| 1. |
- Alm, Henrik, et al.
(författare)
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Exposure to brominated flame retardant PBDE-99 affects cytoskeletal protein expression in the neonatal mouse cerebral cortex
- 2008
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Ingår i: Neurotoxicology. - : Elsevier BV. - 0161-813X .- 1872-9711. ; 29:4, s. 628-637
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Tidskriftsartikel (refereegranskat)abstract
- Polybrominated diphenyl ethers (PBDEs) are environmental contaminants found in human and animal tissues worldwide. Neonatal exposure to the flame retardant 2,2', 4,4',5-pentabromodiphenyl ether (PBDE-99) disrupts normal brain development in mice, and results in disturbed spontaneous behavior in the adult. The mechanisms underlying the late effects of early exposure are not clear. To gain insight into the initial neurodevelopmental damage inflicted by PBDE-99, we investigated the short-term effects of PBDE-99 on protein expression in the developing cerebral cortex of neonatal mice, and the cytotoxic and apoptotic effects of PBDE-99 in primary cultures of fetal rat cortical cells. We used two-dimensional difference gel electrophoresis (2D-DIGE) to analyze protein samples isolated from the cortex of NMRI mice 24h after exposure to a single oral dose of 12 mg/kg PBDE-99 on post-natal day 10. Protein resolution was enhanced by sample pre-fractionation. In the cell model, we determined cell viability using the trypan blue exclusion assay, and apoptosis using immunocytochemical detection of cleaved caspase-3. We determined the identity of 111 differentially expressed proteins, 32 (29%) of which are known to be cytoskeleton-related. Similar to previous findings in the striatum, we found elevated levels of the neuron growth-associated protein Gap43 in the cortex. In cultured cortical cells, a high concentration of PBDE-99 (30 microM) induced cell death without any apparent increase in caspase-3 activity. These results indicate that the permanent neurological damage induced by PBDE-99 during the brain growth spurt involve detrimental effects on cytoskeletal regulation and neuronal maturation in the developing cerebral cortex.
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| 2. |
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| 3. |
- Alm, Henrik, et al.
(författare)
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In Vitro Neurotoxicity of PBDE-99 : Immediate and Concentration-Dependent Effects on Protein Expression in Cerebral Cortex Cells
- 2010
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 9:3, s. 1226-1235
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Tidskriftsartikel (refereegranskat)abstract
- Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants in various consumer products. Pre- and postnatal exposure to congeners of PBDEs disrupts normal brain development in rodents. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to analyze concentration-dependent differences in protein expression in cultured cortical cells isolated from rat fetuses (GD 21) after 24 h exposure to PBDE-99 (3, 10, or 30 muM). Changes on a post-translational level were studied using a 1 h exposure to 30 muM PBDE-99. The effects of 24 h exposure to 3 and 30 muM PBDE-99 on mRNA levels were measured using oligonucleotide microarrays. A total of 62, 46, and 443 proteins were differentially expressed compared to controls after 24 h of exposure to 3, 10, and 30 muM PDBE-99, respectively. Of these, 48, 43, and 238 proteins were successfully identified, respectively. We propose that the biological effects of low-concentration PBDE-99 exposure are fundamentally different than effects of high-concentration exposure. Low-dose PBDE-99 exposure induced marked effects on cytoskeletal proteins, which was not correlated to cytotoxicity or major morphological effects, suggesting that other more regulatory aspects of cytoskeletal functions may be affected. Interestingly, 0.3 and 3 muM, but not 10 or 30 muM increased the expression of phosphorylated (active) Gap43, perhaps reflecting effects on neurite extension processes.
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| 4. |
- Alm, Henrik, et al.
