| 1. |
- Essand, Magnus, et al.
(författare)
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Identification and characterization of a novel splicing variant of vesicular monoamine transporter 1
- 2005
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Ingår i: Journal of Molecular Endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 35:3, s. 489-501
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Tidskriftsartikel (refereegranskat)abstract
- Vesicular monoamine transporter 1 (VMAT1) is an integral protein in the membrane of secretory vesicles of neuroendocrine and endocrine cells that allows the transport of biogenic monoamines, such as serotonin, from the cytoplasm into the secretory vesicles. The full-length VMAT1 transcript is produced from 16 exons. We have identified and characterized an alternatively spliced form of VMAT1 that lacks exon 15, the next to last exon of VMAT1. The new form was therefore denoted VMAT1Delta15. Exon 15 does not contain an even multiple of three nucleotides. As a consequence, there is a shift of reading frame, and exon 16 is translated in an alternative reading frame, yielding a novel protein with a shorter and unrelated C-terminus compared with the native VMAT1 protein. VMAT1 and VMAT1Delta15 mRNAs are simultaneously expressed in normal and neoplastic neuroendocrine cells of the GI tract. However, VMAT1 expression is always higher than VMAT1Delta15 expression. We prove that VMAT1Delta15 is not localized in large, dense core vesicles as the native form but in the endoplasmic reticulum. Furthermore, while VMAT1 can take up serotonin, VMAT1Delta15 cannot, indicating different functions for the two forms of VMAT1.
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| 2. |
- Ferletta, Maria, et al.
(författare)
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Canine mammary tumors contain cancer stem-like cells and form spheroids with an embryonic stem cell signature
- 2011
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Ingår i: International Journal of Developmental Biology. - : UPV/EHU Press. - 0214-6282 .- 1696-3547. ; 55:7-9, s. 791-799
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the presence of tentative stem-like cells in the canine mammary tumor cell line CMT-U229. This cell line is established from an atypical benign mixed mammary tumor, which has the property of forming duct-like structures in collagen gels. Stem cells in mammary glands are located in the epithelium; therefore we thought that the CMT-U229 cell line would be suitable for detection of tentative cancer stem-like cells. Side population (SP) analyses by flow cytometry were performed with cells that formed spheroids and with cells that did not. Flow cytometric, single sorted cells were expanded and re-cultured as spheroids. The spheroids were paraffin embedded and characterized by immunohistochemistry. SP analyses showed that spheroid forming cells (retenate) as well as single cells (filtrate) contained SP cells. Sca1 positive cells were single cell sorted and thereafter the SP population increased with repeated SP analyses. The SP cells were positively labeled with the cell surface-markers CD44 and CD49f (integrin alpha6); however the expression of CD24 was low or negative. The spheroids expressed the transcription factor and stem cell marker Sox2, as well as Oct4. Interestingly, only peripheral cells of the spheroids and single cells were positive for Oct4 expression. SP cells are suggested to correspond to stem cells and in this study, we have enriched for tentative tumor stem-like cells derived from a canine mammary tumor. All the used markers indicate that the studied CMT-U229 cell line contains SP cells, which in particular have cancer stem-like cell characteristics.
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| 3. |
- Fred, Charlotta, et al.
