| 1. |
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| 2. |
- Agaton, C., et al.
(författare)
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Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues
- 2003
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 2, s. 405-
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Tidskriftsartikel (refereegranskat)abstract
- Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.
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| 3. |
- Ahmadian, Afshin, et al.
(författare)
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Analysis of the p53 tumor suppressor gene by pyrosequencing
- 2000
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 28:1, s. 140-
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Tidskriftsartikel (refereegranskat)abstract
- Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one ol both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Further-more, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.
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| 4. |
- Aizman, O., et al.
(författare)
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Ouabain, a steroid hormone that signals with slow calcium oscillations
- 2001
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 98:23, s. 13420-13424
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Tidskriftsartikel (refereegranskat)abstract
- The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca2+](i)) oscillations that elicit activation of the transcription factor, NF-KB. Partial inhibition of Na,K-ATPase using low extracellular K+ and depolarization of cells did not have these effects. Incubation of cells in Ca2+-free media, inhibition of voltage-gated calcium channels, inositol triphosphate receptor antagonism, and redistribution of actin to a thick layer adjacent to the plasma membrane abolished [Ca2+](i) oscillations, indicating that they were caused by a concerted action of inositol triphosphate receptors and capacitative calcium entry via plasma membrane channels. Blockade of ouabain-induced [C-a2+](i) oscillations prevented activation of NF-kappaB. The results demonstrate a new mechanism for steroid signaling via plasma membrane receptors and underline a novel role for the steroid hormone, ouabain, as a physiological inducer of [Ca2+](i) oscillations involved in transcriptional regulation in mammalian cells.
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| 5. |
- Bandmann, Nina, et al.
(författare)
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Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 79:2, s. 161-172
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Tidskriftsartikel (refereegranskat)abstract
- The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitioning of the enzyme in PEG-salt based aqueous two-phase bioseparation systems. Seven different cutinase lipase variants were constructed containing various C-terminal peptide extensions including tryptophan rich peptide tags ((WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positively ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified cutinase variants were stably produced in Escherichia coli as secreted to the periplasm from which they were efficiently purified by IgG-affinity chromatography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants. Partitioning experiments performed in a PEG 4000/sodium phosphate aqueous two-phase system showed that for variants containing either (WP)(2) or (WP)(4) peptide extensions, 10- to 70-fold increases in the partitioning to the PEG rich top-phase were obtained, when compared to the wild type enzyme. An increased partitioning was also seen for cutinase variants tagged with both tryptophans and charged amino acids, whereas the effect of solely charged peptide extensions was relatively small. In addition, when performing partitioning experiments from cell disintegrates, the (WP)(4)-tagged cutinase showed a similarly high PEG-phase partitioning, indicating that the effect from the peptide tag was unaffected by the background of the host proteins. Taken together, the results show that the partitioning of the recombinantly produced cutinase model enzyme could be significantly improved by relatively minor genetic engineering and that the effects observed for purified proteins are retained also in an authentic whole cell disintegrate system. The results presented should be of general interest also for the improvement of the partitioning properties of other industrially interesting proteins including bulk enzymes.
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| 6. |
- Blomstergren, A., et al.
(författare)
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Cooperative oligonucleotides in purification of cycle sequencing products
- 2000
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 29:2, s. 352-
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Tidskriftsartikel (refereegranskat)abstract
- Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid copurification of non extended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.
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| 7. |
- Bäck, Torbjörn, et al.
(författare)
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A TOF-PET system for educational purposes
- 2002
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Ingår i: Nuclear Instruments and Methods in Physics Research Section A. - : Elsevier. - 0168-9002 .- 1872-9576. ; 477, s. 82-82
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Tidskriftsartikel (refereegranskat)abstract
- A TOF-PET system has been designed and constructed for educational purposes. The aim of this system is to demonstrate the possibilities of positron emission tomography in general and the time-of-flight method in particular to the students of various courses at the Royal Institute of Technology, Stockholm, Sweden. The set-up consists of 48 small BaF2 crystals coupled to fast photomultipliers placed in a ring geometry. The signals of the photomultipliers are fed into fast constant fraction discriminators (CFD). The outputs of these are directed to a specially designed logic VME unit. which combines the CFD signals of 6 neighbouring channels to one signal by adding a different delay to each channel. The logic circuitry produces a prompt pulse for each event that serves as the start pulse for the 8-channel fast TDC. The delayed pulses act as the stop pulses for the TDC. In a computer. the measured times are converted into information about which the photomultipliers fired with the difference in the time of flight. The set-up is described and the results are presented.
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| 8. |
- Bäck, Torbjörn, et al.