(författare)
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Proteomic evaluation of neonatal exposure to 2,2,4,4,5-pentabromodiphenyl ether
- 2006
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Ingår i: Journal of Environmental Health Perspectives. - : Environmental Health Perspectives. - 0091-6765 .- 1552-9924. ; 114:2, s. 254-259
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Tidskriftsartikel (refereegranskat)abstract
- Exposure to the brominated flame retardant 2,2 ,4,4 ,5-pentabromodiphenyl ether (PBDE-99) during the brain growth spurt disrupts normal brain development in mice and results in disturbed spontaneous behavior in adulthood. The neurodevelopmental toxicity of PBDE-99 has been reported to affect the cholinergic and catecholaminergic systems. In this study we use a proteomics approach to study the early effect of PBDE-99 in two distinct regions of the neonatal mouse brain, the striatum and the hippocampus. A single oral dose of PBDE-99 (12 mg/kg body weight) or vehicle was administered to male NMRI mice on neonatal day 10, and the striatum and the hippocampus were isolated. Using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), we found 40 and 56 protein spots with significantly (p < 0.01) altered levels in the striatum and the hippocampus, respectively. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS) to determine the protein identity of 11 spots from the striatum and 10 from the hippocampus. We found that the levels of proteins involved in neurodegeneration and neuroplasticity (e.g., Gap-43/neuromodulin, stathmin) were typically altered in the striatum, and proteins involved in metabolism and energy production [e.g., alpha-enolase; gamma-enolase; ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit (Atp5b); and alpha-synuclein] were typically altered in the hippocampus. Interestingly, many of the identified proteins have been linked to protein kinase C signaling. In conclusion, we identify responses to early exposure to PBDE-99 that could contribute to persistent neurotoxic effects. This study also shows the usefulness of proteomics to identify potential biomarkers of developmental neurotoxicity of organohalogen compounds.
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| 5. |
- Brännström, Mats, 1958, et al.
(författare)
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Reproductive, obstetric, and long-term health outcome after uterus transplantation: results of the first clinical trial
- 2022
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Ingår i: Fertility and Sterility. - : Elsevier BV. - 0015-0282 .- 1556-5653. ; 118:3, s. 576-585
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Tidskriftsartikel (refereegranskat)abstract
- Objective: To evaluate reproductive, obstetric, and long-term health of the first completed study of uterus transplantation (UTx). Design: Prospective. Setting: University hospital. Patient(s): Nine live donor UTx procedures were conducted and seven were successful. Donors, recipients, and children born were observed. Intervention(s): In vitro fertilization was performed with embryo transfer (ET) of day 2 or day 5 embryos in natural cycles. Pregnancies and growth trajectory of the children born were observed. Health-related quality of life, psychosocial outcome, and medical health of donors and recipients were evaluated by questionnaires. Main Outcome Measure(s): The results of in vitro fertilization, pregnancies, growth of children, and long-term health of patients were reported. Result(s): Six women delivered nine infants, with three women giving birth twice (cumulative birth rates of 86% and 67% in surgically successful and performed transplants, respectively). The overall clinical pregnancy rate (CPR) and live birth rate (LBR) per ET were 32.6% and 19.6%, respectively. For day 2 embryos, the CPR and LBR per ET were 12.5% and 8.6%, respectively. For day 5 embryos, the CPR and LBR per ET were 81.8% and 45.4%, respectively. Fetal growth and blood flow were normal in all pregnancies. Time of delivery (median in full pregnancy weeks + days [ranges]) by cesarean section and weight deviations was 35 + 3 (31 + 6 to 38 + 0) and -1% (-13% to 23%), respectively. Three women developed preeclampsia and four neonates acquired respiratory distress syndrome. All children were healthy and followed a normal growth trajectory. Measures of long-term health in both donors and recipients were noted to be favorable. When UTx resulted in a birth, scores for anxiety, depression, and relationship satisfaction were reassuring for both the donors and recipients. Conclusion(s): The results of this first complete UTx trial show that this is an effective infertility treatment, resulting in births of healthy children and associated with only minor psychological and medical long-term effects for donors and recipients. Clinical Trial Registration Number: NCT02987023.
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| 6. |
- Jergil, Måns, et al.
