Chemico-Biological Interactions 238 (2015) 91–92
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Letter to the Editor
Conjugate products of pyocyanin–glutathione reactions Rajkumar Cheluvappa a,⇑, Rajaraman Eri b a b
Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, NSW, Australia Mucosal Biology Laboratory, School of Human Life Sciences, University of Tasmania, Launceston, TAS, Australia
a r t i c l e
i n f o
Article history: Received 2 May 2015 Accepted 2 June 2015 Available online 12 June 2015 Keywords: Pyocyanin Glutathione Catalase Superoxide dismutase Hydrogen peroxide Conjugate
a b s t r a c t This ‘‘Letter to the Editor’’ is a ‘‘gentle but purposeful rejoinder’’ to specific comments made in pages 36– 37 of your Muller and Merrett (2015) [1] publication regarding the data presented in our Cheluvappa et al. (2008) [2] paper. Our rebuttal topics include the effect of oxygen on the pyocyanin–glutathione reaction, relevance of reaction-duration to pathophysiology, rationale of experiments, veracity of statements germane to molecular-structure construction, and correction of hyperbole. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
Dear Dr. Dietrich, and Respected Editors, This is a ‘‘gentle but purposeful rejoinder’’ to specific comments made in pages 36–37 of your Muller et al. (2015) publication [1] regarding the data presented in our Cheluvappa et al. (2008) paper [2]. Your Muller et al., 2015 paper [1] claims that, ‘‘It was previously claimed by Cheluvappa et al. [39] that pyocyanin reacts with GSH in a non-enzymatic manner to produce a conjugate with specific physico-chemical properties and an identical structure to that presented in Fig. 6 (also hyperlinked as such) of the present study.’’ There is no ‘‘conjugate structure’’ presented in Fig. 6 of your Muller et al. (2015) paper [1]. Your Muller et al., 2015 paper [1] also claims that, ‘‘. . .we identified an ion by mass spectrometry with of m/z = (sic) 516, consistent with a pyocyanin–GSH conjugate (Fig. 5), whereas similar data were not presented in the previous study.’’
Abbreviations: GSH, reduced glutathione; NMR, Nuclear Magnetic Resonance; P. aeruginosa, Pseudomonas aeruginosa. ⇑ Corresponding author at: Inflammation and Infection Research Centre, School of Medical Sciences, Wallace Wurth Building, University of New South Wales, Gate 9 High Street, Sydney, NSW 2052, Australia. Tel.: +61 0406 0406 20; fax: +61 02 9385 1389. E-mail address: [email protected] (R. Cheluvappa). http://dx.doi.org/10.1016/j.cbi.2015.06.002 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
In our Cheluvappa et al. (2008) paper [2], ‘‘similar’’ data (‘‘via structure identification’’) was indeed presented in the form of NMR analysis (Fig. 5) on page 576; and a structure-proposal (Fig. 7) on page 577. While contesting the reliability of the molecular structure-construction (if possible) is one thing; stating that ‘‘. . .similar data were not presented in the previous study’’ is patently untrue. In your Muller et al. (2015) paper [1], it was claimed that, ‘‘In particular, it was claimed that oxygen was essential for the reaction between pyocyanin and GSH, with hydrogen peroxide a critical intermediate, but no mechanism was provided as to how this might occur. As their experiments were performed under aerobic conditions with incubations (sic) periods of several days, it may be assumed that the hydrogen peroxide arose from the autoxidation of GSH in their system, which may have led to non-specific oxidation of pyocyanin over the extended incubation period.’’ Although no mechanistic overview was postulated in our Cheluvappa et al. (2008) work [2], Fig. 1 (E, F, G, and H) on page 574 depict time-course experiments involving GSH and pyocyanin under aerobic or oxygen-deprived conditions. These data candidly demonstrate that the reaction was much faster under aerobic conditions than vice versa. Therefore, the totally speculative statement, ‘‘it may be assumed that the hydrogen peroxide arose from the autoxidation of GSH in their system’’, may only be partially true.
