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The interaction of cardanol derivatives on the activity of PDR pumps in Saccharomyces cerevisiae
S52
Abstracts / Journal of Biotechnology 131S (2007) S48–S52
observed for HRP glassified beads than for the lyophilized protein (25%). We also achieved higher stability during release for HRP glassified beads. The polymer microspheres obtained were more homogeneous and smooth for the HRP glass beads than for the lyophilized HRP. Glassification of proteins can be an alternative dehydration method that can be used for protein encapsulation in polymer microspheres for controlled delivery systems.
References Costantino, H.R., Langer, R., Klibanov, A.M., 1994. Pharm. Res. 11, 21–29. Griebenow, K., Klibanov, A.M., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 10969–10976. Hageman, M.J., 1988. Drug Dev. Ind. Pharm. 14, 2047–2070. Pearlman, R., Nguyen, T., 1992. J. Pharm. Pharmacol. 44 (Suppl. 1), 178–185.
doi:10.1016/j.jbiotec.2007.07.085 doi:10.1016/j.jbiotec.2007.07.084 8. Mechanism of moisture induced solid phase aggregation of pharmaceutical proteins
9. The interaction of cardanol derivatives on the activity of PDR pumps in Saccharomyces cerevisiae Bartosiewicz ∗ , Anna
Giselle Flores ∗ , Myriam Diaz, Jose Rodriguez, Kai Griebenow
Dominika Lukaszewicz
University of Puerto Rico Rio Piedras, Puerto Rico, United States
Wroclaw University Faculty of Biotrchnology, Wroclaw, Poland
Solid protein pharmaceuticals often suffer from instability problems hampering their successful therapeutic application. Upon storage, proteins may become partially hydrated and detrimental moisture induced aggregation is observed (Costantino et al., 1994; Pearlman and Nguyen, 1992; Hageman, 1988). The extent of protein aggregation has been related to the amount of water sorbed and often a bell-shaped curve is observed when plotting the amount of aggregates formed versus the relative humidity used to incubate the protein. One frequently formulated hypothesis in this context is that partial hydration influences protein dynamics and thus unfolding in the solid state (Costantino et al., 1994; Griebenow and Klibanov, 1995). Simply put, increased hydration leads to increased protein dynamics allowing for unfolding and aggregation. The decrease in protein aggregation at very high relative humidity in this model is explained by protein refolding. However, thus far, no solid data on protein structure in partially hydrated states after incubation under defined humidity conditions have been published and thus the hypothesis has not verified. To test the hypothesis, lyophilized insulin powder was incubated in humidity chambers at various relative humidities. Formation of buffer insoluble aggregates was monitored for these samples for weeks. To detect unfolding/refolding events occurring to the solid protein powder, these were subjected to analysis by Fourier-transform infrared spectroscopy and circular dichroism (CD) after 48 h of incubation (after this time the system is in complete equilibrium). We employed an FTIRmicroscope using a diamond cell to avoid exposing the moist protein powders to the high pressure encountered in the formation of a KBr pellets usually employed in such studies on solid protein powders. Furthermore, to avoid artifacts by water vapor and sorbed water, the amide III region (1220–1330 cm−1 ) was used. Results obtained show that insulin forms aggregates upon storage in humidity chambers. Preliminary analysis of the FTIR data currently point in the direction that the hypothesis explaining the bell-shaped curve is correct, but further experiments are currently ongoing to verify this including experiments such as H/D exchange.
Krasowska, Marcin
Phenolic lipids are natural amphiphilic long-chain homologues of phenol, resorcinol and catechol occuring in numerous plants and microorganisms. They are non-isoprenoid compounds that consist of a phenolic structure and a long aliphatic hydrocarbon chain (of varying length and saturation) attached to it. They exhibit high affinity for lipid bilayers and biological membranes and are able to modify the activity of membrane enzymes (AChE, PLA2, or Ca2+ -ATPase). Differences in the chemical structure of phenolic lipids are reflected in different properties of these compounds. Efflux pumps are present in all living cells and participate in the detoxifying process by expelling various harmful compounds and xenobiotics. The function of active efflux systems is to transport drugs through the membrane and limit the intracellular accumulation of toxic compounds. This export is energized by ATP hydrolysis or by an ion antiport mechanism that contributes to the membrane energy state. To prevent this resistance, effective inhibitors of efflux pumps are sought. These compounds either specifically interact with MDR proteins while others influence the supply of energy for MDR pumps by affecting cellular ATP production or the ion-motive force driving the pumps what inhibits pumps activity, e.g. by influencing membrane phospholipids and increasing the permeability of the membrane for small non-electrolytes and cations. The properties of semisynthetic derivatives of a phenolic lipid, cardanol, and their effect on ABC transporters in S. cerevisiae are presented. The influence of semisynthetic derivatives of cardanol on yeast PDR proteins activity was studied by spectrofluorimetric method using the potentiometric fluorescence probe diS-C3(3). The probe is expelled from S. cerevisiae by ABC pumps and can conveniently be used for studying their performance. Two pump-competent S. cerevisiae strains and different pump-free mutant strains were used to check the effect of the studied compounds on the activity of major ABC transporters. Luciferase assay was used to studied the changing of ATP cellular level under the influence of semisynthetic compounds. doi:10.1016/j.jbiotec.2007.07.086
Abstracts / Journal of Biotechnology 131S (2007) S48–S52
observed for HRP glassified beads than for the lyophilized protein (25%). We also achieved higher stability during release for HRP glassified beads. The polymer microspheres obtained were more homogeneous and smooth for the HRP glass beads than for the lyophilized HRP. Glassification of proteins can be an alternative dehydration method that can be used for protein encapsulation in polymer microspheres for controlled delivery systems.