(författare)
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Exploring Transcriptional Response toValproic Acid and Valproic Acid Analogs in Human Embryonic Stem Cells
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Annan publikation (populärvet., debatt m.m.)abstract
- Developmental toxicity is a major concern for manufacturers of new pharmaceuticals,and current testing requires many laboratory animals. Human embryonic stem (hES)cells, potentially being close in function to cells in the developing embryo, mayprovide a technology for classification of candidate drugs in the early phase of toxicityevaluation. Altered gene expression in such system may be predictive of teratogenicproperties of a substance if important gene regulatory pathways are affected, and mayhence be used as appropriate endpoint. In the present study we used the pluripotenthES cell line SA002 (Cellartis AB), and microarrays to profile the response tovalproic acid (VPA), a known human teratogen causing increased risk of e.g. spinabifida and cognitive disorders in exposed embryos We also investigated three closelyrelated VPA analogs with differing in vivo teratogenicity in mice as well as histonedeacetylase (HDAC) inhibition, a proposed teratogenic mechanism of VPA. hEScells in an undifferentiated state were exposed for 24 h to either 1 mM VPA, 0.25mM or 0.5 mM (S)-2-pentyl-4-pentynoic acid a more potent teratogen and HDACinhibitor than VPA, 1 mM 3-propyl-heptanoic acid, a potent teratogen but not anHDAC inhibitor, 1 mM 2-ethyl-4-methyl-pentanoic acid, a non-teratogen and non-HDAC inhibitor, or 0.1% DMSO. Gene expression was subsequently profiled usingCodelink Human Whole Genome BioArrays. We found the HDAC inhibitors tostrongly deregulate largely the same genes. Further, a concordance of altered geneontology groups, predominantly neurogenic processes, was evident between all theteratogenic substances. Also, comparison with mouse ES cells showed an overlap ofderegulated genes as well as species specific gene to be deregulated.
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| 7. |
- Jergil, Måns, 1975-, et al.
(författare)
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Short-Time Gene Expression Response to Valproic Acid and Valproic Acid analogs in Mouse Embryonic Stem Cells
- 2011
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Ingår i: Toxicological Sciences. - : Oxford University Press (OUP). - 1096-6080 .- 1096-0929. ; 121:2, s. 328-342
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Tidskriftsartikel (refereegranskat)abstract
- The prediction of potential developmental toxicity in vitro could be based ontoxicogenomic endpoints a short time after exposure in cultured embryo-derived celllines. Our previous microarray studies in P19 mouse embryonal carcinoma cells andmouse embryos have indicated that the teratogen valproic acid (VPA), an inducerof neural tube defects, deregulates the expression of a large number of genes, manyof which have critical roles in neural tube formation and closure. In this study weexposed undifferentiated R1 mouse embryonic stem (ES) cells to VPA and VPA analogto define genes whose expression responses may be related to teratogenic potential.After 6 h of exposure, RNA samples were subjected to microarray analysis usingCodeLinkTM Mouse Whole Genome Bioarrays. VPA (1 mM) and the teratogenic VPAanalog (S)-2-pentyl-4-pentynoic (0.25 mM or 0.5 mM) deregulate a large numberof genes, whereas for the non-teratogenic (and potentially pharmacologically active)analog 2-ethyl-4-methyl-pentanoic acid (1 mM) the expression of only a few geneswas affected. Biological process ontology groups related to embryonic development,morphogenesis, and cell behavior were overrepresented among the affected teratogentarget genes. Multivariate analysis indicated that as few as five genes (out of ~2500array probes correlating with the separation) could separate the data set accordingto teratogenicity. Genes deregulated by the two teratogens showed a substantialoverlap with genes previously found to be deregulated by VPA in P19 cells and mouseembryos. A panel of candidate genes was defined as potential markers predictiveof teratogenicity and evaluated through TaqMan low density array analysis. Theteratogens butyrate and trichostatin A, which like VPA and (S)-2-pentyl-4-pentynoicacid are known histone deacetylase (HDAC) inhibitors, induced similar responsesas these two teratogens for a large subset of markers. This indicates that HDACinhibition may be a major mechanism by which VPA induces gene deregulation andpossibly teratogenicity. Other teratogenic compounds tested had no effect on thepanel of selected markers, indicating that they may not be predicitive of teratogenicityfor compounds acting through other mechanisms than VPA.
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| 8. |
- Jergil, Måns, et al.