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R. Cheluvappa, R. Eri / Chemico-Biological Interactions 238 (2015) 91–92
Your Muller et al. (2015) paper [1] claims that, ‘‘. . .a number of very substantial discrepancies exist between the present and the former study.’’, and that, ‘‘. . .the reaction of the present study proceeded in a quantifiable manner over the course of a few hours not several days as previously described.’’ It was frankly posited on page 572 of our Cheluvappa et al. (2008) publication [2], that ‘‘Both Muller (2002) [3] and O’Malley et al. (2004) [4] reported the results of experiments involving pyocyanin and GSH only that were incubated for less than an hour. Yet in CF patients, Pseudomonas aeruginosa, secreting pyocyanin, lingers for long periods of time causing recurrent remissions and exacerbations. We simulated this scenario by demonstrating a reaction between pathophysiologically relevant concentrations of pyocyanin and GSH over a period of days, rather than minutes or hours. In our study, we show a reaction of pyocyanin with GSH in phosphate buffer (pH 7.4), at 37 °C, observed over a period of days under pathophysiologically relevant conditions. . .’’ This excerpt (previous bullet-point) from our Cheluvappa et al., 2008 publication [2] sets a prolonged pathophysiologicallyrelevant (CF) reaction as a deliberate and predetermined premise, in contrast to ‘‘other pathophysiological scenarios’’. So why is this even an issue, when the differences in pathophysiological scenarios have not been compared and contrasted? Your Muller et al. (2015) paper [1] also claims that, ‘‘. . .a number of very substantial discrepancies exist between the (repeated line-segment) two studies and many of the claims made by Cheluvappa et al. [39] could not be substantiated. The discussion pertaining to the inferences from/implications of, the data presented in our Cheluvappa et al. (2008) work [2] was reasonably and constrainedly within the speculative ambit of the data, as demonstrated by my systematic refutation earlier.
Therefore, your Muller et al. (2015) paper’s [1] grandiose claim that ‘‘many of the claims made by Cheluvappa et al. [39] could not be substantiated’’ could not be substantiated. I sincerely hope that you would publish this letter for the benefit of your readers; and for the kind hypothetical benefit of a counter-rebuttal. Thank you very much. Sincerely Yours, Rajkumar Cheluvappa Rajaraman Eri 2015 May 2 Transparency Document The Transparency document associated with this article can be found in the online version.
Acknowledgement Funding sources not relevant. References [1] M. Muller, N.D. Merrett, Mechanism for glutathione-mediated protection against the Pseudomonas aeruginosa redox toxin, pyocyanin, Chem. Biol. Interact. 232 (2015) 30–37. [2] R. Cheluvappa, R. Shimmon, M. Dawson, S.N. Hilmer, D.G. Le Couteur, Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione, Acta Biochim. Pol. 55 (2008) 571–580. [3] M. Muller, Pyocyanin induces oxidative stress in human endothelial cells and modulates the glutathione redox cycle, Free Rad. Biol. Med. 33 (2002) 1527– 1533. [4] Y.Q. O’Malley, K.J. Reszka, D.R. Spitz, G.M. Denning, B.E. Britigan, Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells, Am. J. Physiol. Lung Cell. Mol. Physiol. 287 (2004) L94– L103.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Letter to the Editor
Conjugate products of pyocyanin–glutathione reactions Rajkumar Cheluvappa a,⇑, Rajaraman Eri b a b
Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, NSW, Australia Mucosal Biology Laboratory, School of Human Life Sciences, University of Tasmania, Launceston, TAS, Australia
a r t i c l e
i n f o
Article history: Received 2 May 2015 Accepted 2 June 2015 Available online 12 June 2015 Keywords: Pyocyanin Glutathione Catalase Superoxide dismutase Hydrogen peroxide Conjugate
a b s t r a c t This ‘‘Letter to the Editor’’ is a ‘‘gentle but purposeful rejoinder’’ to specific comments made in pages 36– 37 of your Muller and Merrett (2015) [1] publication regarding the data presented in our Cheluvappa et al. (2008) [2] paper. Our rebuttal topics include the effect of oxygen on the pyocyanin–glutathione reaction, relevance of reaction-duration to pathophysiology, rationale of experiments, veracity of statements germane to molecular-structure construction, and correction of hyperbole. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
Dear Dr. Dietrich, and Respected Editors, This is a ‘‘gentle but purposeful rejoinder’’ to specific comments made in pages 36–37 of your Muller et al. (2015) publication [1] regarding the data presented in our Cheluvappa et al. (2008) paper [2]. Your Muller et al., 2015 paper [1] claims that, ‘‘It was previously claimed by Cheluvappa et al. [39] that pyocyanin reacts with GSH in a non-enzymatic manner to produce a conjugate with specific physico-chemical properties and an identical structure to that presented in Fig. 6 (also hyperlinked as such) of the present study.’’ There is no ‘‘conjugate structure’’ presented in Fig. 6 of your Muller et al. (2015) paper [1]. Your Muller et al., 2015 paper [1] also claims that, ‘‘. . .we identified an ion by mass spectrometry with of m/z = (sic) 516, consistent with a pyocyanin–GSH conjugate (Fig. 5), whereas similar data were not presented in the previous study.’’
