An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

Mechanistic studies on aromatase and related C---C bond cleaving P-450 enzymes

Some P-450 systems, notably aromatase and 14-demethylase catalyse not only the hydroxylate reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C---C bond cleavage process. All these reactions occur at the same active site. A somewhat analogous situation is noted with 17-hydroxylase-17,20-lyase that participates in hydroxylation as well as C---C bond cleavage process. The C---C bond cleavage reactions catalysed by the above enzymes conform to the general equation:

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It is argued that all three types of reaction catalyzed by these enzymes may be viewed as variations on a common theme. In P-450 dependent hydroxylation the initially formed Fe^III---O---O^. species is converted into Fe^III---O---OH and the heterolysis of the oxygen—oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example Fe^V = O ↔ (+^.)Fe^IV = O ↔ Fe^IV---O^.. One of these, Fe^IV---O^. behaves like an alkoxyl radical and participates in hydrogen abstraction from C---H bond to produce Fe^IV---OH and carbon radical. The latter is then quenched by the delivery of hydroxyl radical from Fe^IV---OH. The latter species may thus be regarded as a carrier of hydroxyl radical. We have proposed that the C---C bond cleavage reaction occurs through the participation of the Fe^III---O---OH species that is trapped by the electrophilic property of the carbonyl compound giving a peroxide adduct that fragments to produce an acyl—carbon cleavage. Scientific developments leading up to this conclusion are considered. In the first author's views,

“The study of mechanisms is not a scientific but a cultural activity. Mechanisms do not aim at an absolute truth but are intended to be a “running” commentary on the status of knowledge in a field. As the structural knowledge in a field advances Mechanisms evolve to take note of the new findings. Just as a constructive “running” commentary provides the stimulus for higher standards of performance, so Mechanisms call for better and firmer structural information from their practitioners”.     

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer ¹⁸F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (¹⁸F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using ¹⁸F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using ¹⁸F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment ¹⁸F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate ¹⁸F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced ¹⁸F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment ¹⁸F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.     

Essential roles of core starvation-stress response loci in carbon-starvation-inducible cross-resistance and hydrogen peroxide-inducible adaptive resistance to oxidative challenge in Salmonella typhimurium

The starvation-stress response (SSR) of Salmonella typhimurium encompasses the physiological changes that occur upon starvation for an essential nutrient, e.g. C-source. A subset of SSR genes, known as core SSR genes, are required for the long-term starvation survival of the bacteria. Four core SSR loci have been identified in S. typhimuriumrpoSstiAstiB, and stiC. Here we report that in S. typhimurium C-starvation induced a greater and more sustainable cross-resistance to oxidative challenge (15 mM hydrogen peroxide (H₂O₂) for 40 min) than either N- or P-starvation. Of the four core SSR loci, only rpoS and stiC mutants exhibited a defective C-starvation-inducible cross-resistance to H₂O₂ challenge. Interestingly, (unadapted) log-phase S. typhimurium rpoS and stiA mutants were very sensitive to oxidative challenge. Based on this, we determined if these core SSR loci were important for H₂O₂ resistance developed during a 60 min adaptive exposure to 60 μM H₂O₂ (adapted cells). Both unadapted and adapted rpoS and stiA mutants were hypersensitive to a H₂O₂ challenge. In addition, a stiB mutant exhibited normal adaptive resistance for the first 20 mins of H₂O₂ challenge but then rapidly lost viability, declining to a level of about 1.5% of the wild-type strain. The results of these experiments indicate that: (i) the rpoS and stiC loci are essential for the development of C-starvation-inducible cross-resistance to oxidative challenge, and (ii) the rpoSstiA, and, in a delayed effect, stiB loci are needed for H₂O₂-inducible adaptive resistance to oxidative challenge. Moreover, we found that both stiA and stiB are induced by a 60 μM H₂O₂ exposure, but only stiA was regulated (repressed) by (reduced form) OxyR.     

An oxygenase enzyme system for Trametes versicolor

Abstract An oxygenase enzyme was isolated from the basidiomycete fungus Trametes versicolor , that is capable of attacking lignin and a large number of di- and tri-substituted benzene rings containing at least one hydroxy group. This enzyme system was produced late in the growth cycle without the requirement for any inducer. This non-selective enzyme system is thermophilic and operates at pH 3–5 in the presence of air or oxygen. The action of this enzyme system caused the loss of UV absorption in ferulic acid solution, the formation of hydroxy muconic semialdehyde from catechol, and transformation with the production of CO₂ from a number of hydroxy aromatics as well as lignin.     

Neurotoxicity,drugs of abuse,and the CuZn-superoxide dismutase transgenic mice

Administration of methamphetamine (METH) to animals causes loss of DA terminals in the brain. The manner by which METH causes these changes in neurotoxicity is not known. We have tested the effects of this drug in copper/zinc (CuZn)-superoxide dismutase transgenic (SOD Tg) mice, which express the human CuZnSOD gene. In nontransgenic (non-Tg) mice, acute METH administration causes significant decreases in DA and dihydroxyphenylacetic acid (DOPAC) in the striata of non-Tg mice. In contrast, there were no significant decreases in striatal DA in the SOD Tg mice. The effects of METH on DOPAC were also attenuated in SOD Tg mice. Chronic METH administration caused decreases in striatal DA and DOPAC in the non-Tg mice, but not in the SOD-Tg mice. Similar studies were carried out with 1-methyl-1,2,3,6-tetrahydropyridine (MPTP), which also causes striatal DA and DOPAC depletion. As in the case of METH, MPTP causes marked depletion of DA and DOPAC in the non-Tg mice, but not in the SOD Tg mice. These results suggest that the mechanisms of toxicity of both METH and MPTP involve superoxide radical formation.