstract: During the massive mucllage event in the northern Adriatic Sea in July 1991 samples of macroaggregate were fixed in different ways: with formaldehyde, deep frozen and freeze-dried. Conventional microscopy (light and epifluorescence) revealed different autotrophic species embedded in gelatinous matl-ix. Cyanobacteria and heterotrophic bacteria were also identified. Scanning confocal laser microscopy (SCLMI and fluorescent molec~~ la r p obes (the lectins concanavalin A and UEA-I) showed wall-free cytoplasm and particulate polysacchar~des leaklng from the envelopes of broken cells in the matrix. The extensive cell lysis was supported by the observation of cytoplasn1-free cytoskeletons, stained by the molecular probe phalloidin High concentrations of triglycerides (30Y0 of total lipids) and free fatty aclds (22'2%) along with very low concentrations of phospholipids ( 2 % ) also indicated massive cell degradation in freeze-dried material. The mucllage observations were compared with those of a natural plankton community grown under hlgh nutrlent conditions using the same techniques. Free polysaccharides were observed as globular flocs (marine snow) during in situ enrichment experiments and inti-acellular polysaccharides as carbon storage materials In autotrophic organisms. No strings, filaments, layers, cell lysis or lipid classes indicating strong cell biodeterioration were observed in a 1 mo controlled experiment during an algal bloom.

Journal Article
TL;DR: It is shown that serum withdrawal activates a succession of initial events that are similar to those defined as 'apoptosis', i.e. nuclear condensation and membrane blebbing, which are accompanied or rapidly followed by cell lysis and disruption of mitochondria, both of which are characteristic of necrosis.
Abstract: AKR-2B cells disintegrate after serum removal. After a delay of approximately 90 minutes, cell death began and reached after six hours a plateau of 40-50% remaining living cells. We used time-lapse video microscopy to monitor dynamic structural changes and to measure the time span of individual cells to die. The first change was the rapid appearance of membrane blebs. Membrane vesicles were rapidly extruded and reintegrated by the cell. This highly dynamic process of an affected cell stopped after 80+/-20 minutes with its death. Conductivity measurements showed that at that time the membrane was electrically permeable. By using fluorescence double staining with propidium iodide and Hoechst 33258, we show that membrane leakage leading to disintegration is accompanied, and for some cells preceded, by nuclear condensation. The energy state of the intact cells was monitored by measuring the intracellular ATP content which remained high (6 mM) throughout the entire time of investigation. Mitochondrial potential was determined by rhodamine 123 fluorescence in parallel to the measurement of membrane permeability via uptake of propidium iodide and lead to the detection of a cell population that exhibits a high mitochondrial potential and an uptake of propidium iodide indicating a membrane disruption of cells which still have a high energy charge. It is shown by electron microscopy that mitochondria were swollen and damaged in parallel to nuclear condensation. There was no DNA fragmentation as shown by two independent methods. Addition of the ICE-like protease inhibitor tyr-val-ala-asp-chloromethylketone immediately after serum starvation lead to an almost complete survival of the cells up to 6 hours. A pronounced protection was still observed after 24 hours, suggesting an involvement of this type of protease in the onset of cell death after serum removal. Apparently, serum withdrawal activates a succession of initial events that are similar to those defined as 'apoptosis', i.e. nuclear condensation and membrane blebbing. These steps are, however, accompanied or rapidly followed by cell lysis and disruption of mitochondria, both of which are characteristic of necrosis.

Journal ArticleDOI
TL;DR: The results suggest that activation or deregulation of the atl gene products at localized sites where formation of new cross wall was disturbed by PCG causes small defects in the cell wall in situ, eventually leading to general autolysis.
Abstract: We investigated the cell surface localization of the atl gene products of Staphylococcus aureus exposed to a lytic concentration (4 MIC) of penicillin G (PCG) by means of immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold conjugates reacting with antigen-antibody complex localized at sites of defects of the cell wall at the nascent cross wall. Anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G inhibited the decreased turbidity caused by PCG-induced lysis and the formation of defects in the wall. The autolysis-defective mutant, S. aureus RUSAL2 (atl::Tn551), exposed to 4 MIC of PCG resisted autolysis and formation of the wall defect. These results suggest that activation or deregulation of the atl gene products at localized sites where formation of new cross wall was disturbed by PCG causes small defects in the cell wall in situ, eventually leading to general autolysis.

Journal ArticleDOI
TL;DR: To elucidate the molecular architecture of the membrane pore formed by γ‐hemolysin, the pore complex was solubilized with 2% sodium dodecyl sulfate, separation from erythrocyte membrane proteins by sucrose gradient ultracentrifugation, and observed the isolated complex under an electron microscope.

Journal ArticleDOI
01 Mar 1997-Genetics
TL;DR: Results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.
Abstract: The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.

Journal ArticleDOI
TL;DR: In this paper, the role of Ipa proteins in induction of membrane ruffles upon entry and lysis of the endosome membrane thereafter was investigated and it was shown that IpaC and IpaB interact with lipid vesicles.

Journal ArticleDOI
TL;DR: The results suggest that the resistance of gram-negative bacteria to the action of hydrophobic antibiotics was caused by a low permeability of the outer cell membranes, which may be reduced by the simultaneous application of antibiotic and ultrasound.

Journal ArticleDOI
TL;DR: The virus which appeared to be a prawn baculovirus was named prawn white spot bacULovirus (PWSBV) due to the white spots which appeared on the inside surface of the crust of infected prawns.

Journal ArticleDOI
15 Oct 1997-Langmuir
TL;DR: Dioctadecyldimethylammonium bromide (DODAB), a liposome-forming synthetic amphiphile, kills Escherichia coli, Salmonella thyphimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in the micromolar range of DODAB concentrations.
Abstract: Dioctadecyldimethylammonium bromide (DODAB), a liposome-forming synthetic amphiphile, kills Escherichia coli, Salmonella thyphimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in the micromolar range of DODAB concentrations. For the four species at cell concentrations higher than 107 bacteria/mL in the interaction mixtures, 5 μM DODAB, and 5 h of interaction time between bacteria and vesicles, 0% survival (no counts for viable cells) was obtained. The mechanism of cell death does not involve cell lysis or vesicle rupture as evaluated from measurements of cell leakage of phosphorylated compounds and from a vesicle disruption assay. The isolated external membrane of E. coli and DODAB cationic small vesicles do interact to yield an increase in the electrophoretic mobility of ghosts as a function of DODAB concentration. Surface charge for the ghosts becomes zero over the micromolar range of DODAB concentrations. Thus vesicle adhesion to the external membrane of the bacteria is certainly the first int...

Journal ArticleDOI
TL;DR: It is proposed that these EA values are characteristic of the plasma membrane relatively unperturbed by cytoskeletal interactions, such that these sperm cells' ability to respond to osmotic stress encountered during freezing and thawing is especially sensitive to cryodamage.

Journal ArticleDOI
TL;DR: The re-examination of the role of choline in autolytic cell wall degradation using the choline-independent S. pneumoniae strain R6Cho is reported, indicating that, in sharp contrast to the case of cell walls, the amidase degradation of teichoic acid-free peptidoglycan did not require the presence of ch Caroline residues in the substrate.
Abstract: Streptococcus pneumoniae has an auxotrophic requirement for choline, and choline residues that incorporate into the wall and membrane teichoic acids are intimately involved with the control of autolytic phenomena of this bacterium We report here the re-examination of the role of choline in autolytic cell wall degradation using the choline-independent S pneumoniae strain R6Cho− recovered from a heterologous cross with DNA from Streptococcus oralis S pneumoniae Cho− cultured in choline-free medium grew with normal generation time but formed long chains, failed to undergo stationary-phase autolysis, and was also resistant to lysis induced by deoxycholate or penicillin Cell walls produced under these conditions had reduced phosphorus content, contained no choline residues detectable by nuclear magnetic resonance, and had reduced binding capacity for the pneumococcal autolytic amidase, and complete hydrolysis of such walls by the amidase required prolonged incubation with high concentrations of t