(författare)
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Hemoglobin adducts and micronuclei in rodents after treatment with isoprene monoxide or butadiene monoxide
- 2005
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Ingår i: Mutation Research. - : Elsevier B.V.. - 1383-5742 .- 1388-2139. ; 585:1-2, s. 21-32
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Tidskriftsartikel (refereegranskat)abstract
- 1,3-Butadiene and isoprene (2-methyl-1,3-butadiene) are chemically related substances that are carcinogenic to rodents. The overall aim of this work is to elucidate the role of the genotoxic action of diepoxide metabolites in the carcinogenesis of the dialkenes. In vivo doses of the diepoxide metabolites were measured through reaction products with hemoglobin (Hb adducts) in studies of induced micronuclei (MN) in rodents. In the reaction with N-terminal valine in Hb, diepoxybutane and isoprenediepoxide form ring-closed adducts, pyrrolidines [N,N-(2,3-dihydroxy-1,4-butadiyl)valine and N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valine, respectively]. The method applied for Hb-adduct measurement is based on tryptic degradation of the protein and liquid chromatography electrospray ionisation tandem mass spectrometry (LC–ESI-MS/MS) analysis. Mice were given single i.p. injections of the monoepoxides of butadiene and isoprene, 1,2-epoxy-3-butene or 1,2-epoxy-2-methyl-3-butene, respectively. Rats were treated in the same way with 1,2-epoxy-3-butene. In mice pyrrolidine adduct levels increased with increasing administered doses of the monoepoxides. The in vivo dose of diepoxybutane was on average twice as high (0.29 ± 0.059 mMh) as the in vivo dose of isoprenediepoxide (0.15 ± 0.053 mMh) per administered dose (mmol/kg body weight) of the monoepoxides. In mice the genotoxic effects of the two monoepoxides, measured as the increase in the frequencies of micronuclei (MN), were approximately linearly correlated to the in vivo doses of the diepoxides (except at the highest dose of diepoxybutane). In rats the pyrrolidine-adduct levels from diepoxybutane were below the limit of quantification at all administered doses of 1,2-epoxy-3-butene and no significant increase was observed in the frequency of MN. Measurement of the ring-closed adducts to N-termini in Hb by the applied method permits analysis of in vivo doses of diepoxybutane and isoprenediepoxide, which may be further used for the elucidation of the mechanisms of carcinogenesis of butadiene and isoprene.
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| 4. |
- Grawe, S., et al.
(författare)
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The immersion freezing behavior of ash particles from wood and brown coal burning
- 2016
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Ingår i: Atmospheric Chemistry and Physics. - : Copernicus GmbH. - 1680-7316 .- 1680-7324. ; 16:21, s. 13911-13928
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Tidskriftsartikel (refereegranskat)abstract
- It is generally known that ash particles from coal combustion can trigger ice nucleation when they interact with water vapor and/or supercooled droplets. However, data on the ice nucleation of ash particles from different sources, including both anthropogenic and natural combustion processes, are still scarce. As fossil energy sources still fuel the largest proportion of electric power production worldwide, and biomass burning contributes significantly to the global aerosol loading, further data are needed to better assess the ice nucleating efficiency of ash particles. In the framework of this study, we found that ash particles from brown coal (i.e., lignite) burning are up to 2 orders of magnitude more ice active in the immersion mode below -32 degrees C than those from wood burning. Fly ash from a coal-fired power plant was shown to be the most efficient at nucleating ice. Furthermore, the influence of various particle generation methods on the freezing behavior was studied. For instance, particles were generated either by dispersion of dry sample material, or by atomization of ash-water suspensions, and then led into the Leipzig Aerosol Cloud Interaction Simulator (LACIS) where the immersion freezing behavior was examined. Whereas the immersion freezing behavior of ashes from wood burning was not affected by the particle generation method, it depended on the type of particle generation for ash from brown coal. It was also found that the common practice of treating prepared suspensions in an ultrasonic bath to avoid aggregation of particles led to an enhanced ice nucleation activity. The findings of this study suggest (a) that ash from brown coal burning may influence immersion freezing in clouds close to the source and (b) that the freezing behavior of ash particles may be altered by a change in sample preparation and/or particle generation.
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| 5. |
- Gustafsson, Karin, et al.
(författare)
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The Src Homology 2 Protein Shb Promotes Cell Cycle Progression In Murine Hematopoietic Stem Cells By Regulation Of Focal Adhesion Kinase Activity
- 2013
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 319:12, s. 1852-1864
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Tidskriftsartikel (refereegranskat)abstract
- The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shbdeficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time.
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| 6. |
- Göransson, Jenny, et al.
(författare)
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Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2, s. e31068-
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Tidskriftsartikel (refereegranskat)abstract
- Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.
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| 7. |
- Kotova, Natalia, et al.
(författare)
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A novel micronucleus in vitro assay utilizing human hematopoietic stem cells
- 2015
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Ingår i: Toxicology in Vitro. - : Elsevier BV. - 0887-2333 .- 1879-3177. ; 29:7, s. 1897-1905
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Tidskriftsartikel (refereegranskat)abstract
- The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed.
