Gary D. Grant

Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa

Pyocyanin has recently emerged as an important virulence factor produced by Pseudomonas aeruginosa. The redox-active tricyclic zwitterion has been shown to have a number of potential effects on various organ systems in vitro, including the respiratory, cardiovascular, urological, and central nervous systems. It has been shown that a large number of the effects to these systems are via the formation of reactive oxygen species. The limitations of studies are, to date, focused on the localized effect of the release of pyocyanin (PCN). It has been postulated that, given its chemical properties, PCN is able to readily cross biological membranes, however studies have yet to be undertaken to evaluate this effect. This review highlights the possible manifestations of PCN exposure; however, most studies to date are in vitro. Further high quality in vivo studies are needed to fully assess the physiological manifestations of PCN exposure on the various body systems.

Caffeine, cycling performance, and exogenous CHO oxidation: a dose-response study.

PURPOSE This study investigated the effects of a low and moderate caffeine dose on exogenous CHO oxidation and endurance-exercise performance. METHODS Nine trained and familiarized male cyclists (mean +/- SD: 29.4 +/- 4.5 yr, 81.3 +/- 10.8 kg body weight [BW], 183.8 +/- 8.2 cm, V O2peak = 61.7 +/- 4.8 mL.kg.min) undertook three trials, with training and high CHO diet being controlled. One hour before exercise, subjects ingested capsules containing placebo and 1.5 or 3 mg.kg BW of caffeine using a double-blind administration protocol. Trials consisted of 120 min steady-state cycling at approximately 70% V O2peak, immediately followed by a 7-kJ.kg BW time trial (TT). During exercise, subjects were provided with fluids containing C-glucose every 20 min to determine exogenous CHO oxidation. RESULTS No significant TT performance improvements were observed during caffeine-containing trials (mean +/- SD: placebo = 30 min 25 s +/- 3 min 10 s; 1.5 mg.kg BW = 30 min 42 s +/- 3 min 41 s; and 3 mg.kg BW = 29 min 51 s +/- 3 min 38 s). Furthermore, caffeine failed to significantly alter maximal exogenous CHO oxidation (maximal oxidation rates: placebo = 0.95 +/- 0.2 g.min; 1.5 mg.kg BW = 0.92 +/- 0.2 g.min; and 3 mg.kg BW = 0.96 +/- 0.2 g.min). CONCLUSION Low and moderate doses of caffeine have failed to improve endurance performance in fed, trained subjects.

The effects of different doses of caffeine on endurance cycling time trial performance

Abstract This study investigated the effects of two different doses of caffeine on endurance cycle time trial performance in male athletes. Using a randomised, placebo-controlled, double-blind crossover study design, sixteen well-trained and familiarised male cyclists (Mean ± s: Age = 32.6 ± 8.3 years; Body mass = 78.5 ± 6.0 kg; Height = 180.9 ± 5.5 cm [Vdot]O2peak = 60.4 ± 4.1 ml · kg−1 · min−1) completed three experimental trials, following training and dietary standardisation. Participants ingested either a placebo, or 3 or 6 mg · kg−1 body mass of caffeine 90 min prior to completing a set amount of work equivalent to 75% of peak sustainable power output for 60 min. Exercise performance was significantly (P < 0.05) improved with both caffeine treatments as compared to placebo (4.2% with 3 mg · kg−1 body mass and 2.9% with 6 mg · kg−1 body mass). The difference between the two caffeine doses was not statistically significant (P = 0.24). Caffeine ingestion at either dose resulted in significantly higher heart rate values than the placebo conditions (P < 0.05), but no statistically significant treatment effects in ratings of perceived exertion (RPE) were observed (P = 0.39). A caffeine dose of 3 mg · kg−1 body mass appears to improve cycling performance in well-trained and familiarised athletes. Doubling the dose to 6 mg · kg−1 body mass does not confer any additional improvements in performance.

Cyclic Dipeptides in the Induction of Maturation for Cancer Therapy

Studies have suggested a possible form of therapy based on the use of maturation‐inducing compounds to induce differentiation of neoplastic cells and stimulate faster recovery of the normal cell population. The study of the effects of nine cyclic dipeptides on biochemical markers of differentiation implicated their potential to induce differentiation. Studies were undertaken to determine the specificity of these agents for HT‐29 cell cultures as well as the identification of the signal transduction pathways affected by these agents inducing the differential gene expression observed in the cells.

Molecular Mechanisms Underlying the Effects of Statins in the Central Nervous System

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, commonly referred to as statins, are widely used in the treatment of dyslipidaemia, in addition to providing primary and secondary prevention against cardiovascular disease and stroke. Statins’ effects on the central nervous system (CNS), particularly on cognition and neurological disorders such as stroke and multiple sclerosis, have received increasing attention in recent years, both within the scientific community and in the media. Current understanding of statins’ effects is limited by a lack of mechanism-based studies, as well as the assumption that all statins have the same pharmacological effect in the central nervous system. This review aims to provide an updated discussion on the molecular mechanisms contributing to statins’ possible effects on cognitive function, neurodegenerative disease, and various neurological disorders such as stroke, epilepsy, depression and CNS cancers. Additionally, the pharmacokinetic differences between statins and how these may result in statin-specific neurological effects are also discussed.