(författare)
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Valproic acid-induced deregulation in vitro of genes associated in vivo with neural tube defects
- 2009
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Ingår i: Toxicological Sciences. - : Oxford University Press (OUP). - 1096-6080 .- 1096-0929. ; 108:1, s. 132-148
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Tidskriftsartikel (refereegranskat)abstract
- The utility of an in vitro system to search for molecular targets and markers of developmental toxicity was explored, using microarrays to detect genes susceptible to deregulation by the teratogen valproic acid (VPA) in the pluripotent mouse embryonal carcinoma cell line P19. Total RNA extracted from P19 cells cultured in the absence or presence of 1, 2.5, or 10mM VPA for 1.5, 6, or 24 h was subjected to replicated microarray analysis, using CodeLink UniSet I Mouse 20K Expression Bioarrays. A moderated F-test revealed a significant VPA response for 2972 (p < 10(-3)) array probes (19.4% of the filtered gene list), 421 of which were significant across all time points. In a core subset of VPA target genes whose expression was downregulated (68 genes) or upregulated (125 genes) with high probability (p < 10(-7)) after both 1.5 and 6 h of VPA exposure, there was a significant enrichment of the biological process Gene Ontology term transcriptional regulation among downregulated genes, and apoptosis among upregulated, and two of the downregulated genes (Folr1 and Gtf2i) have a knockout phenotype comprising exencephaly, the major malformation induced by VPA in mice. The VPA-induced gene expression response in P19 cells indicated that approximately 30% of the approximately 200 genes known from genetic mouse models to be associated with neural tube defects may be potential VPA targets, suggestive of a combined deregulation of multiple genes as a possible mechanism of VPA teratogenicity. Gene expression responses related to other known effects of VPA (histone deacetylase inhibition, G(1)-phase cell cycle arrest, induction of apoptosis) were also identified. This study indicates that toxicogenomic responses to a teratogenic compound in vitro may correlate with known in vitro and in vivo effects, and that short-time (< or =6 h) exposures in such an in vitro system could provide a useful component in mechanistic studies and screening tests in developmental toxicology.
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| 9. |
- Kultima, Kim, et al.
(författare)
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Cadmium-induced gene expression changes in the mouse embryo, and the influence of pretreatment with zinc
- 2006
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Ingår i: Reproductive Toxicology. - : Elsevier BV. - 0890-6238 .- 1873-1708. ; 22:4, s. 636-646
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Tidskriftsartikel (refereegranskat)abstract
- Cadmium (Cd) administered to female C57BL/6 mice on gestation day 8 induces a high incidence of anterior neural tube defects (exencephaly). This adverse effect can be attenuated by maternal pretreatment with zinc (Zn). In this study we used replicated microarray analysis and real-time PCR to investigate gene expression changes induced in the embryo 5 and 10h after maternal Cd exposure in the absence or presence of Zn pretreatment. We report nine genes with a transcriptional response induced by Cd, none of which was influenced by Zn pretreatment, and two genes induced only by combined matemal Cd exposure and Zn pretreatment. We discuss the results in relation to the possibility that Cd is largely excluded from the embryo, that the teratogenic effects of Cd may be secondary to toxicity in extraembryonic tissues, and that the primary protective role of Zn may not be to reverse Cd-induced transcription in the embryo.
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| 10. |
- Kultima, Kim, et al.
(författare)
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Early transcriptional responses in mouse embryos as a basis for selection of molecular markers predictive of valproic acid teratogenicity
- 2010
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Ingår i: Reproductive Toxicology. - : Elsevier BV. - 0890-6238 .- 1873-1708. ; 30:3, s. 457-468
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Tidskriftsartikel (refereegranskat)abstract
- Cell-based in vitro assays would potentially reduce animal testing in preclinical drug development. Mouse embryos exposed to the teratogenic drug valproic acid (VPA) in utero for 1.5, 3 or 6h on gestational day 8 were analyzed using microarrays. Significant effects on gene expression were observed already at 1.5h, and 85 probes were deregulated across all time points. To find transcriptional markers of VPA-induced developmental toxicity, the in vivo data were compared to previous in vitro data on embryonal carcinoma P19 cells exposed to VPA for 1.5, 6 or 24h. Maximal concordance between embryos and cells was at the 6-h time points, with 163 genes showing similar deregulation. Developmentally important Gene Ontology terms, such as "organ morphogenesis" and "tube development" were overrepresented among putative VPA target genes. The genes Gja1, Hap1, Sall2, H1f0,Cyp26a1, Fgf15, Otx2, and Lin7b emerged as candidate in vitro markers of potential VPA-induced teratogenicity.
