Pathways of Resistance to Thymineless Death in Escherichia coli and the Function of UvrD

Thymineless death (TLD) is the rapid loss of viability in bacterial, yeast, and human cells starved of thymine. TLD is the mode of action of common anticancer drugs and some antibiotics. TLD in Escherichia coli is accompanied by blocked replication and chromosomal DNA loss and recent work identified activities of recombination protein RecA and the SOS DNA-damage response as causes of TLD. Here, we examine the basis of hypersensitivity to thymine deprivation (hyper-TLD) in mutants that lack the UvrD helicase, which opposes RecA action and participates in some DNA repair mechanisms, RecBCD exonuclease, which degrades double-stranded linear DNA and works with RecA in double-strand-break repair and SOS induction, and RuvABC Holliday-junction resolvase. We report that hyper-TLD in ∆uvrD cells is partly RecA dependent and cannot be attributed to accumulation of intermediates in mismatch repair or nucleotide-excision repair. These data imply that both its known role in opposing RecA and an additional as-yet-unknown function of UvrD promote TLD resistance. The hyper-TLD of ∆ruvABC cells requires RecA but not RecQ or RecJ. The hyper-TLD of recB cells requires neither RecA nor RecQ, implying that neither recombination nor SOS induction causes hyper-TLD in recB cells, and RecQ is not the sole source of double-strand ends (DSEs) during TLD, as previously proposed; models are suggested. These results define pathways by which cells resist TLD and suggest strategies for combating TLD resistance during chemotherapies

Radiosensitization of hypoxic tumour cells by S-nitroso-N-acetylpenicillamine implicates a bioreductive mechanism of nitric oxide generation

The radiosensitizing activity of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, was assessed in a model of non-metabolic hypoxia achieved in an atmosphere of 95% nitrogen–5% carbon dioxide. A 10 min preincubation of hypoxic EMT-6 cells (10 × 106 ml−1) with 0.1 and 1 mM SNAP before radiation resulted in an enhancement ratio of 1.6 and 1.7 respectively. The level of spontaneous NO release, measured by a NO specific microsensor, correlated directly with the concentration of SNAP and was enhanced 50 times in the presence of cells. Dilution of the cell suspension from 10 to 0.1 × 106 ml−1 resulted in a 16-fold decline in NO release, but only a twofold decrease in radiosensitization was observed. Preincubation of hypoxic cells with SNAP for 3 min up to 30 min caused an increasing radiosensitizing effect. Extended preincubation of 100 min led to the loss of radiosensitization although the half-life of SNAP is known to be 4–5 h. Taken together, these observations suggest that SNAP generates NO predominantly by a bioreductive mechanism and that its biological half-life is unlikely to exceed 30 min. The lack of correlation between free NO radical and radiosensitizing activity may reflect a role of intracellular NO adducts which could contribute to radiosensitization as well. © 1999 Cancer Research Campaig

Unauthorized Horizontal Spread in the Laboratory Environment: The Tactics of Lula, a Temperate Lambdoid Bacteriophage of Escherichia coli

We investigated the characteristics of a lambdoid prophage, nicknamed Lula, contaminating E. coli strains from several sources, that allowed it to spread horizontally in the laboratory environment. We found that new Lula infections are inconspicuous; at the same time, Lula lysogens carry unusually high titers of the phage in their cultures, making them extremely infectious. In addition, Lula prophage interferes with P1 phage development and induces its own lytic development in response to P1 infection, turning P1 transduction into an efficient vehicle of Lula spread. Thus, using Lula prophage as a model, we reveal the following principles of survival and reproduction in the laboratory environment: 1) stealth (via laboratory material commensality), 2) stability (via resistance to specific protocols), 3) infectivity (via covert yet aggressive productivity and laboratory protocol hitchhiking). Lula, which turned out to be identical to bacteriophage phi80, also provides an insight into a surprising persistence of T1-like contamination in BAC libraries