Pyocyanin-induced toxicity in A549 respiratory cells is causally linked to oxidative stress

Pyocyanin, a virulence factor produced by Pseudomonas aeruginosa, has many damaging effects on mammalian cells. Several lines of evidence suggest that this damage is primarily mediated by its ability to generate ROS and deplete host antioxidant defence mechanisms. However, a causal role for oxidative stress has not yet been demonstrated conclusively. Parallel measures of ROS production, antioxidant levels and cytotoxicity provide convincing evidence that pyocyanin-induced cytotoxicity in A549 respiratory cells is mediated by acute ROS production and subsequent oxidative stress. Pyocyanin increased ROS levels in A549 cells as measured by the fluorescent H(2)O(2) probes Amplex Red and DCFH-DA. These effects were attenuated by the antioxidant N-acetylcysteine. Furthermore, pyocyanin-induced depletion of intracellular GSH levels 24h after exposure was also prevented by pre-treatment of cells with NAC. Under these conditions, NAC protected cells against pyocyanin-induced cytotoxicity as measured by resazurin reduction to resorufin and viable cell counts, strongly supporting a causal role for oxidative stress. Finally, we also show that pyocyanin-induced activation of the immune and inflammatory transcription factor NF-κB in A549 cells is likely mediated by increased ROS. This increased understanding of mechanisms underlying pyocyanin-induced cytotoxicity may ultimately lead to better strategies for reducing the virulence associated with chronic P. aeruginosa infection.

Caffeine withdrawal and high-intensity endurance cycling performance

Abstract In this study, we investigated the impact of a controlled 4-day caffeine withdrawal period on the effect of an acute caffeine dose on endurance exercise performance. Twelve well-trained and familiarized male cyclists, who were caffeine consumers (from coffee and a range of other sources), were recruited for the study. A double-blind placebo-controlled cross-over design was employed, involving four experimental trials. Participants abstained from dietary caffeine sources for 4 days before the trials and ingested caspulses (one in the morning and one in the afternoon) containing either placebo or caffeine (1.5 mg · kg−1 body weight · day−1). On day 5, capsules containing placebo or caffeine (3 mg · kg−1 body weight) were ingested 90 min before completing a time trial, equivalent to one hour of cycling at 75% peak sustainable power output. Hence the study was designed to incorporate placebo–placebo, placebo–caffeine, caffeine–placebo, and caffeine–caffeine conditions. Performance time was significantly improved after acute caffeine ingestion by 1:49 ± 1:41 min (3.0%, P = 0.021) following a withdrawal period (placebo–placebo vs. placebo–caffeine), and by 2:07 ± 1:28 min (3.6%, P = 0.002) following the non-withdrawal period (caffeine–placebo vs. caffeine–caffeine). No significant difference was detetcted between the two acute caffeine trials (placebo–caffeine vs. caffeine–caffeine). Average heart rate throughout exercise was significantly higher following acute caffeine administration compared with placebo. No differences were observed in ratings of perceived exertion between trials. A 3 mg · kg−1 dose of caffeine significantly improves exercise performance irrespective of whether a 4-day withdrawal period is imposed on habitual caffeine users.

Forecasting elasmobranch survival following exposure to severe stressors.

Current fishing practices and habitat degradation in most of the worlds oceans pose significant threats to marine fish including elasmobranchs. The accurate prediction of survival probability for elasmobranchs subjected to prolonged immobilisation and diminished oxygen availability during capture and a vulnerable state post-release, is reliant on selecting a reliable set of biomarkers to profile as well as using them to design pre-release interventions which minimise elasmobranch death. The purpose of this review is: i) to make a case for the need to develop new biomarkers to use in conjunction with blood chemistry; ii) to briefly present the survival strategies used by other vertebrates subjected to diminished oxygen iii) to discuss new approaches to forecasting the effect that altered physiological and biochemical markers have on long-term survival with a particular emphasis on oxidative stress, the adenylate energy charge, heat shock protein expression and the capacity for repair, so that a more detailed profile of the qualities of elasmobranch survivorship can be constructed. In addition, the review will discuss the relevance of biomarkers to field samples as well as their incorporation into laboratory based research, aimed at providing physiological and biochemical data to inform conservation management.

A review of the bioactivity of coffee, caffeine and key coffee constituents on inflammatory responses linked to depression

Coffee is a widely consumed beverage containing numerous biologically active constituents predominantly belonging to the polyphenol and alkaloid classes. It has been established that coffee has a beneficial effect on numerous disease states including depression. A number of prospective and retrospective cohort studies have assessed the effects of coffee consumption on the relative risk of developing major depressive disorder in humans. These studies have identified an inverse relationship between the consumption of caffeinated coffee and the risk of developing depression. Caffeine, chlorogenic acid, ferulic acid and caffeic acid, all important constituents of coffee, have been shown to possess biological activities that highlight a possible mechanistic link to the pathology of depression. This review aims to assess the evidence from the biological evaluation of these constituents of coffee on markers of inflammation associated with depression in in vitro and in vivo models of inflammation, neuroinflammation and depression. The ability of bioactive coffee constituents to modulate the parameters of neuroinflammation has been shown with caffeine having strong antioxidant properties in vitro, chlorogenic acid and caffeic acid having strong anti-inflammatory and antioxidant properties in vitro and ferulic acid having activities in in vivo animal models of depression.

Effects of Pseudomonas Aeruginosa Virulence Factor Pyocyanin on Human Urothelial Cell Function and Viability

PURPOSE We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. MATERIALS AND METHODS RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. RESULTS The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. CONCLUSIONS Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection.