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Find video protocols related to scientific articles indexed in Pubmed.
SIRT1-mediated deacetylation of CRABPII regulates cellular retinoic acid signaling and modulates embryonic stem cell differentiation.
Mol. Cell
PUBLISHED: 08-21-2014
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Retinoid homeostasis is critical for normal embryonic development. Both the deficiency and excess of these compounds are associated with congenital malformations. Here we demonstrate that SIRT1, the most conserved mammalian NAD?-dependent protein deacetylase, contributes to homeostatic retinoic acid (RA) signaling and modulates mouse embryonic stem cell (mESC) differentiation in part through deacetylation of cellular retinoic acid binding protein II (CRABPII). We show that RA-mediated acetylation of CRABPII at K102 is essential for its nuclear accumulation and subsequent activation of RA signaling. SIRT1 interacts with and deacetylates CRABPII, regulating its subcellular localization. Consequently, SIRT1 deficiency induces hyperacetylation and nuclear accumulation of CRABPII, enhancing RA signaling and accelerating mESC differentiation in response to RA. Consistently, SIRT1 deficiency is associated with elevated RA signaling and development defects in mice. Our findings reveal a molecular mechanism that regulates RA signaling and highlight the importance of SIRT1 in regulation of ESC pluripotency and embryogenesis.
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Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency, the PEX5 mRNA and protein levels and induction of peroxisomal deficiency.
Histochem. Cell Biol.
PUBLISHED: 07-14-2014
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The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine(®) 2000, FuGENE 6, HiPerFect(®), INTERFERin™, RiboJuice™) as well as microporation using the Neon™ Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0-70 %), to the reduction of PEX5 mRNA (by 90 vs. 0-50 %) and PEX5 protein levels (by 70 vs. 0-50 %). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3' fragment (15 %) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded to transiently knockdown PEX5 mRNA and its protein level leading to functional consequences similar as observed in peroxisome deficiencies.
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Acute epididymitis induces alterations in sperm protein composition.
Fertil. Steril.
PUBLISHED: 02-26-2014
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To use a proteomic approach to evaluate possible postinflammatory alterations in the protein composition of motile sperm in patients 3 months after acute epididymitis.
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BDNF and its TrkB receptor in human fracture healing.
Ann. Anat.
PUBLISHED: 02-07-2014
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Fracture healing is a physiological process of repair which proceeds in stages, each characterized by a different predominant tissue in the fracture gap. Matrix reorganization is regulated by cytokines and growth factors. Neurotrophins and their receptors might be of importance to osteoblasts and endothelial cells during fracture healing. The aim of this study was to examine the presence of brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor (TrkB) during human fracture healing. BDNF and TrkB were investigated in samples from human fracture gaps and cultured cells using RT-PCR, Western blot, and immunohistochemistry. Endothelial cells and osteoblastic cell lines demonstrated a cytoplasmic staining pattern of BDNF and TrkB in vitro. At the mRNA level, BDNF and TrkB were expressed in the initial and osteoid formation phase of human fracture healing. In the granulation tissue of fracture gap, both proteins--BDNF and TrkB--are concentrated in endothelial and osteoblastic cells at the margins of woven bone suggesting their involvement in the formation of new vessels. There was no evidence of BDNF or TrkB during fracture healing in chondrocytes of human enchondral tissue. Furthermore, BDNF is absent in mature bone. Taken together, BDNF and TrkB are involved in vessel formation and osteogenic processes during human fracture healing. The detection of BDNF and its TrkB receptor during various stages of the bone formation process in human fracture gap tissue were shown for the first time. The current study reveals that both proteins are up-regulated in human osteoblasts and endothelial cells in fracture healing.
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The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies.
Histochem. Cell Biol.
PUBLISHED: 07-29-2013
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Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.
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Peroxisomes in dental tissues of the mouse.
Histochem. Cell Biol.
PUBLISHED: 07-21-2013
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Patients with mild forms of peroxisomal biogenesis disorders show facial dysmorphism and exhibit dentition problems accompanied by enamel hypoplasia. However, no information is available on the role of peroxisomes in dental and paradontal tissues. Therefore, we studied the distribution of these organelles, their protein composition and the expression of corresponding genes during dental development and in mature decalcified teeth in mice. Perfusion-fixed heads of mice of different developmental stages (E13.5 to adult) were cut in sagittal direction into two halves and embedded in paraffin for serial sectioning and subsequent peroxidase-based immunohistochemistry or double-immunofluorescence preparations. Frozen, unfixed heads of newborn mice were used for cryosectioning and subsequent laser-assisted microdissection of ameloblasts and odontoblasts, RNA isolation and RT-PCR analysis. Our results revealed the presence of peroxisomes already in the bud stage of dental development. An increase in peroxisome abundance was noted during differentiation of ameloblasts and odontoblasts with the highest number of organelles in Tomes processes of mature ameloblasts. A strong heterogeneity of peroxisomal enzyme content developed within differentiated dental cell types. A drastic down-regulation of catalase in maturing ameloblasts was noted in contrast to high levels of lipid metabolizing enzymes in peroxisomes of these cells. As known from the literature, differentiated ameloblasts are more prone to oxidative damage which could be explained by the low catalase levels inside of this cell type.
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Mammalian SOD2 is exclusively located in mitochondria and not present in peroxisomes.
Histochem. Cell Biol.
PUBLISHED: 05-08-2013
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Superoxide dismutases (SODs) are metalloenzymes that belong to the essential antioxidant enzyme systems of virtually all oxygen-respiring organisms. SODs catalyze the dismutation of highly reactive superoxide radicals into hydrogen peroxide and molecular oxygen. For the subcellular localization of the manganese superoxide dismutase (SOD2) in eukaryotic cells, a dual mitochondrial localization and peroxisomal localization were proposed in the literature. However, our own observation from immunofluorescence preparations of human and mouse tissues suggested that SOD2 serves as an excellent marker protein for mitochondria but never co-localized with peroxisomes. To clarify whether our observations were correct, we have carefully reinvestigated the subcellular localization of SOD2 using sensitive double-immunofluorescence methods on frozen and paraffin sections as well as in cell culture preparations. In addition, ultrastructural analyses were performed with post-embedding immunoelectron microscopy on LR White sections as well as labeling of ultrathin cryosections with various immunogold techniques. In all morphological experiments, the SOD2 localization was compared to one of the catalase, a typical marker protein for peroxisomes, solely localized in these organelles. Moreover, biochemical subcellular fractions of mouse liver was used to isolate enriched organelles and highly purified peroxisomal fractions for Western blot analyses of the exact subcellular distributions of SOD2 and catalase. All results with the various methodologies, tissues, and cell types used revealed that catalase and SOD2 were always confined to distinct and separate subcellular compartments. SOD2 was unequivocally in mitochondria, but never present in peroxisomes. Furthermore, our results are supported by accumulating database information on organelle proteomes that also indicate that SOD2 is a pure mitochondrial protein.
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Deletion of a single allele of the Pex11? gene is sufficient to cause oxidative stress, delayed differentiation and neuronal death in mouse brain.
Dis Model Mech
PUBLISHED: 10-04-2011
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Impaired neuronal migration and cell death are commonly observed in patients with peroxisomal biogenesis disorders (PBDs), and in mouse models of this diseases. In Pex11?-deficient mice, we observed that the deletion of a single allele of the Pex11? gene (Pex11?(+/-) heterozygous mice) caused cell death in primary neuronal cultures prepared from the neocortex and cerebellum, although to a lesser extent as compared with the homozygous-null animals (Pex11?(-/-) mice). In corresponding brain sections, cell death was rare, but differences between the genotypes were similar to those found in vitro. Because PEX11? has been implicated in peroxisomal proliferation, we searched for alterations in peroxisomal abundance in the brain of heterozygous and homozygous Pex11?-null mice compared with wild-type animals. Deletion of one allele of the Pex11? gene slightly increased the abundance of peroxisomes, whereas the deletion of both alleles caused a 30% reduction in peroxisome number. The size of the peroxisomal compartment did not correlate with neuronal death. Similar to cell death, neuronal development was delayed in Pex11?(+/-) mice, and to a further extent in Pex11?(-/-) mice, as measured by a reduced mRNA and protein level of synaptophysin and a reduced protein level of the mature isoform of MAP2. Moreover, a gradual increase in oxidative stress was found in brain sections and primary neuronal cultures from wild-type to heterozygous to homozygous Pex11?-deficient mice. SOD2 was upregulated in neurons from Pex11?(+/-) mice, but not from Pex11?(-/-) animals, whereas the level of catalase remained unchanged in neurons from Pex11?(+/-) mice and was reduced in those from Pex11?(-/-) mice, suggesting a partial compensation of oxidative stress in the heterozygotes, but a failure thereof in the homozygous Pex11?(-/-) brain. In conclusion, we report the alterations in the brain caused by the deletion of a single allele of the Pex11? gene. Our data might lead to the reconsideration of the clinical treatment of PBDs and the common way of using knockout mouse models for studying autosomal recessive diseases.
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Brutons tyrosine kinase is required for TLR-dependent heme oxygenase-1 gene activation via Nrf2 in macrophages.
J. Immunol.
PUBLISHED: 06-15-2011
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Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation and provides cytoprotection against oxidative stress by its products carbon monoxide and biliverdin. More recently, HO-1 has also been shown to exert immunomodulatory functions via cell type-specific anti-inflammatory effects in myeloid/macrophage cells. In the current study, it is demonstrated that Brutons tyrosine kinase (Btk), the gene of which is mutated in the human immunodeficiency X-linked agammaglobulinemia, is involved in the upregulation of HO-1 gene expression via TLR signaling in macrophages. The specific Btk inhibitor LFM-A13 blocked HO-1 induction by the classical TLR4 ligand LPS in cell cultures of RAW264.7 monocytic cells and primary mouse alveolar macrophages. Moreover, upregulation of HO-1 gene expression was abrogated in LPS-stimulated alveolar macrophages from Btk(-/-) mice. Transfection studies with luciferase reporter gene constructs demonstrated that LPS-dependent induction of HO-1 promoter activity was attenuated by pharmacological Btk inhibition and by an overexpressed dominant-negative mutant of Btk. This induction was mediated by the transcription factor Nrf2, which is a master regulator of the antioxidant cellular defense. Accordingly, nuclear translocation of Nrf2 in LPS-treated macrophages was reduced by Btk inhibition. The generation of reactive oxygen species, but not that of NO, was involved in this regulatory pathway. Btk-dependent induction of HO-1 gene expression was also observed upon macrophage stimulation with ligands of TLR2, TLR6, TLR7, and TLR9, suggesting that Btk is required for HO-1 gene activation by major TLR pathways.
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Germ cells of male mice express genes for peroxisomal metabolic pathways implicated in the regulation of spermatogenesis and the protection against oxidative stress.
Histochem. Cell Biol.
PUBLISHED: 06-07-2011
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Peroxisomes are organelles with main functions in the metabolism of lipids and of reactive oxygen species. Within the testis, they have different functional profiles depending on the cell types. A dysfunction of peroxisomes interferes with regular spermatogenesis and can lead to infertility due to spermatogenic arrest. However, so far only very little is known about the functions of peroxisomes in germ cells. We have therefore analyzed the peroxisomal compartment in germ cells and its alterations during spermatogenesis by fluorescence and electron microscopy as well as by expression profiling of peroxisome-related genes in purified cell populations isolated from mouse testis. We could show that peroxisomes are present in all germ cells of the germinal epithelium. During late spermiogenesis, the peroxisomes form large clusters that are segregated from the spermatozoa into the residual bodies upon release from the germinal epithelium. Germ cells express genes for proteins involved in numerous metabolic pathways of peroxisomes. Based on the expression profile, we conclude that newly identified functions of germ cell peroxisomes are the synthesis of plasmalogens as well as the metabolism of retinoids, polyunsaturated fatty acids and polyamines. Thus, germ cell peroxisomes are involved in the regulation of the homeostasis of signaling molecules regulating spermatogenesis and they contribute to the protection of germ cells against oxidative stress.
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PEX13 deficiency in mouse brain as a model of Zellweger syndrome: abnormal cerebellum formation, reactive gliosis and oxidative stress.
Dis Model Mech
PUBLISHED: 10-19-2010
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Delayed cerebellar development is a hallmark of Zellweger syndrome (ZS), a severe neonatal neurodegenerative disorder. ZS is caused by mutations in PEX genes, such as PEX13, which encodes a protein required for import of proteins into the peroxisome. The molecular basis of ZS pathogenesis is not known. We have created a conditional mouse mutant with brain-restricted deficiency of PEX13 that exhibits cerebellar morphological defects. PEX13 brain mutants survive into the postnatal period, with the majority dying by 35 days, and with survival inversely related to litter size and weaning body weight. The impact on peroxisomal metabolism in the mutant brain is mixed: plasmalogen content is reduced, but very-long-chain fatty acids are normal. PEX13 brain mutants exhibit defects in reflex and motor development that correlate with impaired cerebellar fissure and cortical layer formation, granule cell migration and Purkinje cell layer development. Astrogliosis and microgliosis are prominent features of the mutant cerebellum. At the molecular level, cultured cerebellar neurons from E19 PEX13-null mice exhibit elevated levels of reactive oxygen species and mitochondrial superoxide dismutase-2 (MnSOD), and show enhanced apoptosis together with mitochondrial dysfunction. PEX13 brain mutants show increased levels of MnSOD in cerebellum. Our findings suggest that PEX13 deficiency leads to mitochondria-mediated oxidative stress, neuronal cell death and impairment of cerebellar development. Thus, PEX13-deficient mice provide a valuable animal model for investigating the molecular basis and treatment of ZS cerebellar pathology.
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Heme oxygenase-1 as a therapeutic target in inflammatory disorders of the gastrointestinal tract.
World J. Gastroenterol.
PUBLISHED: 07-02-2010
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Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. HO-1 not only protects against oxidative stress and apoptosis, but has received a great deal of attention in recent years because of its potent anti-inflammatory functions. Studies with HO-1 knockout animal models have led to major advances in the understanding of how HO-1 might regulate inflammatory immune responses, although little is known on the underlying mechanisms. Due to its beneficial effects the targeted induction of this enzyme is considered to have major therapeutic potential for the treatment of inflammatory disorders. This review discusses current knowledge on the mechanisms that mediate anti-inflammatory protection by HO-1. More specifically, the article deals with the role of HO-1 in the pathophysiology of viral hepatitis, inflammatory bowel disease, and pancreatitis. The effects of specific HO-1 modulation as a potential therapeutic strategy in experimental cell culture and animal models of these gastrointestinal disorders are summarized. In conclusion, targeted regulation of HO-1 holds major promise for future clinical interventions in inflammatory diseases of the gastrointestinal tract.
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Heme oxygenase-1 and iron in liver inflammation: a complex alliance.
Curr Drug Targets
PUBLISHED: 05-19-2010
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Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. HO-1 has potent antioxidant and also anti-inflammatory functions, the underlying mechanisms of which are not well understood. Together with antioxidant carbon monoxide and biliverdin, HO produces reactive iron, which unambiguously connect this enzyme with the iron metabolism and its potential toxicity. A link between HO-1 and iron homeostasis has been demonstrated in HO-1 knockout mice, which develop major hemosiderosis in solid organs such as liver and kidney. Moreover, genetic HO-1 deficiency causes a chronic inflammatory condition in these animals. As the liver plays a crucial role for the bodys iron homeostasis (eg. via secretion of the iron regulatory hormone hepcidin) and also for systemic inflammation, hepatic HO-1 may be important for the regulation of both systems. In particular, cell-specific functions of HO-1 in liver tissue macrophages (Kupffer cells) might be of major significance, because these cells play a key role in iron recycling during erythrophagocytosis and also in the control of hepatic and systemic inflammatory responses. This review discusses the current knowledge on interactions of HO-1 with iron metabolism in the context of systemic as well as hepatic inflammatory disorders. Recent advances in the understanding of the functional role of HO-1 in inflammatory liver diseases, namely viral hepatitis, alcoholic liver disease and non-alcoholic steatohepatitis are summarized. Finally, it is highlighted how targeted modulation of HO-1 may provide specific protection in these inflammatory disorders.