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| 11. |
- Kultima, Kim, 1975-, et al.
(författare)
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Early transcriptional responses in mouse embryos as a basis for selection of molecular markers predictive of valproic acid teratogenicity
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Annan publikation (refereegranskat)abstract
- Cell-based in vitro assays would potentially reduce animal testing in preclinical drugdevelopment. Mouse embryos exposed to the teratogenic drug valproic acid (VPA)in utero for 1.5, 3 or 6 h on gestational day 8 were analyzed using microarrays.Significant effects on gene expression were observed as early as 1.5 h, and 85 probeswere deregulated across all time points. To find transcriptional markers of VPAinduceddevelopmental toxicity, the in vivo data were compared to previous in vitrodata on embryonal carcinoma P19 cells exposed to VPA for 1.5, 6 or 24 h. Maximalconcordance between embryos and cells was at the 6-h time points, with 163 genesshowing similar deregulation. Developmentally important Gene Ontology terms, suchas “morphogenesis” and “tube development” were overrepresented among putativeVPA target genes. The genes Gja1, Hap1, Sall2, H1f0, Cyp26a1, Fgf15, Otx2, andLin7b emerged as candidate in vitro markers of potential VPA-induced teratogenicity.
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| 12. |
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| 13. |
- Kultima, Kim, et al.
(författare)
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Valproic acid teratogenicity: a toxicogenomics approach
- 2004
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Ingår i: Journal of Environmental Health Perspectives. - 0091-6765 .- 1552-9924. ; 112:12, s. 1225-1235
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Tidskriftsartikel (refereegranskat)abstract
- Embryonic development is a highly coordinated set of processes that depend on hierarchies of signaling and gene regulatory networks, and the disruption of such networks may underlie many cases of chemically induced birth defects. The antiepileptic drug valproic acid (VPA) is a potent inducer of neural tube defects (NTDs) in human and mouse embryos. As with many other developmental toxicants however, the mechanism of VPA teratogenicity is unknown. Using microarray analysis, we compared the global gene expression responses to VPA in mouse embryos during the critical stages of teratogen action in vivo with those in cultured P19 embryocarcinoma cells in vitro. Among the identified VPA-responsive genes, some have been associated previously with NTDs or VPA effects [vinculin, metallothioneins 1 and 2 (Mt1, Mt2), keratin 1-18 (Krt1-18)], whereas others provide novel putative VPA targets, some of which are associated with processes relevant to neural tube formation and closure [transgelin 2 (Tagln2), thyroid hormone receptor interacting protein 6, galectin-1 (Lgals1), inhibitor of DNA binding 1 (Idb1), fatty acid synthase (Fasn), annexins A5 and A11 (Anxa5, Anxa11)], or with VPA effects or known molecular actions of VPA (Lgals1, Mt1, Mt2, Id1, Fasn, Anxa5, Anxa11, Krt1-18). A subset of genes with a transcriptional response to VPA that is similar in embryos and the cell model can be evaluated as potential biomarkers for VPA-induced teratogenicity that could be exploited directly in P19 cell-based in vitro assays. As several of the identified genes may be activated or repressed through a pathway of histone deacetylase (HDAC) inhibition and specificity protein 1 activation, our data support a role of HDAC as an important molecular target of VPA action in vivo.
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| 14. |
- Parichy, D M, et al.
(författare)
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Genetic analysis of steel and the PG-M/versican-encoding gene AxPG as candidates for the white (d) pigmentation mutant in the salamander Ambystoma mexicanum.