Journal ArticleDOI
TL;DR: An interaction between NO and H2O2 which leads to a markedly enhanced cytotoxic activity, in part, via induction of apoptosis is indicated and it is suggested that poly(ADP-ribosylation) and subsequent NAD+ depletion mediate, at least in parts, this cytot toxic activity.
Abstract: We examined whether NO and H2O2 could interact in inducing DNA fragmentation and cell death. H2O2 and the NO-releasing compounds sodium nitroprusside (SNP) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) by themselves elicited lysis of YAC-1 murine lymphoma cells in a concentration-dependent manner. Exposure of the cells to a combination of sublytic concentrations of SNP (0.78 mM) plus H2O2 (7.8 microM) or SNAP (0.18 mM) plus H2O2 (7.8 microM) resulted in cell death which is mediated, in part, through apoptosis. Evidence for this direction is provided by fluorescence microscopic evaluation of the cells, which revealed the presence of changes in nuclear morphology characteristic of apoptosis in 30-40% of lymphoma cells and by the specific pattern of internucleosomal DNA fragmentation detected by gel electrophoresis. The cytotoxic effect of SNP plus H2O2 could be effectively inhibited by either oxyhaemoglobin, which binds NO, or catalase, which eliminates H2O2. Partial protection from SNP-plus-H2O2-induced cell lysis was observed with the poly(ADP-ribose) polymerase inhibitors, nicotinamide and 3-aminobenzamide, parallelling their ability to reverse depletion of cellular NAD+ pools. These results indicate an interaction between NO and H2O2 which leads to a markedly enhanced cytotoxic activity, in part, via induction of apoptosis and suggest that poly(ADP-ribosylation) and subsequent NAD+ depletion mediate, at least in part, this cytotoxic activity.

Journal ArticleDOI
TL;DR: It is concluded that cytotoxicity is not linked to attack from free radicals formed outside the cells, and the loss of cell colony forming ability is also linked to damage of cellular membranes.
Abstract: Intense ultrasound beams may have the potential to treat malignant tumours when combined with sensitizers, often called sonodynamic agents. Some of these agents, e.g., the porphyrins, are currently used for photodynamic therapy. However, the experimental evidence for ultrasound activation of sensitizers is inconsistent. This paper attempts to discover whether they yield of free radicals such as .OH and .H, which are produced by transient cavitation, could explain the killing of Chinese hamster ovary (CHO) cells in vitro with and without sonodynamic agents. CHO cells were irradiated with ultrasound beams in phosphate-buffered saline or in growth medium, and the immediate cell lysis and loss of cell colony forming ability were measured. Under our specific conditions, in which the standing wave patterns were minimized, a general correlation was observed between the transient cavitation, free radical production, and cytotoxicity. However, the yield of free radicals was much too small to explain the cell killing observed. We conclude that cytotoxicity is not linked to attack from free radicals formed outside the cells. In our experiments, immediate cell lysis is closely linked to the transient cavitation, which is known to produce shear forces that disrupt cellular membranes. We hypothesize that the loss of cell colony forming ability is also linked to damage of cellular membranes.

Journal ArticleDOI
TL;DR: Cryotechniques, morphological and immunocytochemical findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration.
Abstract: Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer. Gram-negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180-187] found that lysozyme per se is able to kill some Gram-negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.

Journal ArticleDOI
TL;DR: Findings indicate that the formation of giant DNA fragments is a specific characteristic of cells responding to oxidative stress, and it may be an initial event that leads to cell death.

Journal ArticleDOI
TL;DR: Results suggest that at least two different autolytic enzymes are present in the autolytics L. lactis subsp.
Abstract: Two highly autolytic Lactococcus lactis subsp. cremoris strains (CO and 2250) were selected and analyzed for their autolytic properties. Both strains showed maximum lysis when grown in M17 broth containing a limiting concentration of glucose (0.4 to 0.5%) as the carbohydrate source. Lysis did not vary greatly with pH or temperature but was reduced when strains were grown on lactose or galactose. Growth in M17 containing excess glucose (1%) prevented autolysis, although rapid lysis of L. lactis subsp. cremoris CO did occur in the presence of 1% glucose if sodium fluoride (an inhibitor of glycolysis) was added to the medium. Maximum cell lysis in a buffer system was observed early in the stationary phase, and for CO, two pH optima were observed for log-phase and stationary-phase cells (6.5 and 8.5, respectively). Autolysins were extracted from the cell wall fraction of each strain by using either 4% sodium dodecyl sulfate (SDS), 6 M guanidine hydrochloride, or 4 M lithium chloride, and their activities were analyzed by renaturing SDS-polyacrylamide gel electrophoresis on gels containing Micrococcus luteus or L. lactis subsp. cremoris CO cells as the substrate. More than one lytic band was observed on each substrate, with the major band having an apparent molecular mass of 48 kDa for CO. Each lytic band was present throughout growth and lysis. These results suggest that at least two different autolytic enzymes are present in the autolytic L. lactis subsp. cremoris strains. The presence of the lactococcal cell wall hydrolase gene, acmA (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrikman, J. Bacteriol. 177:1554-1563, 1995), in strains 2250 and CO was confirmed by Southern hybridization. Analysis of an acmA deletion mutant of 2250 confirmed that the gene was involved in cell separation and had a role in cell lysis.