| 91. |
- Wu, Siqin, et al.
(författare)
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TGF-beta enforces senescence in Myc-transformed hematopoietic tumor cells through induction of Mad1 and repression of Myc activity
- 2009
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 315:18, s. 3099-3111
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Tidskriftsartikel (refereegranskat)abstract
- Inhibition of tumor growth factor (TGF)-beta-mediated cell cycle exit is considered an important tumorigenic function of Myc oncoproteins. Here we found that TGF-beta1 enforced G(1) cell cycle arrest and cellular senescence in human U-937 myeloid tumor cells ectopically expressing v-Myc, which contains a stabilizing mutation frequently found in lymphomas. This correlated with induced expression of the Myc antagonist Mad1, resulting in replacement of Myc for Mad1 at target promoters, reduced histone acetylation and strong repression of Myc-driven transcription. The latter was partially reversed by histone deacetylase (HDAC) inhibitors, consistent with involvement of Mad1. Importantly, knockdown of MAD1 expression prevented TGF-beta1-induced senescence, underscoring that Mad1 is a crucial component of this process. Enforced Mad1 expression sensitized U-937-myc cells to TGF-beta and restored phorbol ester-induced cell cycle exit, but could not alone induce G(1) arrest, suggesting that Mad1 is required but not sufficient for cellular senescence. Our results thus demonstrate that TGF-beta can override Myc activity despite a stabilizing cancer mutation and induce senescence in myeloid tumor cells, at least in part by induction of Mad1. TGF-beta-induced senescence, or signals mimicking this pathway, could therefore potentially be explored as a therapeutic principle for treating hematopoietic and other tumors with deregulated MYC expression.
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| 92. |
- Wöhri, Annemarie, 1976, et al.
(författare)
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A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization
- 2008
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 16:7, s. 1003-1009
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Tidskriftsartikel (refereegranskat)abstract
- A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.
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| 93. |
- Öberg, Anna, 1979, et al.
(författare)
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Barriers for industrial implementation of in-process monitoring of weld penetration for quality control
- 2017
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Ingår i: The International Journal of Advanced Manufacturing Technology. - : Springer Science and Business Media LLC. - 0268-3768 .- 1433-3015. ; 91:5-8, s. 2427-2434
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Tidskriftsartikel (refereegranskat)abstract
- The research conducted sheds a light on the question why robust in-process monitoring and adaptive control are not fully implemented in the welding industry. In the research project FaRoMonitA, the possibilities to monitor the weld quality during welding have been investigated. Research conducted in this area has merely focused on technical issues investigated in a laboratory environment. To advance the research front and release some barriers related to industrial acceptance, the studies conducted in this paper have been both quantitative and qualitative in form of experiments combined with an interview study. The quality property weld penetration depth was chosen for in-process monitoring to evaluate the industrial relevance and applicability. A guaranteed weld penetration depth is critical for companies producing parts influenced by fatigue. The parts studied were fillet welds produced by gas metal arc welding. The experiments show that there is a relationship between final penetration depth and monitored arc voltage signals and images captured by CMOS vision and infrared cameras during welding. There are still technical issues to be solved to reach a robust solution. The interview study indicates that soft issues, like competence and financial aspects, are just as critical.
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| 94. |
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| 95. |
- Öberg, Fredrik, et al.
(författare)
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Acoustical and perceptual influence of duplex stringing in grand pianos
- 2012
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Ingår i: Journal of the Acoustical Society of America. - : Acoustical Society of America (ASA). - 0001-4966 .- 1520-8524. ; 131:1, s. 856-871
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Tidskriftsartikel (refereegranskat)abstract
- This study investigates the acoustical and perceptual influence of the string parts outside the speaking length in grand pianos (front and rear duplex strings). Acoustical measurements on a grand piano in concert condition were conducted, measuring the fundamental frequencies of all main and duplex strings in the four octaves D4-C8. Considerable deviations from the nominal harmonic relations between the rear duplex and main string frequencies, as described by the manufacturer in a patent, were observed. Generally the rear duplex strings were tuned higher than the nominal harmonic relations with average and median deviations approaching _50 cent. Single keys reached +190 and -100 cent. The spread in deviation from harmonic relations within trichords was also substantial with average and median values around 25 cent, occasionally reaching 60 cent. Contributions from both front and rear duplex strings were observed in the bridge motion and sound. The audibility of the duplex strings was studied in an ABX listening test. Complete dampening of the front duplex was clearly perceptible both for an experiment group consisting of musicians and a control group with naive subjects. The contribution from the rear duplex could also be perceived, but less pronounced.