- 1999
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Ingår i: Development, Genes and Evolution. - 0949-944X .- 1432-041X. ; 209:6, s. 349-56
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Tidskriftsartikel (refereegranskat)abstract
- Vertebrate non-retinal pigment cells are derived from neural crest (NC) cells, and several mutations have been identified in the Mexican axolotl Ambystoma mexicanum (Ambystomatidae) that affect the development of these cell lineages. In "white" (d) mutant axolotls, premigratory NC cells differentiate as pigment cells, yet fail to disperse, survive, or both, and this leads to a nearly complete absence of pigment cells in the skin. Previous studies revealed that d affects pigment cell development non-autonomously, and have reported differences between white and wild-type axolotls in the structure and composition of the extracellular matrix through which NC and pigment cells migrate. Here we test the correspondence of d and two candidate genes: steel and AxPG. In amniotes, Steel encodes the cytokine Steel factor (mast cell growth factor; stem cell factor; kit ligand), which is expressed along the migratory pathways of melanocyte precursors and is required by these cells for their migration and survival; mammalian Steel mutants resemble white mutant axolotls in having a deficit or complete absence of pigment cells. In contrast, AxPG encodes a PG-M/versican-like proteoglycan that may promote the migration of A. mexicanum pigment cells, and AxPG expression is reduced in white mutant axolotls. We cloned a salamander orthologue of steel and used a partial genetic linkage map of Ambystoma to determine the genomic locations of steel, AxPG, and d. We show that the three genes map to different linkage groups, excluding steel and AxPG as candidates for d.
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| 15. |
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| 16. |
- Scholz, Birger, et al.
(författare)
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Sex-dependent gene expression in early brain development of chicken embryos
- 2006
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Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 7, s. 12-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:Differentiation of the brain during development leads to sexually dimorphic adult reproductive behavior and other neural sex dimorphisms. Genetic mechanisms independent of steroid hormones produced by the gonads have recently been suggested to partly explain these dimorphisms.RESULTS:Using cDNA microarrays and real-time PCR we found gene expression differences between the male and female embryonic brain (or whole head) that may be independent of morphological differentiation of the gonads. Genes located on the sex chromosomes (ZZ in males and ZW in females) were common among the differentially expressed genes, several of which (WPKCI-8, HINT, MHM non-coding RNA) have previously been implicated in avian sex determination. A majority of the identified genes were more highly expressed in males. Three of these genes (CDK7, CCNH and BTF2-P44) encode subunits of the transcription factor IIH complex, indicating a role for this complex in neuronal differentiation.CONCLUSION:In conclusion, this study provides novel insights into sexually dimorphic gene expression in the embryonic chicken brain and its possible involvement in sex differentiation of the nervous system in birds.
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| 17. |
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| 18. |
- Stigson, Michael, et al.
(författare)
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Large disulfide-stabilized proteoglycan complexes are synthesized by the epidermis of axolotl embryos.
- 1991
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Ingår i: Archives of Biochemistry and Biophysics. - 0003-9861 .- 1096-0384. ; 290:2, s. 391-6
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans (PGs) synthesized by the epidermis during stages crucial to the subepidermal migration of neural crest cells in the trunk of the axolotl (Ambystoma mexicanum, Urodela, Amphibia) embryo were studied. The glycosaminoglycan chains were biosynthetically labeled with [35S]sulfate in vitro during a period corresponding to the onset of migration. After extraction with guanidine HCl, the radiolabeled PGs were separated according to size by molecular-sieve chromatography on Sepharose CL-2B under dissociative conditions. This resulted in the separation of high-molecular-weight PGs, which eluted in the void volume, and low-molecular-weight PGs, eluting in a broad peak with a mean Kav of 0.7. The large PGs were also found to elute in the void volume when chromatographed on a Sephacryl S-1000 column. The low-molecular-weight PGs contained heparan sulfate and chondroitin sulfate (CS) and were not further characterized. The glycosaminoglycan component of the high-molecular-weight PG was completely degraded by chondroitinase ABC, while a large portion was resistant to chondroitinase AC, indicating the presence of dermatan sulfate (DS). These CS/DS chains were of unusually large size (Mr approximately 150,000) as estimated by chromatography on Sepharose CL-4B, relating the elution position to hyaluronan standards. Moreover, the chains were found to have a lower surface charge density than standard CS, and may therefore be undersulfated. After reduction and alkylation the high-molecular-weight PGs were included on both Sepharose CL-2B and Sephacryl S-1000 columns, eluting at Kav 0.2 and 0.4, respectively. Hence, the high-molecular-weight material appears to consist of large PG complexes, stabilized by intermolecular disulfide bonds. A CS/DSPG of similar size as the reduced monomeric form of the high-molecular-weight PG was found in small amounts in the total extract of 35S-labeled material.
