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At least 1,009 records · Page 56Linked to original sources
PubMed1978

In vivo synthesis and properties of uracil-containing DNA.

T4 bacteriophage DNA containing as much as 30% of its thymine replaced by uracil can be synthesised in Escherichia coli deficient in both dUTPase and uracil--DNA glycosidase. This uracil-containing DNA is competent for RNA transcription, and can be packaged into phage which are viable, if the host cells are deficient in uracil--DNA glycosidase activity. If the host cells are not deficient in this glycosidase activity the infecting phage DNA is rapidly attacked, resulting in more than 50% acid-solubilisation of the DNA. The infected cells are inefficiently killed, presumably because of very limited, if any, expression of the phage DNA. These results indicate that this replacement of thymine by uracil in DNA does not seriously impair the biological functionality of T4 DNA, provided the DNA is not subjected to the breakdown (repair) pathway initiated by uracil--DNA glycosidase.

Bacteriolysis↗
PubMed2002

A bacteriolytic agent that detects and kills Bacillus anthracis.

The dormant and durable spore form of Bacillus anthracis is an ideal biological weapon of mass destruction. Once inhaled, spores are transported by alveolar macrophages to lymph nodes surrounding the lungs, where they germinate; subsequent vegetative expansion causes an overwhelming flood of bacteria and toxins into the blood, killing up to 99% of untreated victims. Natural and genetically engineered antibiotic-resistant bacilli amplify the threat of spores being used as weapons, and heighten the need for improved treatments and spore-detection methods after an intentional release. We exploited the inherent binding specificity and lytic action of bacteriophage enzymes called lysins for the rapid detection and killing of B. anthracis. Here we show that the PlyG lysin, isolated from the gamma phage of B. anthracis, specifically kills B. anthracis isolates and other members of the B. anthracis 'cluster' of bacilli in vitro and in vivo. Both vegetative cells and germinating spores are susceptible. The lytic specificity of PlyG was also exploited as part of a rapid method for the identification of B. anthracis. We conclude that PlyG is a tool for the treatment and detection of B. anthracis.

Amino Acid Sequence↗
PubMed2004

Old dogma, new tricks--21st Century phage therapy.

As antibiotic resistant bacteria threaten a public health crisis, biotechnology is turning to bacteriophages, nature's tiniest viruses. But can phage therapy overcome its historical baggage?

Bacterial Infections↗
PubMed1998

Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes.

Eukaryotic expression vectors can be delivered to macrophages using attenuated self-destructing Listeria monocytogenes. L. monocytogenes cells are preferentially lysed in the host cell macrophage cytosol by the production of a PactA-dependent Listeria-specific phage lysin. Efficient expression of the cloned reporter genes by the macrophages and subsequent antigen presentation were achieved after the delivery of eukaryotic expression vectors by the attenuated suicide L. monocytogenes strain. After delivery by L. monocytogenes plasmid DNAs were found to integrate into the macrophage cell's genome at a frequency of about 10(-7).

Animals↗
PubMed2005

Predation on prokaryotes in the water column and its ecological implications.

The oxic realms of freshwater and marine environments are zones of high prokaryotic mortality. Lysis by viruses and predation by ciliated and flagellated protists result in the consumption of microbial biomass at approximately the same rate as it is produced. Protist predation can favour or suppress particular bacterial species, and the successful microbial groups in the water column are those that survive this selective grazing pressure. In turn, aquatic bacteria have developed various antipredator strategies that range from simply 'outrunning' protists to the production of highly effective cytotoxins. This ancient predator-prey system can be regarded as an evolutionary precursor of many other interactions between prokaryotic and eukaryotic organisms.

Animals↗
PubMed2005

Bacteriocins: developing innate immunity for food.

Bacteriocins are bacterially produced antimicrobial peptides with narrow or broad host ranges. Many bacteriocins are produced by food-grade lactic acid bacteria, a phenomenon which offers food scientists the possibility of directing or preventing the development of specific bacterial species in food. This can be particularly useful in preservation or food safety applications, but also has implications for the development of desirable flora in fermented food. In this sense, bacteriocins can be used to confer a rudimentary form of innate immunity to foodstuffs, helping processors extend their control over the food flora long after manufacture.

Amino Acid Sequence↗
PubMed2003

Optimizing alkaline lysis for DNA plasmid recovery.

Optimization of the alkaline lysis (P2) and neutralization (N3) steps in the recovery of DNA plasmids was pursued. Experiments were conducted at the test-tube and 5-litre scales with 3 kb (pUC18) and 20 kb (pQR150) plasmids. The scale and degree of mixing/shear did not affect the optimum yield of supercoiled plasmid during the P2 step, but did effect the time required for the optimum to be achieved. This optimum time for P2 at the large scale was longer (8-9 min), especially when a low-shear impeller was used. Also, when the yield of supercoiled plasmid reached a maximum during the P2 step, the purity (percentage of plasmids in the supercoiled form) simultaneously reached a minimum. As the duration of the N3 step increased from 1 to 6 min, the yield of the supercoiled plasmids remained fairly constant, provided that a lowshear impeller was used. The neutralized (post-N3) plasmid solution was shear-sensitive; however, mixing with a Rushton turbine in a tank (maximum energy dissipation rate in the mixing tank, epsilon (max), 12 m(2)/s(3); mixing-tank power consumption/volume of mixing tank, 2.0 W/m(3)) for 5-10 min resulted in a slight decrease in supercoiled plasmid and a notable increase in genomic DNA concentrations. The loss of the larger 20 kb plasmid (20%) was more than for the 3 kb plasmid. Finally, preparing the cells for alkaline lysis with lysozyme or low-pressure homogenization did not increase the plasmid yield. Furthermore, the homogenizer broke up the genomic DNA into fragments that followed through the entire Qiagen prep with the plasmids as impurities.

Alkalies↗
PubMed2003

Large-scale capture and partial purification of plasmid DNA using anion-exchange membrane capsules.

The demand for larger quantities of high-purity plasmids continues to increase. Substantial quantities of plasmid DNA are needed to support viral and plasmid-based gene-therapy programmes, including drug delivery, preclinical and clinical studies and production of DNA vaccines. Reliance on fermentation, which generates large lysate volumes, for producing the needed quantities of plasmid DNA is becoming more widespread. Development of an efficient capture-unit operation for very large plasmid DNA molecules from these large lysate volumes can present a considerable challenge. Use of conventional chromatographic beaded media for plasmid capture is problematic, owing to their restrictive pores, which limits access of the large DNA molecules to only those binding sites on the beads' outer surface. Anion-exchange membranes, on the other hand, have large convective pores that allow the plasmid DNA to readily access all of the membrane's anionic binding sites, even at very high flow rates. The convenience of anion-exchange membranes, configured in ready-to-use capsules, can greatly simplify large-scale plasmid purification strategies. The effectiveness of membrane-based technology for the capture of a pCAT reporter plasmid from large volumes of clarified Escherichia coli lysate was demonstrated. The captured and eluted plasmid pool was found to have been concentrated 10-fold with a reduction in endotoxin of four orders of magnitude.

Adsorption↗
PubMed2004

Evaluation of disruption methods for the release of intracellular recombinant protein from Escherichia coli for analytical purposes.

The aim of the present study was to find disruption methods that allow fast and reproducible measurement of intracellular recombinant proteins with potential for on-line application. Production of rhSOD (recombinant human superoxide dismutase) by Escherichia coli was used as a model. Three methods of cell disruption, sonication, osmotic shock and chemical treatment using a non-ionic surfactant, were critically compared with respect to efficiency and reproducibility of the release of rhSOD. The release of the recombinant protein was monitored by (i) measurement of the protein content in cell-culture extracts using an SPR (surface plasmon resonance) biosensor, and (ii) assaying the enzyme activity with a colorimetric reagent using a spectrophotometer. Disruption by the non-ionic surfactant showed the best performance in terms of simplicity, reproducibility and efficiency of sample treatment. The surfactant did not interfere with the rhSOD binding to the antibody immobilized on the SPR chip or with the rhSOD activity assay. When comparing the two detection methods during monitoring of an E. coli cultivation, comparable results were obtained.

Bacteriolysis↗
PubMed1966

Ribonucleic acid synthesis by Escherichia coli C 3000/L after infection by the ribonucleic acid coliphage ZIK/1, and properties of the coliphage-induced double-stranged ribonucleic acid.

1. The efficiency of extracting nucleic acids from Escherichia coli after five methods of obtaining cell lysis was determined. 2. The recovery of various nucleic acid species isolated after chromatography on methylated albumin-coated kieselguhr was also examined. 3. Double-stranded coliphage-induced RNA was isolated from infected bacteria and its resistance to ribonuclease digestion under various conditions determined. 4. The involvement of double-stranded RNA during the infection process was demonstrated. 5. The time-course of the syntheses in infected cells of double-stranded RNA, DNA, single-stranded coliphage and 16s ribosomal RNA, transfer RNA and ribosomal 23s RNA was examined. 6. It was demonstrated that the syntheses of DNA, transfer RNA and ribosomal RNA decreased 10-15min. after infection. 7. Synthesis of coliphage RNA commenced 10-15min. after infection and double-stranded RNA was also synthesized from about 10min. after coliphage adsorption.

Bacteriolysis↗
PubMed1967

Intracellular protein breakdown in non-growing cells of Escherichia coli.

1. When Escherichia coli leu(-) was incubated at 35 degrees in a medium based on minimal medium, but with the omission of phosphate ions, or glucose, or NH(4) (+) ions and leucine, intracellular protein was degraded at a rate of about 5%/hr. in each case. If Mg(2+) ions were omitted, however, the rate of degradation was 2.9%/hr. 2. Under certain conditions of incubation, protein degradation was inhibited. The inhibitor was neither NH(4) (+) ions nor amino acids, and its properties were not those of a protein, but it might be an unstable species of RNA. 3. Although a large part of the cell protein was degraded at about 5%/hr. during starvation of NH(4) (+) ions and leucine, some proteins were lost at more rapid rates, whereas others were lost at lower rates or not at all. 4. In particular, beta-galactosidase activity was lost at about 8%/hr. during starvation of NH(4) (+) ions and leucine, whereas d-serine-deaminase and alkaline-phosphatase activities were stable. During starvation of Mg(2+) ions, all three enzyme activities were stable.

Alkaline Phosphatase↗
PubMed1968

The purification and properties of two staphylolytic enzymes from Streptomyces griseus.

1. Two staphylolytic enzymes have been purified from cultures of a soil isolate of Streptomyces griseus. 2. The purified enzymes were shown to be basic proteins of low molecular weight. Each enzyme released N-acetylmuramic acid reducing groups from the cell walls of Staphylococcus aureus. 3. The enzymes lysed whole staphylococci best at higher pH values and lower ionic strengths than when the substrate was isolated cell walls or purified mucopeptide. 4. Added teichoic acid did not inhibit the enzymes, but it formed an ethanol-precipitable complex with them. 5. The possibility that teichoic acid on the surface of whole cells prevents the access of the enzymes to their mucopeptide substrate is discussed.

Bacterial Proteins↗