(författare)
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An educational tool for demonstrating the TOF-PET technique
- 2001
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Ingår i: Nuclear Instruments and Methods in Physics Research Section A. - : AMERICAN PHYSICAL SOC. - 0168-9002 .- 1872-9576. ; 471:1-2, s. 200-204
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Tidskriftsartikel (refereegranskat)abstract
- A detector system for positron emission tomography with time-of-flight capability has been built to serve as an educational tool for undergraduate students. The set-up consists of 48 BaF2 scintillator crystals, each coupled to a fast photo-multiplier tube, mounted in a circular geometry. The analogue detector pulses are handled by fast constant fraction discriminators. A dedicated unit reduces the 48 channels to eight channels via delay-fine encoding, and the signals are then fed to an eight channel fast time-to-digital converter. A VME processor sorts the events and sends them to a workstation where the coincident events are extracted. The time resolution of the detectors together with fast VME based electronics allows for time-of-flight measurements to improve on the signal-to-noise ratio in the, reconstructed images. The system can be used for different types of exercises for the students, varying from the fundamentals of scintillator detectors to advanced image reconstruction. The set-up is described and some results are presented. (C) 2001 Elsevier Science B.V. All rights reserved.
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| 9. |
- Ehn, M., et al.
(författare)
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Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein
- 2001
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 22:1, s. 120-127
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli (E. coli) single-stranded binding protein (SSB) is a valuable protein for various biotechnical applications, such as PCR and DNA sequencing, Here we describe an efficient expression and purification scheme where the tendency of SSB to aggregate at low salt concentration and high protein concentration is avoided. The method contains fewer steps of purification and results in high protein yield, compared to previous published protocols. In our protocol, cells are harvested after cultivation overnight and SSB is isolated by ammonium sulfate precipitation followed by anion-exchange chromatography. The yield from a 2-liter fed-batch fermenter is 2 g protein, which is higher than all production methods for SSB earlier reported, Moreover, the two classical isolation steps combined in the purification scheme are robust, cost-efficient, and suitable for scaling up. The resulting SSB is pure and a correctly folded tetramer with an apparent binding to single-stranded DNA with a K-D of 10(-8) M, as determined by surface plasmon resonance.
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| 10. |
- Eklund, M., et al.
(författare)
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Anti-idiotypic protein domains selected from protein A-based affibody libraries
- 2002
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 48:3, s. 454-462
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Tidskriftsartikel (refereegranskat)abstract
- Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fe binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K.) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.
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| 11. |
- Graslund, S., et al.
(författare)
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Single-vector three-frame expression systems for affinity-tagged proteins
- 2002
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 215:1, s. 139-147
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Tidskriftsartikel (refereegranskat)abstract
- An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.
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| 12. |
- Gulich, S., et al.
(författare)
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Stability towards alkaline conditions can be engineered into a protein ligand
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:2, s. 169-178
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Tidskriftsartikel (refereegranskat)abstract
- One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting of replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.
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| 13. |
- Gustafsson, A. C., et al.
(författare)
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Screening and scanning of single nucleotide polymorphisms in the pig melanocortin 1 receptor gene (MC1R) by pyrosequencing
- 2001
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Ingår i: Animal Biotechnology. - 1049-5398 .- 1532-2378. ; 12:2, s. 145-153
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Tidskriftsartikel (refereegranskat)abstract
- The increasing interest in the discovery and characterization of single nucleotide polymorphisms (SNPs) emphasis the need for high-throughput and cost effective scoring methods. Pyrosequencing is a novel method for screening SNPs. In this study we examine breed specific SNPs in the pig melanocortin I receptor gene (MC1R), some causing coat color phenotypes. A total of fifteen pigs representing eight breeds and crosses were analyzed by pyrosequencing. In addition to nine previously known SNPs, we also detected one new missense mutation by pyrosequencing. We here show that the SNPs were readily scored using standard reaction conditions. Insertions as well as substitutions were unambiguously detected and all genotypes were resolved in terms of homo- and heterozygozity.
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| 14. |
- Hertzberg, M., et al.
(författare)
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A transcriptional roadmap to wood formation
- 2001
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 98:25, s. 14732-14737
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Tidskriftsartikel (refereegranskat)abstract
- The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.
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| 15. |
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| 16. |
- Jonasson, P., et al.
(författare)
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Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 76:03-feb, s. 215-226
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Tidskriftsartikel (refereegranskat)abstract
- An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.
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| 17. |
- Kaessmann, H., et al.