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| 8. |
- Kotova, Natalia, 1975-
(författare)
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Biomarkers for DNA damage in human biomonitoring
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Genomic DNA in humans is constantly exposed to different kinds of damage. Therefore, it is desirable to implement methods for detecting and measuring of inflicted body burden. Human biomonitoring (HBM) can here be a useful tool as a link between environmental exposure and disease outcome. The present thesis aims to monitor DNA damage in humans by studies on: 1) urinary thymidine dimer (T=T) as a novel biomarker (BM) of human exposure to UV-light; 2) enumeration of variants in HPRT gene in human peripheral blood lymphocytes by developing a sensitive flow cytometric (FCM) analysis; 3) the impact of dietary habits on genomic stability in vegetarians and omnivores in terms of micronuclei (MN) induction detected by FCM. Urinary T=T was quantified by a 32P-postlabeling technique, the kinetics of T=T excretion was studied and the method was validated by delivering controlled UV-doses. The major conclusion was that the amount of urinary T=T was determined by the UV dose, and hence T=T can be used as a BM of UV exposure. Moreover, a new approach for rapid and sensitive enumeration of HPRT-variants by FCM was developed. The obtained HPRT-frequencies were comparable to those previously published by others. Finally, the FCM assay for MN enumeration was applied to study effects of dietary habits in vegetarians and omnivores. The main finding was that vegetarians had significantly lower MN frequencies compared to omnivores. In summary, the applied BMs and respective methods have high sensitivity and/or throughput possibility which are important factors considered in HBM.
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| 9. |
- Kotova, Natalia, et al.
(författare)
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Differences in micronucleus frequency and acrylamide adduct levels with hemoglobin between vegetarians and non-vegetarians
- 2015
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Ingår i: European Journal of Nutrition. - : Springer Science and Business Media LLC. - 1436-6207 .- 1436-6215. ; 54:7, s. 1181-1190
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Tidskriftsartikel (refereegranskat)abstract
- Nutrients and food constituents can prevent or contribute to genotoxicity. In this study, the possible influence of a vegetarian/non-vegetarian diet on genotoxic effects was investigated in 58 non-smoking healthy vegetarians (V) and non-vegetarians (NV), age 21-37 years from the Stockholm area in Sweden. Physical activity and dietary habits were similar in both groups, with the exception of the intake of meat and fish. Using flow cytometry, we determined the formation of micronuclei (MN) in transferrin-positive immature peripheral blood reticulocytes (Trf-Ret) (Total: n = 53; V: n = 27; NV: n = 26). Dietary exposure to acrylamide was measured through hemoglobin (Hb) adducts in peripheral erythrocytes (Total: n = 53; V: n = 29; NV: n = 24). Hb adducts of both acrylamide and its genotoxic metabolite glycidamide were monitored as a measure of the corresponding in vivo doses. Our data demonstrated that compared with the non-vegetarians, the vegetarians exhibited lower frequencies of MN (fMN) in the Trf-Ret (p < 0.01, Student's t test). A multivariate analysis demonstrated that there was no association between the fMN and factors such as age, sex, intake of vitamins/minerals, serum folic acid and vitamin B12 levels, physical activity, and body mass index. The mean Hb adduct levels of acrylamide and glycidamide showed no significant differences between vegetarians and non-vegetarians. Furthermore, there were no significant relationships between the adduct levels and fMN in the individuals. The ratio of the Hb adduct levels from glycidamide and acrylamide, however, showed a significant difference (p < 0.04) between the two groups. These data suggest that the vegetarian diet might be beneficial in lowering genomic instability in healthy individuals. The measured Hb adduct levels indicate that the total intake of acrylamide does not differ between the two studied groups and does not contribute to the observed difference in fMN, although an influence of the diet on the metabolic rates of acrylamide was indicated. In addition, the observed significant difference in the background fMN in the two groups demonstrated that the MN analysis method has a sensitivity applicable to the biomonitoring of human lifestyle factors.
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| 10. |
- Kotova, Natalia, 1975-, et al.
