- Email: [email protected]
Addition of a histone deacetylase inhibitor increases recombinant protein expression in plant cell cultures
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
between constipation and an excessive intake of high-fat diet (HFD) has been speculated. In addition, some anti-oxidants have been reported to improve the state of constipation. However, the mechanism behind these phenomena is not clear yet. Aim: The aim of this study is to investigate whether HFD causes constipation and to clarify the mechanism behind this phenomenon, focusing on the involvement of oxidative stress. Method: Male 6-week-old C57BL/6 mice were randomly divided into two groups; feeding HFD and normal diet. Fecal pellet numbers, fecal pellet water content and colon transit time were measured to evaluate whether mice are in the state of constipation. Colonic malondialdehyde (MDA) density was measured as oxidative stress marker. Result: Compared to mice feeding normal diet, fecal pellet numbers were less, colon transit time was longer, and the MDA density was higher in HFD feeding mice. Conclusion: HFD might cause constipation via oxidative stress dependent mechanism, and the therapeutic strategy targeting oxidative stress may be effective.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.443
P-297
Addition of a histone deacetylase inhibitor increases recombinant protein expression in plant cell cultures
S135
P-298
Quality peroxynitrite can be produced in any laboratory Romina Medeiros Raúl Chiozzone, Graciela Borthagaray Facultad de Química, Universidad de la República, Uruguay
Peroxynitrite is a reactive oxygen and nitrogen specie generated in vivo when nitric oxide reacts with superoxide, being a strong oxidant and nitrating agent of biomolecules. Its preparations are used frequently in biochemical reactions, being reported many methods for its synthesis. Most of them are potentially dangerous and require special and expensive equipment. In the present work we propose a simple, fast, low cost synthesis method, with high yield and low content of contaminants; based on Hugues and Nicklin 1968. The method requires hydrogen peroxide, sodium nitrite, hydrochloric acid and sodium hydroxide as reagents, and material commonly available in any laboratory. The remaining hydrogen peroxide was removed with manganese dioxide which was also prepared in house from potassium permanganate, ethanol and water. The concentration of peroxynitrite was determined spectrophotometrically, of hydrogen peroxide with a horseradish peroxidase-based assay, and of nitrite with the Griess method. Peroxynitrite was obtained with an approximate 80% yield, which decreased to approximately 70% after treatment with manganese dioxide, a hydrogen content lower than the assay detection limit (50 μM) and a nitrite concentration of less than 5%.
E-mail address: [email protected] Rita B. Santos, Ana Sofia Pires, Rita Abranches http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.445 Plant Cell Biology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Oeiras, Portugal P-299 Plant cell cultures are an attractive platform for the production of complex recombinant proteins, with lower costs than mammalian cells. A major drawback, hindering the establishment of plant cell suspensions as an industrial platform, is the low yields obtained thus far. Histone acetylation is associated with increased transcription levels. It is expected that the use of histone deacetylase inhibitors (HDACi) would result in an increase in mRNA and protein levels. This hypothesis was tested by adding HDACi to a cell line of the model legume Medicago truncatula expressing a recombinant human protein. Histone deacetylase inhibition and histone H3 acetylation levels were studied, and the effect of HDACi on gene expression and recombinant protein levels was assessed by digital PCR. HDACi were able to inhibit histone deacetylase activity resulting in increased histone H3 acetylation. Higher levels of transgene expression and protein accumulation were observed. This is the first report describing HDACi as inducers of recombinant protein expression in plant cell suspensions as well as the use of digital PCR in these biological systems. This study paves the way for employing epigenetic strategies to improve the final yields of recombinant proteins produced by plant cell cultures.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.444
Short-term regulation of Aquaporin-5 by combined phosphorylation and pH A.F. Mósca 1,2, C. Rodrigues 1,2, A.P. Martins 2, G. Soveral 1,2 1
iMed.ULisboa – Research Institute of Medicines, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal 2 Dept. Bioquímica e Biologia Humana, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
Aquaporin-5 (AQP5) is a membrane water channel widely distributed in human tissues that was found up-regulated in different tumors and considered implicated in carcinogenesis. AQP5 phosphorylation was reported to increase its expression and trafficking, resulting in increased membrane abundance. Interestingly, AQP5 is mostly found phosphorylated in tumor tissues. However, the effects of phosphorylation on channel activity as well as its sensitivity to extracellular acidification have not been investigated so far. In this work, using a yeast model of heterologous expression, we investigated the effect of protein phosphorylation and pH sensitivity as a mechanism of AQP5 short-term regulation. We observed that external acidification alone (pH 5) does not affect AQP5 activity. However, AQP5 phosphorylation induced by intracellular cAMP renders the protein channel prone to pH sensing, resulting in marked increase in water permeability when pH is raised to 7.4. The mechanism of gating may involve intracellular phosphorylation of AQP5 with consequent change in protein conformation and channel pore widening. In this new