(författare)
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Extensive linkage disequilibrium in small human populations in Eurasia
- 2002
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Ingår i: American Journal of Human Genetics. - : Elsevier BV. - 0002-9297 .- 1537-6605. ; 70:3, s. 673-685
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Tidskriftsartikel (refereegranskat)abstract
- The extent of linkage disequilibrium (LD) was studied in two small food-gathering populations-Evenki and Saami- and two larger food-producing populations-Finns and Swedes-in northern Eurasia. In total, 50 single-nucleotide polymorphisms (SNPs) from five genes were genotyped using real-time pyrophosphate DNA sequencing, whereas 14 microsatellites were genotyped in two X-chromosomal regions. In addition, hypervariable region I of the mtDNA was sequenced to shed light on the demographic history of the populations. The SNP data, as well as the microsatellite data, reveal extensive levels of LD in Evenki and Saami when compared to Finns and Swedes. mtDNA-sequence variation is compatible with constant population size over time in Evenki and Saami but indicates population expansion in Finns and Swedes. Furthermore, the similarity between Finns and Swedes in SNP allele- and haplotype-frequency distributions indicate that these two populations may share a recent common origin. These findings suggest that populations such as the Evenki and the Saami, rather than the Finns, may be particularly suited for the initial coarse mapping of common complex diseases.
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| 18. |
- Kowalewski, Jacob M., et al.
(författare)
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Generation of Slow Calcium oscillations in Spatial Cell Models Driven by Store Operated Calcium Entry
- 2004
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Konferensbidrag (refereegranskat)abstract
- Calcium signaling is involved in many cellular processes ranging from fertilization to apoptosis. We have created two general mathematical models to simulate calcium transport between the endoplasmic reticulum (ER), the cytosol and the extracellular environment. Active in this process is inositol-1,4,5-trisphosphate (InsP3). The behavior of the changes in calcium concentration was studied. The first model is compartmental and uses ordinary differential equations. The second one is a spatial model, which uses partial differential equations. The tools used in constructing these models were MATLAB and Virtual Cell. Store operated calcium entry can be observed in experiments with living cells when the sarco(endo)plasmic reticulum calcium-ATPase (SERCA) pumps are blocked by cyclopiazonic acid (CPA) or thapsigargin. We have developed a phenomenological model of store operated calcium channels (SOC). This model involves a diffusible messenger that leaves the ER and binds to a channel in the plasma membrane. Many parameters in the models are not fully known. A basic assumption is that the parameters can be estimated from the fact that a cell at rest has an almost constant level of calcium. Results from both the compartmental and the spatial simulations show that InsP3 can cause the cytosolic calcium level to oscillate at frequencies between 1 and 5 mHz. Experimental studies have shown than calcium oscillations in this frequency range are present in renal cells. The SERCA blocking simulations show good similarity with experimental results. The results also show that the high surface-to-volume ratio of the ER is important for causing oscillations.
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| 19. |
- Larsson, M., et al.
(författare)
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Expression profile viewer (ExProView) : A software tool for transcriptome analysis
- 2000
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Ingår i: Genomics. - : Elsevier BV. - 0888-7543 .- 1089-8646. ; 63:3, s. 341-353
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Tidskriftsartikel (refereegranskat)abstract
- A software tool, Expression Profile Viewer (ExProView), for analysis of gene expression profiles derived from expressed sequence tags (ESTs) and SAGE (serial analysis of gene expression) is presented. The software visualizes a complete set of classified transcript data in a two-dimensional array of dots, a virtual chip, in which each dot represents a known gene as characterized in the transcript databases Expressed Gene Anatomy Database or UniGene. The virtual chip display can be changed between representations of different conceptual systems for gene/protein classification and grouping. Four alternative projections are currently available: (i) cellular role, (ii) subcellular compartment, (iii) chromosome localization, and (iv) total UniGene display. However, the chip can be adapted to any other desired layout. By selecting dots, further information about the represented genes is obtained from the local database and WWW links. The software thus provides a visualization of global mRNA expression at the descriptive level and guides in the exploration of patterns of functional expression, while maintaining direct access to detailed information on each individual gene. To evaluate the software, public EST and SAGE gene expression data obtained from the Cancer Genome Anatomy Project at the National Center for Biotechnology Information were analyzed and visualized. A demonstration of the software is available at http://www.biochem.kth. se/exproview/.
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| 20. |
- Larsson, M., et al.
