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- Gyllenhammar, Irina, et al.
(författare)
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Influence of contaminated drinking water on perfluoroalkyl acid levels in human serum - A case study from Uppsala, Sweden
- 2015
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Ingår i: Environmental Research. - : Elsevier BV. - 0013-9351 .- 1096-0953. ; 140, s. 673-683
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Tidskriftsartikel (refereegranskat)abstract
- In 2012 a contamination of drinking water with perfluoroalkyl acids (PFAAs) was uncovered in the City of Uppsala, Sweden. The aim of the present study was to determine how these substances have been distributed from the contamination source through the groundwater to the drinking water and how the drinking water exposure has influenced the levels of PFAAs in humans over time. The results show that PFAA levels in groundwater measured 2012-2014 decreased downstream from the point source, although high Sigma PFAA levels (> 100 ng/L) were still found several kilometers from the point source in the Uppsala aquifer. The usage of aqueous film forming fire-fighting foams (AFFF) at a military airport in the north of the city is probably an important contamination source. Computer simulation of the distribution of PFAA-contaminated drinking water throughout the City using a hydraulic model of the pipeline network suggested that consumers in the western and southern parts of Uppsala have received most of the contaminated drinking water. PFAA levels in blood serum from 297 young women from Uppsala County, Sweden, sampled during 1996-1999 and 2008-2011 were analyzed. Significantly higher concentrations of perfluorobutane sulfonic acid (PFBS) and perfluorohexane sulfonic acid (PFHxS) were found among women who lived in districts modeled to have received contaminated drinking water compared to unaffected districts both in 1996-1999 and 2008-2011, indicating that the contamination was already present in the late 1990s. Isomer-specific analysis of PFHxS in serum showed that women in districts with contaminated drinking water also had an increased percentage of branched isomers. Our results further indicate that exposure via contaminated drinking water was the driving factor behind the earlier reported increasing temporal trends of PFBS and PFHxS in blood serum from young women in Uppsala.
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| 3. |
- Gyllenhammar, Irina, et al.
(författare)
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Perfluoroalkyl acids in serum from nursing women living in an area in Sweden with drinking water contamination
- 2011
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Under perioden 1996 till 2011 har Livsmedelsverket samlat in blodserum från förstföderskor i Uppsala län. Ett av syftena med studierna är att undersöka hur halterna av vissa persistenta organiska miljögifter (POP) förändras med tiden. I denna rapport utvärderas blodserumnivåer för 14 perfluoroalkylsyror; (PFAA) varav tio perfluoroalkylkarboxylsyror (PFCA), fyra perfluoroalkansulfonsyror (PFSA) och perfluorooktansulfonamiden FOSA , från prover tagna 1996-1999 och 2008-2011 (n=297). Syftet med studien var att utvärdera om det finns signifikanta skillnader i blodnivåer av PFAA mellan kvinnor som bor inom olika områden i Uppsala stad, för att därigenom försöka bedöma om dricksvattenexponering påverkar nivåerna av PFAA i blod. Undersökningar av PFAA i grundvattenbrunnar och dricksvatten i Uppsala har tidigare visat att kontaminerat vatten främst distribuerats till de södra delarna av Uppsala. Kontaminerade brunnar har nu tagits ur produktion. Högre blodnivåer av perflurohexansulfonsyra (PFHxS) och perfluorbutansulfonsyra (PFBS) hittades i främst det södra området av Uppsala, både 1996-1999 (ej PFBS) och 2008-2011, vilket tyder på att konsumtion av dricksvatten är en viktig källa för exponering. Halterna av PFHxS var i allmänhet högre i Uppsala stad 2008-2011 än 1996-1999, men ej bland deltagare boende utanför Uppsala. Liknande resultat sågs för PFBS, vilket antyder att kontaminerat dricksvatten ligger bakom de ökande blodhalter av substanserna som tidigare observerats i Uppsala. Vi såg också samband mellan fiskkonsumtion och ökade nivåer av PFOS från kvinnor 2008-2011, vilket indikerar att fiskkonsumtion som exponeringskälla har ökat i betydelse sen 1990-talet.
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- Kotova, Irina, et al.
