Direct Quantification of Inorganic Polyphosphate in Microbial Cells Using 4 '-6-Diamidino-2-Phenylindole (DAPI)

Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.

Direct Contact as a Moderator of Extended Contact Effects: Cross-Sectional and Longitudinal Impact on Outgroup Attitudes, Behavioral Intentions, and Attitude Certainty

Cross-group friendships (the most effective form of direct contact) and extended contact (i.e., knowing ingroup members who have outgroup friends) constitute two of the most important means of improving outgroup attitudes. Using cross-sectional and longitudinal samples from different intergroup contexts, this research demonstrates that extended contact is most effective when individuals live in segregated neighborhoods having only few, or no, direct friendships with outgroup members. Moreover, by including measures of attitudes and behavioral intentions the authors showed the broader impact of these forms of contact, and, by assessing attitude certainty as one dimension of attitude strength, they tested whether extended contact can lead not only to more positive but also to stronger outgroup orientations. Cross-sectional data showed that direct contact was more strongly related to attitude certainty than was extended contact, but longitudinal data showed both forms of contact affected attitude certainty in the long run.

Reaction of elemental chalcogens with imidazolium acetates to yield imidazole-2-chalcogenones: direct evidence for ionic liquids as proto-carbenes

Mechanistic analysis of the reaction between elemental sulfur or selenium and 1,3-dialkylimidazolium acetate ionic liquids, in the absence of an external base or solvent, affords evidence for the equilibrium presence of carbene species in these ionic liquids. It demonstrates the potential to control, through anion selection, the concentration of carbene in stable ionic liquids.

Acetaldehyde Production in the Direct Ethanol Fuel Cell: Mechanistic Elucidation by Density Functional Theory

This study employs density functional theory (DFT) calculations to examine the mechanism by which acetaldehyde is formed on platinum in a typical direct ethanol fuel cell (DEFC). A pathway is found involving the formation of a strongly hydrogen-bonded complex between adsorbed ethanol and the surface hydroxyl (OH) species, followed by the facile alpha-dehydrogenation of ethanol, with spontaneous weakening of the hydrogen bond in favor of adsorbed acetaldehyde and water. This mechanism is found to be comparably viable on both the close-packed surface and the monatomic steps. Comparison of further reactions on these two sites strongly indicates that the steps act as net removers of acetaldehyde from the product stream, while the flat surface acts as a net producer.

Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts, We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600-800 cells were targeted with between 1 and 15 helium ions (effectively, alpha -particles). The charged particles were delivered through the centre of the nucleus with an accuracy of +/- 2 mum and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80-100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell, Similar results of a 2-3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2-3-fold increase in the damage level in an unexposed quadrant of the dish, This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved. (C) 2001 Cancer Research Campaign.

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using GO-G, confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser(15) (p53(Ser15)), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologicafly distinct subpool of p53(Ser15) is ATM dependent and resistant to 26S-proteasomal degradation. p53(Ser15) colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear micro-beam irradiation studies confirm p53 S,,15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser(15) or Ser(18) phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21(WAF) decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis. (Cancer Res 2005; 65(23): 10810-21).