(författare)
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High-throughput protein expression of cDNA products as a tool in functional genomics
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:2, s. 143-157
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Tidskriftsartikel (refereegranskat)abstract
- A proteomics approach has been developed aimed to allow high throughput analysis of protein products expressed from cDNA fragments (expressed sequence tags, ESTs). The concept relies on expression of gene products to generate specific antibodies for protein analysis, such as immunolocalization of the proteins on cellular and subcellular level. To evaluate the system, 55 cDNA clones with predominantly unknown function were selected from a mouse testis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 mg l(-1) were obtained for 49 of the clones. Five clones were selected for immunization and all yielded functional antibodies that gave specific staining in Western blot screening of samples from various cell types. Furthermore, extensive immunolocalization information on subcellular level was obtained for three of the five clones. All generated data were stored in a relational database, and are made available through a web-interface (http://www.biochem.kth.se/multiscale/), which also provides relevant links and allows homology searches from the original sequences. The possibility to allow analysis of gene products from whole genomes using this 'localization proteomics' approach is discussed.
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| 21. |
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| 22. |
- Linhult, M., et al.
(författare)
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Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin
- 2002
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 11:2, s. 206-213
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple a-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using, circular dichroism. (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.
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| 23. |
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| 24. |
- Miyakawa-Naito, A., et al.
(författare)
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Cell signaling Microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 17:4, s. A43-A43
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Tidskriftsartikel (refereegranskat)abstract
- Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na, K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na, K-ATPase and inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer ( FRET) measurements, we detected a close spatial proximity between Na, K-ATPase and InsP(3)R. Ouabain significantly enhanced FRET between Na, K-ATPase and InsP(3)R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na, K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.
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| 25. |
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| 26. |
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| 27. |
- Ronnmark, J., et al.
(författare)
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Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol
- 2003
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 281:02-jan, s. 149-160
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant immunoconjugates constitute a novel class of immunoassay reagents produced by genetic fusion between an antigen recognizing moiety and a reporter enzyme or fluorescent protein, obviating the need for chemical coupling. In this work, we describe the construction, Escherichia coli production and characterization of recombinant beta-galactosidase (beta-gal)-based immunoconjugates directed to human imrnunoglobulin A (IgA). As the antigen recognizing moieties, either monovalent or dimeric (head-to-tail) versions of an IgA-specific affibody (Z(IgA1)) were used, previously selected in vitro from a protein library based on combinatorial engineering of a single staphylococcal protein A domain. To increase the likelihood of proper presentation on the assembled homotetrameric enzyme surface, the affibody moieties were linked to the N-terminus of the enzyme subunits via a heptapeptide linker sequence. The two resulting immunoconjugates Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal, containing four and eight affibody moieties per enzyme, respectively, could be expressed as soluble and proteolytically stable proteins intracellularly in E. coli from where they were purified to high purity by a single anion exchange chromatography step. The yields of immunoconjugates were in the range 200-400 mg/l culture. Biosensor-binding studies showed that both the Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal immunoconjugates were capable of selective IgA-recognition, but with an apparent higher binding affinity for the variant containing divalent affibody moieties, presumably due to avidity effects. The applicability of this class of recombinant immunoconjugates was demonstrated by IgA detection in enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. In addition, using human kidney biopsy samples from a nephropathy patient, IgA depositions in glomeruli could be detected by immunohistochemistry with low background staining of tissue.
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| 28. |
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| 29. |
- Ronnmark, J., et al.
(författare)
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Construction and characterization of affibody-Fc chimeras produced in Escherichia coli
- 2002
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 261:02-jan, s. 199-211
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Tidskriftsartikel (refereegranskat)abstract
- Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human I-G. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture, Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of artificial antibodies was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such artificial antibodies should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.
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- Sterky, Fredrik, et al.
(författare)
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A Populus EST resource for plant functional genomics
- 2004
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 101:38, s. 13951-13956
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Tidskriftsartikel (refereegranskat)abstract
- Trees present a life form of paramount importance for terrestrial ecosystems and human societies because of their ecological structure and physiological function and provision of energy and industrial materials. The genus Populus is the internationally accepted model for molecular tree biology. We have analyzed 102,019 Populus ESTs that clustered into 11,885 clusters and 12,759 singletons. We also provide >4,000 assembled full clone sequences to serve as a basis for the upcoming annotation of the Populus genome sequence. A public web-based EST database (POPULUSDB) provides digital expression profiles for 18 tissues that comprise the majority of differentiated organs. The coding content of Populus and Arabidopsis genomes shows very high similarity, indicating that differences between these annual and perennial angiosperm life forms result primarily from differences in gene regulation. The high similarity between Populus and Arabidopsis will allow studies of Populus to directly benefit from the detailed functional genomic information generated for Arabidopsis, enabling detailed insights into tree development and adaptation. These data will also valuable for functional genomic efforts in Arabidopsis.
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| 31. |
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