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- Hedfalk, Kristina, 1969, et al.
(författare)
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Production, characterization and crystallization of the Plasmodium falciparum aquaporin.
- 2008
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Ingår i: Protein expression and purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 59:1, s. 69-78
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Tidskriftsartikel (refereegranskat)abstract
- The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A+T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18mg/L) and initial attempts at crystallization of the protein yielded several different forms.
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- Karlgren, Sara, 1975, et al.
(författare)
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Conditional Osmotic Stress in Yeast. A system to study transport through aquaglyceroporins and osmostress signaling
- 2005
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Ingår i: Journal of biological chemistry. ; 280:8, s. 7186-7193
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Tidskriftsartikel (refereegranskat)abstract
- The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1{Delta} gpd2{Delta} mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1{Delta} gpd2{Delta} mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-{Delta}1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-{Delta}1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-{Delta}1 showed similarly low permeability for water. The growth defect on polyols in the gpd1{Delta} gpd2{Delta} mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1{Delta} gpd2{Delta} mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.
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- Olsson, C. E., et al.
(författare)
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Feasibility of Mastication-Structure-Sparing Radiotherapy for Head and Neck Cancer
- 2021
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Ingår i: International journal of radiation oncology, biology, physics. - : Elsevier BV. - 1879-355X .- 0360-3016. ; 111:3
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE/OBJECTIVE(S): Although radiation-induced side-effects affecting mastication functionality have been studied in head and neck cancer (HNC) radiotherapy (RT), dose constraints for the associated structures are rarely included during treatment plan optimization. Previous research has identified several radiation dose relationships with mean dose thresholds around 30-40 Gy for masseter muscles, 40-50 Gy for pterygoid muscles, and 15-60 Gy for temporomandibular joint (TMJ) relating to a 10% trismus risk post RT. The purpose of this work was to use a multi-criteria optimization (MCO) approach to investigate to what extent doses to these structures can be lowered without violating existing clinical treatment goals in inverse planning of HNC RT. MATERIALS/METHODS: This exploratory treatment planning study used data from 22 HNC patients treated to 68 Gy without mastication-structure-sparing intent in 2017-2019 at one institute in Sweden. Original volumetric-modulated arc therapy (VMAT) plans were re-activated in the treatment planning system and masseter muscles, pterygoid muscles (medial and lateral), and TMJ were consistently delineated according to a previously reported delineation manual4. Re-planning was done using the MCO function of the treatment planning system with the resulting dose distribution normalized to match the clinical target volume (CTV T) mean dose of the clinical treatment plan. Dose differences between MCO and clinical plans were not allowed to exceed 2 Gy for any original clinical treatment goal unless tolerance doses had been substantially exceeded in the clinical treatment plan. To what extent dose to mastication structures could be lowered without violating existing clinical treatment goals were quantified by group and by patient. RESULTS: Altogether, there were 334 clinical treatment goals in the clinical treatment plans (median=15, range: 7-24 per patient, depending on tumor location), which easily could be met in the corresponding MCO plans. Mean doses to the mastication structures were in most cases below proposed tolerance doses in the clinical plan but could on average be further reduced between 3-5 Gy in the MCO plans (Table). Of the 25/88 patient reductions below 5 Gy (28%), 18/25 (72%) were for the masseter (n=8) and medial pterygoid (n=10) muscles. CONCLUSION: With modern RT, it seems possible to reduce the dose to mastication structures below proposed trismus dose tolerance thresholds for most HNC patients without violating clinical treatment goals. Focusing on masseter and medial pterygoid muscle doses may prove to give the largest benefit in individual cases. Copyright © 2021. Published by Elsevier Inc.
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- Pettersson, Nina, 1974, et al.