(författare)
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A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 268:16, s. 4527-4536
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Tidskriftsartikel (refereegranskat)abstract
- Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity. We have also purified a recombinant His6-tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro. Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter.
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| 5. |
- Kotova, Irina, 1972-
(författare)
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Purification of general RNA polymerase II transcription factors from mouse for studies of proliferation-specific transcription
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Accurate initiation of transcription by RNA polymerase II depends on general transcription factors (GTFs), which include the TATA-binding protein (TBP) and the transcription factors (TF) IIB, IIF, IIE and IIH. In order to reconstitute mouse transcription in vitro, we cloned the genes encoding mouse TFIIB, and both subunits of TFIIE and TFIIF from a mouse cDNA library. TBP and TFIIB were expressed in E.coli, while both subunits of TFIIE and the two subunits of TFIIF were expressed in a baculovirus system. All these factors were purified to > 90% homogeneity. The more complex transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse ascites cells. We have shown that the purified mouse transcription factors are active in a reconstituted RNA polymerase II in vitro transcription assay. The transcription reaction was inhibited by α-amanitine, and dependent on the addition of all the GTFs.Ribonucleotide reductase is a key enzyme in deoxyribonucleotide synthesis. It consists of two subunits, R1 and R2, which are both required for the enzyme activity. Transcription of the R1 and R2 genes is restiricted to the S-phase of the cell cycle, but the mechanisms that control this coordinated expression remain to be identified. We have studied initiation of transcription from the mouse R2 gene using a combination of in vivo reporter gene assays and in vitro transcription assays with crude nuclear extracts or with purified transcription factors. This promoter has an atypical TATA-box and a CCAAT-box that binds the transcription factor NF-Y.We found that a mutation in the R2 CCAAT-box had no effect on the transcription level in in vitro transcription assays reconstituted with pure transcription factors. However, it significantly decreased the level of transcription in similar experiments using crude nuclear extract. We also found that the sequence downstream from the R2 transcription start site (5´-UTR) (from +1 to +17 base pairs relative to transcription start site) is essential for initiation of transcription from this promoter. The presence of the wild type 5´-UTR made the R2 TATA-box redundant. On the other hand, the R2 5´-UTR had a repressing effect on transcription from the mouse R2 promoter. This region contains a palindrome sequence that covers 10 base pairs, and it is partially conserved in the human R2 promoter. Gel shift assays and in vitro transcription experiments using antibodies against mouse TAF4 (=TAF135) demonstrate that TAF4 is a component of the protein complex that interacts with this palindrome region, and suggest involvement of this component of the TFIID complex in negative regulation of the R2 promoter.The Adenovirus Major Late (AdML) promoter is commonly used as a model for studies of transcription initiation and regulation. It is a TATA-box dependent promoter, which also contains an initiator (Inr) element, a CCAAT-box interacting with transcription factor NF-Y, and an E-box binding the upstream stimulatory factor (USF). Using gel shift assays with recombinant NF-Y, USF, and immunopurified human TFIID, we show that binding of USF1 and NF-Y to DNA is not cooperative and that both factors independently facilitate binding of TFIID to the core promoter. The activation domains of NF-Y are expendable for this effect. Negative cofactor (NC2) comprises two subunits, which have a histone-fold structure similar to NF-Y, and represses transcription through formation of an inhibitory complex with TBP. Using an in vitro transcription system based on crude nuclear extracts, we show that NC2 has a negative effect on transcription in the presence of NF-Y or USF1, indicating that the two activators do not act as antirepressors. In vitro transcription using highly purified transcription factors efficiently reproduces repression of transcription by NC2. However, USF1 was inactive and NF-Y had a repressing effect in this system, which suggests that the activator functions of USF and NF-Y depend on cofactors.
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| 6. |
- Kotova, Irina, et al.
(författare)
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Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription
- 2003
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Ingår i: European Journal of Biochemistry. - : Blackwell Publishing. - 0014-2956 .- 1432-1033. ; 270:8, s. 1791-1801
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5'-UTR are present, the mouse R2 TATA-box is dispensable both for unregulated, basal transcription from the R2 promoter and for S-phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAF(II)s. This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box.
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