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| 96. |
- Öberg, Fredrik, 1982
(författare)
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AQUAPORINS: Production Optimization and Characterization
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism – including humans. They are vital molecules and their malfunction can lead to several severe disorders. An increased understanding of their structure, function and regulation is of utmost importance for developing current and future drugs. The first problem to overcome is to acquire the proteins in sufficient amounts to enable characterization. To achieve this, proteins are often produced in a host organism. One of the most successful hosts for recombinant overproduction is the yeast Pichia pastoris. Using this yeast we could obtain exceptional yield of aquaporin 1, whereas some others were below the threshold needed for successful subsequent characterization. In this process, we have established methods allowing fast and accurate determination of the initial production yield. Furthermore, we optimized the yield for low producing targets, enabling studies of proteins previously out of reach, exemplified with human aquaporin 4. Characterization has been performed on aquaporins obtained in sufficient quantities, and the functionality of aquaporin 1, 5 and 10 has been assessed. Furthermore, a glycosylation was found to stabilize the aquaporin 10 tetramer although only a minority of the monomers where modified. Moreover, we used protein crystallography to determine the three dimensional structure of a hAQP5 mutant, providing insight into regulation of the protein by trafficking. Taken together, these results provide insight into factors directing high production of eukaryotic membrane proteins. The subsequent characterization, including functional and structural determination, reveals new knowledge about aquaporin activity and regulation.
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| 97. |
- Öberg, Fredrik, et al.
(författare)
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Cytokine-induced restoration of differentiation and cell cycle arrest in v-Myc transformed U-937 monoblasts correlates with reduced Myc activity
- 2001
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Ingår i: Leukemia. - 0887-6924 .- 1476-5551. ; 15:2, s. 217-227
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Tidskriftsartikel (refereegranskat)abstract
- Deregulated expression of the myc-family of oncogenes in hematopoietic and other cell types plays an important role in tumorigenesis, and results in increased proliferative potential and block of cellular differentiation. We have previously shown that IFN-gamma restores phorbol ester-induced differentiation and cell cycle arrest in v-myc transformed human U-937 monoblasts. To investigate whether other cytokine signals could also abrogate such a block, IL-1, IL-3, IL-4, IL-6, IL-7, IL-10, IL-11, LIF, oncostatin M, M-CSF, G-CSF and GM-CSF, and TGFbeta1, TNF-alpha, IFN-alpha were examined. We show that GM-CSF and IL-6, in combination with the phorbol ester 12-O-tetradecanoyl-phorbol acetate (TPA), restored differentiation and cell cycle arrest. In contrast, treatment by TGFbeta1 +/- TPA resulted in an efficient G1/G0 arrest, but did not appear to induce terminal differentiation. Restoration of differentiation and cell cycle arrest was accomplished despite maintained expression of the v-Myc protein. Our results show that the cytokine-induced signals reduced Myc-dependent transcription of an artificial target promoter/reporter gene construct, correlating in most, but not all, cases with decreased association of v- and c-Myc with its essential partner, Max. Thus, cytokine-induced signals may counteract the activity of deregulated Myc, and contribute to the normalization of differentiation, arrest in the G1/G0 phase of the cell cycle, or both.
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| 98. |
- Öberg, Fredrik, 1982, et al.
(författare)
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Glycosylation increases the thermostability of human aquaporin 10 protein.
- 2011
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 286:36, s. 31915-23
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Tidskriftsartikel (refereegranskat)abstract
- Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.
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| 99. |
- Öberg, Fredrik, 1982, et al.
(författare)
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Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: the importance of codon optimization and clone selection.
- 2011
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Ingår i: Molecular membrane biology. - : Informa UK Limited. - 1464-5203 .- 0968-7688. ; 28:6, s. 398-411
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Tidskriftsartikel (refereegranskat)abstract
- In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.
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| 100. |
- Öberg, Fredrik, 1982, et al.
(författare)
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Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris.
- 2009
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Ingår i: Molecular membrane biology. - : Informa UK Limited. - 1464-5203 .- 0968-7688. ; 26:4, s. 215-27
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are key players in all living cells. To achieve a better understanding of membrane protein function, significant amounts of purified protein are required for functional and structural analyses. Overproduction of eukaryotic membrane proteins, in particular, is thus an essential yet non-trivial task. Hence, improved understanding of factors which direct a high production of eukaryotic membrane proteins is desirable. In this study we have compared the overproduction of all human aquaporins in the eukaryotic host Pichia pastoris. We report quantitated production levels of each homologue and the extent of their membrane localization. Our results show that the protein production levels vary substantially, even between highly homologous aquaporins. A correlation between the extents of membrane insertion with protein function also emerged, with a higher extent of membrane insertion for pure water transporters compared to aquaporin family members with other substrate specificity. Nevertheless, the nucleic acid sequence of the second codon appears to play an important role in overproduction. Constructs containing guanine at the first position of this codon (being part of the mammalian Kozak sequence) are generally produced at a higher level, which is confirmed for hAQP8. In addition, mimicking the yeast consensus sequence (ATGTCT) apparently has a negative influence on the production level, as shown for hAQP1. Moreover, by mutational analysis we show that the yield of hAQP4 can be heavily improved by directing the protein folding pathway as well as stabilizing the aquaporin tetramer.
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