References Costantino, H.R., Langer, R., Klibanov, A.M., 1994. Pharm. Res. 11, 21–29. Griebenow, K., Klibanov, A.M., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 10969–10976. Hageman, M.J., 1988. Drug Dev. Ind. Pharm. 14, 2047–2070. Pearlman, R., Nguyen, T., 1992. J. Pharm. Pharmacol. 44 (Suppl. 1), 178–185.
doi:10.1016/j.jbiotec.2007.07.085 doi:10.1016/j.jbiotec.2007.07.084 8. Mechanism of moisture induced solid phase aggregation of pharmaceutical proteins
9. The interaction of cardanol derivatives on the activity of PDR pumps in Saccharomyces cerevisiae Bartosiewicz ∗ , Anna
Giselle Flores ∗ , Myriam Diaz, Jose Rodriguez, Kai Griebenow
Dominika Lukaszewicz
University of Puerto Rico Rio Piedras, Puerto Rico, United States
Wroclaw University Faculty of Biotrchnology, Wroclaw, Poland
Solid protein pharmaceuticals often suffer from instability problems hampering their successful therapeutic application. Upon storage, proteins may become partially hydrated and detrimental moisture induced aggregation is observed (Costantino et al., 1994; Pearlman and Nguyen, 1992; Hageman, 1988). The extent of protein aggregation has been related to the amount of water sorbed and often a bell-shaped curve is observed when plotting the amount of aggregates formed versus the relative humidity used to incubate the protein. One frequently formulated hypothesis in this context is that partial hydration influences protein dynamics and thus unfolding in the solid state (Costantino et al., 1994; Griebenow and Klibanov, 1995). Simply put, increased hydration leads to increased protein dynamics allowing for unfolding and aggregation. The decrease in protein aggregation at very high relative humidity in this model is explained by protein refolding. However, thus far, no solid data on protein structure in partially hydrated states after incubation under defined humidity conditions have been published and thus the hypothesis has not verified. To test the hypothesis, lyophilized insulin powder was incubated in humidity chambers at various relative humidities. Formation of buffer insoluble aggregates was monitored for these samples for weeks. To detect unfolding/refolding events occurring to the solid protein powder, these were subjected to analysis by Fourier-transform infrared spectroscopy and circular dichroism (CD) after 48 h of incubation (after this time the system is in complete equilibrium). We employed an FTIRmicroscope using a diamond cell to avoid exposing the moist protein powders to the high pressure encountered in the formation of a KBr pellets usually employed in such studies on solid protein powders. Furthermore, to avoid artifacts by water vapor and sorbed water, the amide III region (1220–1330 cm−1 ) was used. Results obtained show that insulin forms aggregates upon storage in humidity chambers. Preliminary analysis of the FTIR data currently point in the direction that the hypothesis explaining the bell-shaped curve is correct, but further experiments are currently ongoing to verify this including experiments such as H/D exchange.
Krasowska, Marcin
Phenolic lipids are natural amphiphilic long-chain homologues of phenol, resorcinol and catechol occuring in numerous plants and microorganisms. They are non-isoprenoid compounds that consist of a phenolic structure and a long aliphatic hydrocarbon chain (of varying length and saturation) attached to it. They exhibit high affinity for lipid bilayers and biological membranes and are able to modify the activity of membrane enzymes (AChE, PLA2, or Ca2+ -ATPase). Differences in the chemical structure of phenolic lipids are reflected in different properties of these compounds. Efflux pumps are present in all living cells and participate in the detoxifying process by expelling various harmful compounds and xenobiotics. The function of active efflux systems is to transport drugs through the membrane and limit the intracellular accumulation of toxic compounds. This export is energized by ATP hydrolysis or by an ion antiport mechanism that contributes to the membrane energy state. To prevent this resistance, effective inhibitors of efflux pumps are sought. These compounds either specifically interact with MDR proteins while others influence the supply of energy for MDR pumps by affecting cellular ATP production or the ion-motive force driving the pumps what inhibits pumps activity, e.g. by influencing membrane phospholipids and increasing the permeability of the membrane for small non-electrolytes and cations. The properties of semisynthetic derivatives of a phenolic lipid, cardanol, and their effect on ABC transporters in S. cerevisiae are presented. The influence of semisynthetic derivatives of cardanol on yeast PDR proteins activity was studied by spectrofluorimetric method using the potentiometric fluorescence probe diS-C3(3). The probe is expelled from S. cerevisiae by ABC pumps and can conveniently be used for studying their performance. Two pump-competent S. cerevisiae strains and different pump-free mutant strains were used to check the effect of the studied compounds on the activity of major ABC transporters. Luciferase assay was used to studied the changing of ATP cellular level under the influence of semisynthetic compounds. doi:10.1016/j.jbiotec.2007.07.086