(författare)
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Flow cytometric determination of HPRT-variantsin human peripheral blood lymphocytes
- 2002
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Ingår i: Mutation research. - 0027-5107 .- 1873-135X. ; 499:1, s. 63-71
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Tidskriftsartikel (refereegranskat)abstract
- Hypoxanthine guanine phosphoribosyl transferase (HPRT) deficient human peripheral blood lymphocytes are usually enumerated either by the cloning assay or by the autoradiographic short-term assay. The short-term approach presented here is based on flow cytometric (FCM) scoring of 6-thioguanine (6-TG) resistant lymphocytes. HPRT-variants are enumerated on the basis of both DNA synthesis (by use of immunofluorescent detection of incorporated 5-bromo-2-deoxyuridine, BrdU) and total DNA content (by propidium iodide (PI) incorporation) of proliferating cells, i.e. the cells must both be labelled with BrdU and reside in late-S or G(2) phase in order to be scored as a HPRT-variant. This approach is combined with a stringent discrimination of false-positive events, minimising occurrence of phenocopies or other non-specifically labelled cells that might falsely be scored as true HPRT-variants. The HPRT-variant frequency (V-f) found by the presented method varied between 0.8 x 10(-5) and 5.8 x 10(-5) for healthy male and female donors aged between 20 and 74 years. There was no significant gender difference in V-f. A strong linear correlation was found between HPRT-variant frequency and age, showing an increase of 0.56 x 10(-6) per year of age (r(2) = 0.62, P < 0.001). The frequencies of false-positive events found showed a mean of 0.22 x 10(-5) in comparison with a pooled mean V-f of 2.87 x 10(-5). There was no significant age effect on the frequency of false events (r(2) = = 0.15, P < 0.095). The method presented here may provide a rapid and sensitive alternative to the autoradiographic technique for the short-term enumeration of HPRT-variants.
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| 11. |
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| 12. |
- Kotova, Natalia, et al.
(författare)
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Genotoxicity of alcohol is linked to DNA replication-associated damage and homologous recombination repair
- 2013
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Ingår i: Carcinogenesis. - : Oxford University Press (OUP). - 0143-3334 .- 1460-2180. ; 34:2, s. 325-330
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Tidskriftsartikel (refereegranskat)abstract
- Although alcohol consumption is related to increased cancer risk, its molecular mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde (AA), the first product of ethanol metabolism, is believed to be responsible for DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. AA-induced DNA damage sites colocalize with the homologous recombination (HR) repair protein RAD51. HR measured in the hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by AA and recombination defective mammalian cells are hypersensitive to AA, clearly demonstrating that HR is essential in the repair of AA-induced DNA damage. Altogether, our data indicate that alcohol genotoxicity related to AA produces replication lesions on DNA triggering HR repair.
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| 13. |
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| 14. |
- Li, Li, et al.
(författare)
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Induction of apoptosis or necrosis in human endometrial cancer cells by 2-Methoxyestradiol
- 2004
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Ingår i: Anticancer Research. - 0250-7005 .- 1791-7530. ; 24:6, s. 3983-3990
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:We investigated the effects of 2methoxyestradiol (2-ME), an endogenous estrogenic metabolite, on human endometrial cancer HEC-1-A and RL-95-2 cell lines.MATERIALS AND METHODS:After exposure of HEC-1-A and RL-95-2 cells to 2-ME, the morphological changes were evaluated by acridine orange staining and transmission electron microscopy. Cell cycle progress, apoptosis and necrosis were assessed by flow cytometry, DNA fragmentation and Western blot.RESULTS:2-ME inhibited cell growth by blocking the S- and G2/M-phase in both cell lines, by inducing apoptosis in HEC-1-A cells and by causing necrosis in RL-95-2 cells. Apoptosis, on HEC-1-A cells, was accompanied by an increased expression of iNOS and STAT1. This apoptotic effect was prevented by the iNOS inhibitor 1400W and eliminated by the caspase inhibitor Z-VAD-FMK. Necrosis, on RL-95-2 cells, was due to a severe disruption of the mitochondrial membrane potential. 2-ME had no significant effect on normal human endometrial cells.CONCLUSION:The data suggest that 2-ME has an antitumor effect on human endometrial carcinoma cells (HEC-1-A and RL-95-2) and may contribute as a new therapeutic agent for endometrial cancer patients.
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| 15. |
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