(författare)
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Aquaporins in yeasts and filamentous fungi
- 2005
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Ingår i: Biology of the Cell. ; 97:7, s. 487-500
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Recension (övrigt vetenskapligt/konstnärligt)abstract
- Recently, genome sequences from different fungi have become available. This information reveals that yeasts and filamentous fungi possess up to five aquaporins. Functional analyses have mainly been performed in budding yeast, Saccharomyces cerevisiae, which has two orthodox aquaporins and two aquaglyceroporins. Whereas Aqy1 is a spore-specific water channel, Aqy2 is only expressed in proliferating cells and controlled by osmotic signals. Fungal aquaglyceroporins often have long, poorly conserved terminal extensions and differ in the otherwise highly conserved NPA motifs, being NPX and NXA respectively. Three subgroups can be distinguished. Fps1-like proteins seem to be restricted to yeasts. Fps1, the osmogated glycerol export channel in S. cerevisiae, plays a central role in osmoregulation and determination of intracellular glycerol levels. Sequences important for gating have been identified within its termini. Another type of aquaglyceroporin, resembling S. cerevisiae Yfl054, has a long N-terminal extension and its physiological role is currently unknown. The third group of aquaglyceroporins, only found in filamentous fungi, have extensions of variable size. Taken together, yeasts and filamentous fungi are a fruitful resource to study the function, evolution, role and regulation of aquaporins, and the possibility to compare orthologous sequences from a large number of different organisms facilitates functional and structural studies
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- Pettersson, Nina, 1974, et al.
(författare)
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Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae
- 2006
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Ingår i: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 50:4, s. 247-255
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Tidskriftsartikel (refereegranskat)abstract
- In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO4, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use
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- Pettersson, Nina, 1974, et al.
(författare)
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Using yeast to study transport and structure-function relationship in aquaglyceroporins
- 2005
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Ingår i: Febs Journal. - 1742-464X .- 1742-4658. ; 272:Suppl. 1, JUL 2005, s. 196-197
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Konferensbidrag (refereegranskat)abstract
- Aquaporins are small membrane proteins that transport water while the closely related aquaglyceroporins also can be permeable to polyols, urea and even arsenic. These substances can pass the pore in both directions by facilitated diffusion. Aquaporins are represented in organisms ranging from archaea to human, and their discovery was awarded the Nobel Prize in chemistry in 2003. Eleven different aquaporins (0–10) have been identified in mammals. Of these, AQP3, 7, 9 and 10 are aquaglyceroporins. They are expressed in a tissue-specific manner and play key roles Abstracts 196 in the regulation of water balance. Examples for relevant applications are transpiration, water retention in the kidneys and glycerol transport following fat metabolism. Aquaglyceroporins are probably also an entry point for arsenic in the liver. To study the function of aquaglyceroporins, we have developed a test system in Saccharomyces cerevisiae. When exposed to a hyperosmotic stress, yeast cells uses glycerol as a compatible solute to regain the turgor pressure decreased by water loss. When aquaglyceroporins are expressed in such cells, they cause sensitivity to hyperosmotic stress, due to glycerol loss through the aquaglyceroporins. When expressed in a strain deficient in glycerol production, the sensitivity of that strain to high levels of certain polyols is suppressed because the polyol can be taken up by the cell through the aquaglyceroporin. We have employed this system of conditional osmotic shock to design a genetic screen which has made it possible to identify residues responsible for the regulation of the yeast aquaglyceroporin Fps1. The genetic screen is being further developed to unravel key residues in channel specificity as well as the mode of action of potential inhibitors. We are also using this system to study mammalian aquaglyceroporins.
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- Sidoux-Walter, Frédéric, et al.
(författare)
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The Saccharomyces cerevisiae aquaporin Aqy1 is involved in sporulation
- 2004
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Ingår i: PNAS. - : Proceedings of the National Academy of Sciences. ; 101:50, s. 17422-17427
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins mediate rapid selective water transport across biological membranes. Elucidation of their precise physiological roles promises important insight into cellular and organismal osmoregulation. The genome of the yeast Saccharomyces cerevisiae encodes two similar but differentially regulated aquaporins. Here, we show that expression of AQY1 is stimulated during sporulation and that the Aqy1 protein is detectable exclusively in spore membranes. When spores are rapidly frozen, those that lack Aqy1 survive better, providing for a functional test of active spore water channels. Under ambient conditions, lack of Aqy1 reduces spore fitness. Because this reduction is independent from germination conditions, Aqy1 may be important during spore formation rather than subsequent maintenance or germination. Indeed, it seems that Aqy1 is degraded after spores have been formed and during germination. Taken together, Aqy1 is developmentally controlled and may play a role in spore maturation, probably by allowing water outflow. Taken together, we demonstrate a functional role of an aquaporin in gametogenesis, as well as in the formation of durable structures such as spores, a role that may have wider biological and medical implications.
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