between constipation and an excessive intake of high-fat diet (HFD) has been speculated. In addition, some anti-oxidants have been reported to improve the state of constipation. However, the mechanism behind these phenomena is not clear yet. Aim: The aim of this study is to investigate whether HFD causes constipation and to clarify the mechanism behind this phenomenon, focusing on the involvement of oxidative stress. Method: Male 6-week-old C57BL/6 mice were randomly divided into two groups; feeding HFD and normal diet. Fecal pellet numbers, fecal pellet water content and colon transit time were measured to evaluate whether mice are in the state of constipation. Colonic malondialdehyde (MDA) density was measured as oxidative stress marker. Result: Compared to mice feeding normal diet, fecal pellet numbers were less, colon transit time was longer, and the MDA density was higher in HFD feeding mice. Conclusion: HFD might cause constipation via oxidative stress dependent mechanism, and the therapeutic strategy targeting oxidative stress may be effective.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.443
P-297
Addition of a histone deacetylase inhibitor increases recombinant protein expression in plant cell cultures
S135
P-298
Quality peroxynitrite can be produced in any laboratory Romina Medeiros Raúl Chiozzone, Graciela Borthagaray Facultad de Química, Universidad de la República, Uruguay
Peroxynitrite is a reactive oxygen and nitrogen specie generated in vivo when nitric oxide reacts with superoxide, being a strong oxidant and nitrating agent of biomolecules. Its preparations are used frequently in biochemical reactions, being reported many methods for its synthesis. Most of them are potentially dangerous and require special and expensive equipment. In the present work we propose a simple, fast, low cost synthesis method, with high yield and low content of contaminants; based on Hugues and Nicklin 1968. The method requires hydrogen peroxide, sodium nitrite, hydrochloric acid and sodium hydroxide as reagents, and material commonly available in any laboratory. The remaining hydrogen peroxide was removed with manganese dioxide which was also prepared in house from potassium permanganate, ethanol and water. The concentration of peroxynitrite was determined spectrophotometrically, of hydrogen peroxide with a horseradish peroxidase-based assay, and of nitrite with the Griess method. Peroxynitrite was obtained with an approximate 80% yield, which decreased to approximately 70% after treatment with manganese dioxide, a hydrogen content lower than the assay detection limit (50 μM) and a nitrite concentration of less than 5%.
E-mail address: [email protected] Rita B. Santos, Ana Sofia Pires, Rita Abranches http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.445 Plant Cell Biology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Oeiras, Portugal P-299 Plant cell cultures are an attractive platform for the production of complex recombinant proteins, with lower costs than mammalian cells. A major drawback, hindering the establishment of plant cell suspensions as an industrial platform, is the low yields obtained thus far. Histone acetylation is associated with increased transcription levels. It is expected that the use of histone deacetylase inhibitors (HDACi) would result in an increase in mRNA and protein levels. This hypothesis was tested by adding HDACi to a cell line of the model legume Medicago truncatula expressing a recombinant human protein. Histone deacetylase inhibition and histone H3 acetylation levels were studied, and the effect of HDACi on gene expression and recombinant protein levels was assessed by digital PCR. HDACi were able to inhibit histone deacetylase activity resulting in increased histone H3 acetylation. Higher levels of transgene expression and protein accumulation were observed. This is the first report describing HDACi as inducers of recombinant protein expression in plant cell suspensions as well as the use of digital PCR in these biological systems. This study paves the way for employing epigenetic strategies to improve the final yields of recombinant proteins produced by plant cell cultures.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.444
Short-term regulation of Aquaporin-5 by combined phosphorylation and pH A.F. Mósca 1,2, C. Rodrigues 1,2, A.P. Martins 2, G. Soveral 1,2 1
iMed.ULisboa – Research Institute of Medicines, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal 2 Dept. Bioquímica e Biologia Humana, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
Aquaporin-5 (AQP5) is a membrane water channel widely distributed in human tissues that was found up-regulated in different tumors and considered implicated in carcinogenesis. AQP5 phosphorylation was reported to increase its expression and trafficking, resulting in increased membrane abundance. Interestingly, AQP5 is mostly found phosphorylated in tumor tissues. However, the effects of phosphorylation on channel activity as well as its sensitivity to extracellular acidification have not been investigated so far. In this work, using a yeast model of heterologous expression, we investigated the effect of protein phosphorylation and pH sensitivity as a mechanism of AQP5 short-term regulation. We observed that external acidification alone (pH 5) does not affect AQP5 activity. However, AQP5 phosphorylation induced by intracellular cAMP renders the protein channel prone to pH sensing, resulting in marked increase in water permeability when pH is raised to 7.4. The mechanism of gating may involve intracellular phosphorylation of AQP5 with consequent change in protein conformation and channel pore widening. In this new