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Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6.
BMC Cell Biol.
PUBLISHED: 02-20-2009
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Peroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes.
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Peroxisomes in airway epithelia and future prospects of these organelles for pulmonary cell biology.
Histochem. Cell Biol.
PUBLISHED: 01-29-2009
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Peroxisomes are intimately involved in the metabolism of reactive oxygen species, in the synthesis of ether lipids and of polyunsaturated fatty acids as well as in the beta-oxidation of bioactive and toxic lipid derivatives. Therefore, the metabolic pathways of this organelle might play an important role in pulmonary biology by protection of inner pulmonary surface epithelia against oxidative stress, induced by the high oxygen levels in the air and/or by regulation of the lipid homeostasis in pulmonary epithelia and the pulmonary surfactant film. In this article, original results on the distribution of peroxisomal marker proteins, involved in the biogenesis, ROS- and lipid-metabolism of this organelle in the bronchiolar epithelium and the alveolar region of the adult human lung in comparison to newborn and adult murine lungs are presented. In addition, we investigated the expression of the PEX11beta-mRNA, encoding a protein involved in peroxisomal division. Our study revealed significant differences in the abundance and distribution of peroxisomal proteins in distinct cell types of the lung and different developmental stages and led to the discovery of species-specific differences in the peroxisomal compartment in pulmonary epithelia between mouse and man. Finally, the structure and general biology of pulmonary airways-with special emphasis on Clara cells-are reviewed and discussed in relation to peroxisomal metabolism and proliferation. Future prospects of peroxisomes and Pex11 proteins for pulmonary cell biology are highlighted.
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Phenotype, differentiation, and function differ in rat and mouse neocortical astrocytes cultured under the same conditions.
J. Neurosci. Methods
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The study of slowly progressing brain diseases in which glial cells play a pathogenic role requires astrocytes that have been cultured for several weeks. We characterized neocortical astrocytes, grown for up to 42 days in vitro (DIV), from newborn rats and mice by indirect immunofluorescence technique, Western blot, and real-time RT-PCR analyses. We obtained highly enriched rat and mouse astrocyte cultures, where most cells were positively stained for the astrocyte markers GFAP, vimentin, and S100?, whereas neuronal and oligodendrocyte markers were undetectable. The protein and mRNA levels of GFAP, vimentin, and nestin were higher in rat than in mouse astrocytes. From 28 to 42 DIV, the levels of vimentin and nestin, but not of GFAP, decreased in both species, with an increase in the vimentin-GFAP ratio of 1.7 for rat, and of 0.9 for mouse astrocytes suggesting that the rat cultures were more differentiated than the mouse cultures, although both remained partially immature. The protoplasmic appearance of the cells, the negative A2B5 immunoreactivity, and the expression of the glutamate transporters GLAST and GLT-1 indicate that the rat and mouse cultures contained mainly type I astrocytes. The protein levels of GLAST and GLT-1 decreased from 28 to 42 DIV in the mouse, but not in the rat astrocytes, suggesting that the rat cultures are suitable for functional studies. Thus, under the same culture conditions, astrocyte cultures from rats and mice differ in phenotype, differentiation, and functionality. This finding should be taken into account when long-lasting glial reaction patterns are being studied.
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Cre-mediated stress affects sirtuin expression levels, peroxisome biogenesis and metabolism, antioxidant and proinflammatory signaling pathways.
PLoS ONE
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Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts), inducing PPAR?/?, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14) as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase). In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2) and a NF-?B activation were noted. TGF?1 and proinflammatory cytokines like IL1, IL6 and TNF? were upregulated and stress-related signaling pathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7) in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt) with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out-mediated effects.
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