Abbreviations: GSH, reduced glutathione; NMR, Nuclear Magnetic Resonance; P. aeruginosa, Pseudomonas aeruginosa. ⇑ Corresponding author at: Inflammation and Infection Research Centre, School of Medical Sciences, Wallace Wurth Building, University of New South Wales, Gate 9 High Street, Sydney, NSW 2052, Australia. Tel.: +61 0406 0406 20; fax: +61 02 9385 1389. E-mail address: [email protected] (R. Cheluvappa). http://dx.doi.org/10.1016/j.cbi.2015.06.002 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
In our Cheluvappa et al. (2008) paper [2], ‘‘similar’’ data (‘‘via structure identification’’) was indeed presented in the form of NMR analysis (Fig. 5) on page 576; and a structure-proposal (Fig. 7) on page 577. While contesting the reliability of the molecular structure-construction (if possible) is one thing; stating that ‘‘. . .similar data were not presented in the previous study’’ is patently untrue. In your Muller et al. (2015) paper [1], it was claimed that, ‘‘In particular, it was claimed that oxygen was essential for the reaction between pyocyanin and GSH, with hydrogen peroxide a critical intermediate, but no mechanism was provided as to how this might occur. As their experiments were performed under aerobic conditions with incubations (sic) periods of several days, it may be assumed that the hydrogen peroxide arose from the autoxidation of GSH in their system, which may have led to non-specific oxidation of pyocyanin over the extended incubation period.’’ Although no mechanistic overview was postulated in our Cheluvappa et al. (2008) work [2], Fig. 1 (E, F, G, and H) on page 574 depict time-course experiments involving GSH and pyocyanin under aerobic or oxygen-deprived conditions. These data candidly demonstrate that the reaction was much faster under aerobic conditions than vice versa. Therefore, the totally speculative statement, ‘‘it may be assumed that the hydrogen peroxide arose from the autoxidation of GSH in their system’’, may only be partially true.
92
R. Cheluvappa, R. Eri / Chemico-Biological Interactions 238 (2015) 91–92
Your Muller et al. (2015) paper [1] claims that, ‘‘. . .a number of very substantial discrepancies exist between the present and the former study.’’, and that, ‘‘. . .the reaction of the present study proceeded in a quantifiable manner over the course of a few hours not several days as previously described.’’ It was frankly posited on page 572 of our Cheluvappa et al. (2008) publication [2], that ‘‘Both Muller (2002) [3] and O’Malley et al. (2004) [4] reported the results of experiments involving pyocyanin and GSH only that were incubated for less than an hour. Yet in CF patients, Pseudomonas aeruginosa, secreting pyocyanin, lingers for long periods of time causing recurrent remissions and exacerbations. We simulated this scenario by demonstrating a reaction between pathophysiologically relevant concentrations of pyocyanin and GSH over a period of days, rather than minutes or hours. In our study, we show a reaction of pyocyanin with GSH in phosphate buffer (pH 7.4), at 37 °C, observed over a period of days under pathophysiologically relevant conditions. . .’’ This excerpt (previous bullet-point) from our Cheluvappa et al., 2008 publication [2] sets a prolonged pathophysiologicallyrelevant (CF) reaction as a deliberate and predetermined premise, in contrast to ‘‘other pathophysiological scenarios’’. So why is this even an issue, when the differences in pathophysiological scenarios have not been compared and contrasted? Your Muller et al. (2015) paper [1] also claims that, ‘‘. . .a number of very substantial discrepancies exist between the (repeated line-segment) two studies and many of the claims made by Cheluvappa et al. [39] could not be substantiated. The discussion pertaining to the inferences from/implications of, the data presented in our Cheluvappa et al. (2008) work [2] was reasonably and constrainedly within the speculative ambit of the data, as demonstrated by my systematic refutation earlier.
Therefore, your Muller et al. (2015) paper’s [1] grandiose claim that ‘‘many of the claims made by Cheluvappa et al. [39] could not be substantiated’’ could not be substantiated. I sincerely hope that you would publish this letter for the benefit of your readers; and for the kind hypothetical benefit of a counter-rebuttal. Thank you very much. Sincerely Yours, Rajkumar Cheluvappa Rajaraman Eri 2015 May 2 Transparency Document The Transparency document associated with this article can be found in the online version.
Acknowledgement Funding sources not relevant. References [1] M. Muller, N.D. Merrett, Mechanism for glutathione-mediated protection against the Pseudomonas aeruginosa redox toxin, pyocyanin, Chem. Biol. Interact. 232 (2015) 30–37. [2] R. Cheluvappa, R. Shimmon, M. Dawson, S.N. Hilmer, D.G. Le Couteur, Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione, Acta Biochim. Pol. 55 (2008) 571–580. [3] M. Muller, Pyocyanin induces oxidative stress in human endothelial cells and modulates the glutathione redox cycle, Free Rad. Biol. Med. 33 (2002) 1527– 1533. [4] Y.Q. O’Malley, K.J. Reszka, D.R. Spitz, G.M. Denning, B.E. Britigan, Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells, Am. J. Physiol. Lung Cell. Mol. Physiol. 287 (2004) L94– L103.