Nature and mental health: An ecosystem service perspective

A growing body of empirical evidence is revealing the value of nature experience for mental health. With rapid urbanization and declines in human contact with nature globally, crucial decisions must be made about how to preserve and enhance opportunities for nature experience. Here, we first provide points of consensus across the natural, social, and health sciences on the impacts of nature experience on cognitive functioning, emotional well-being, and other dimensions of mental health. We then show how ecosystem service assessments can be expanded to include mental health, and provide a heuristic, conceptual model for doing so

In Defence of Political Parties: A Symposium on Jonathan White and Lea Ypi’s The Meaning of Partisanship

A symposium on Jonathan White and Lea Ypi's The Meaning of Partisanship (Oxford University Press, 2016), with replies from the authors

17β-Oestradiol treatment modulates nitric oxide synthase activity in MDA231 tumour with implications on growth and radiation response

The putative oestrogen receptor negative human breast cancer cell line MDA231, when grown as tumours in mice continually receiving 17β-oestradiol, showed substantially increased growth rate when compared to control animals. Further, we observed that 17β-oestradiol treatment could both increase the growth rate of established MDA231 tumours as well as decreasing the time taken for initiating tumour growth. We have also demonstrated that this increase in growth rate is accompanied by a four-fold increase in nitric oxide synthase activity, which was predominantly the inducible form. Inducible-nitric oxide synthase expression in these tumours was confirmed by immunohistochemical analysis and appeared localized primarily in areas between viable and necrotic regions of the tumour (an area that is presumably hypoxic). Prophylactic treatment with the nitric oxide synthase inhibitor nitro-L-arginine methyl ester resulted in significant reduction in this apparent 17β-oestradiol-mediated growth promoting effect. Tumours derived from mice receiving 17β-oestradiol-treatment were characterized by a significantly lower fraction of perfused blood vessels and an indication of an increased hypoxic fraction. Consistent with these observations, 17β-oestradiol-treated tumours were less radio-responsive compared to control tumours when treated with a single radiation dose of 15 Gy. Our data suggests that long-term treatment with oestrogen could significantly alter the tumour oxygenation status during breast tumour progression, thus affecting response to radiotherapy

Offender Theme Analyses in a Crime Narrative: An Applied Approach

There is a great deal of research on the structure of narrative and its mode, and on the narrative positioning and counter positioning of the actor in legal and social contexts. In offender narratives, personal experiences are embedded for observation and analysis of particular realities that contextualize a disposition of the perpetrator being ‘an undergoer’ rather than an ‘effector’ of actions. This is evaluated in the shift from a narrated action to a speaker utterance in prospection and also in anticipation of the criminal act. Using ‘grammatical logic’, it is also possible to demonstrate how the crucial event (the crime) is not a cause, but an effect of a personal theme that encapsulates pattern of circumstances when the narrative outcome in criminal narrative becomes the product of its discursive practices. This is the ‘story of intentionality’ (my term) in crime narratives, characteristically embedded within the 1st the story of crime, the 2nd is the story of investigation [14, 20]. Using techniques from functional grammar and critical stylistics for discourse analysis, I intend to show an effective approach for the search of offender theme that underlies an act of crime. These disciplines provide the analyst with the linguistic material to analyse intersentential cohesion in a chain of semantically linked sentences (in written or spoken discourse) that explore the ways in which things are ‘made to look’ in the structure and functions of the English language. As a case study, I am using an offender narrative from Tony Parker’s book Life After Life: Interviews with Twelve Murderers (1990) showing an effective approach for the search of personal themes underlying the act of crime. Offender theme analyses are also valuable for evaluating the changing nature or development of offender characteristics pre or post crime

Unveiling Novel RecO Distant Orthologues Involved in Homologous Recombination

The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts

Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host

The responses of an anaerobic microorganism, Yersinia intermedia MASE-LG-1 to individual and combined simulated Martian stresses

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today