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| 19. |
- Stigson, Michael, et al.
(författare)
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Molecular targets and early response biomarkers for the prediction of developmental toxicity in vitro
- 2007
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Ingår i: ATLA (Alternatives to Laboratory Animals). - 0261-1929. ; 35:3, s. 335-342
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Tidskriftsartikel (refereegranskat)abstract
- There is an urgent need for new in vitro methods to predict the potential developmental toxicity of candidate drugs in the early lead identification and optimisation process. This would lead to a reduction in the total number of animals required in full-scale developmental toxicology studies, and would improve the efficiency of drug development. However, suitable in vitro systems permitting robust high-throughput screening for this purpose, for the most part, remain to be designed. An understanding of the mechanisms involved in developmental toxicity may be essential for the validation of in vitro tests. Early response biomarkers - even a single one - could contribute to reducing assay time and facilitating automation. The use of toxicogenomics approaches to study in vitro and in vivo models in parallel may be a powerful tool in defining such mechanisms of action and the molecular targets of toxicity, and also for use in finding possible biomarkers of early response. Using valproic acid as a model substance, the use of DNA microarrays to identify teratogen-responsive genes in cell models is discussed. It is concluded that gene expression in P19 mouse embryocarcinoma cells represents a potentially suitable assay system, which could be readily used in a tiered testing system for developmental toxicity testing.
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| 20. |
- Stigson, Michael, et al.
(författare)
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PG-M/versican-like proteoglycans are components of large disulfide-stabilized complexes in the axolotl embryo.
- 1997
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 272:6, s. 3246-53
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Tidskriftsartikel (refereegranskat)abstract
- Large disulfide-stabilized proteoglycan complexes were previously shown to be synthesized by the epidermis of axolotl embryos during stages crucial to subepidermal migration of neural crest cells. We now show that the complexes contain PG-M/versican-like monomers in addition to some other component with low buoyant density. Metabolically 35S-labeled proteoglycans were extracted from epidermal explants and separated by size exclusion chromatography and density equilibrium gradient centrifugation. The complexes, which elute in the void volume on Sepharose CL-2B, were recovered at buoyant density 1.42 g/ml in CsCl gradients, whereas the monomer proteoglycans, which could only be liberated from the complexes by reduction, had a higher buoyant density (1.48 g/ml). The native complexes did not aggregate with hyaluronan. The purified complexes reacted with antibodies against a portion of a cloned PG-M/versican-like axolotl proteoglycan. These antibodies were found to stain the subepidermal matrix of axolotl embryos, suggesting that the proteoglycan complexes are encountered by neural crest cells during subepidermal migration. From Western blot analysis, the core protein of the PG-M/versican-like monomers was found to be of similar size ( approximately 500 kDa) as those of PG-M/versican variants of other species. Another chondroitin sulfate proteoglycan that was present in small amounts in the epidermal extracts was found to be distinctly different from the similarly sized PG-M/versican-like monomers.
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| 21. |
- Stigson, Michael, et al.
(författare)
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Reduced epidermal expression of a PG-M/versican-like proteoglycan in embryos of the white mutant axolotl.
- 1997
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 236:1, s. 57-65
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Tidskriftsartikel (refereegranskat)abstract
- Axolotl embryos have previously been used to study neural crest cell migration. In embryos of the normal wild type, neural crest cells migrate subepidermally to form pigment cells. In the trunk of the white mutant embryo, these cells are unable to migrate, possibly due to an inherited delay in the maturation of the local extracellular matrix. The present investigation reveals a reduced incorporation of [35S]sulfate into PG-M/versican-like proteoglycans synthesized in epidermal explants from the dorsal trunk of white mutant embryos during stages pertinent to migration. This is the major form of proteoglycans in the subepidermal matrix, where they are assembled in large disulfide-stabilized supramolecular complexes. The reduction in [35S]sulfate incorporation is not due to qualitative differences between wild-type and white mutant proteoglycans but is paralleled by a reduced expression of mRNA for the core protein of the PG-M/versican-like proteoglycan. We conclude that a reduced amount of these proteoglycans is produced by the white mutant embryo during the period critical for migration.
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| 22. |
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