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ABSTRACT: Background: Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells.
PLoS ONE 01/2013; 8(1):e55585. · 4.09 Impact Factor
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ABSTRACT: Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells.
The T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium.
Our results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells.
PLoS ONE 01/2013; 8(1):e55585. · 4.09 Impact Factor
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ABSTRACT: Potassium channel tetramerization domain containing 1 (KCTD1) contains a BTB domain, which can facilitate protein-protein interactions that may be involved in the regulation of signaling pathways. Here we describe an expression and purification system that can provide a significant amount of recombinant KCTD1 from Escherichia coli. The cDNA encoding human KCTD1 was amplified and cloned into the expression vector pET-30a(+). The recombinant protein was expressed in E. coli BL21(DE3) cells and subsequently purified using affinity chromatography. To confirm that KCTD1 was correctly expressed and folded, the molecular weight and conformation were analyzed using mass spectroscopy, Western blot, and circular dichroism. Optimizing KCTD1 expression and investigating its secondary structure will provide valuable information for future structural and functional studies of KCTD1 and KCTD family proteins.
Biochemistry (Moscow) 08/2012; 77(8):941-5. · 1.06 Impact Factor
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ABSTRACT: The adult mammalian intestine has long been used as a model to study adult stem cell function and tissue renewal as the intestinal epithelium is constantly undergoing self-renewal throughout adult life. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells located in the crypt. The development of this self-renewal system is, however, poorly understood. A number of studies suggest that the formation/maturation of the adult intestine is conserved in vertebrates and depends on endogenous thyroid hormone (T3). In amphibians such as Xenopus laevis, the process takes place during metamorphosis, which is totally dependent upon T3 and resembles postembryonic development in mammals when T3 levels are also high. During metamorphosis, the larval epithelial cells in the tadpole intestine undergo apoptosis and concurrently, adult epithelial stem/progenitor cells are formed de novo, which subsequently lead to the formation of a trough-crest axis of the epithelial fold in the frog, resembling the crypt-villus axis in the adult mammalian intestine. Here we will review some recent molecular and genetic studies that support the conservation of the development of the adult intestinal stem cells in vertebrates. We will discuss the mechanisms by which T3 regulates this process via its nuclear receptors.
International journal of biological sciences 01/2012; 8(8):1217-24. · 2.70 Impact Factor
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ABSTRACT: Frog metamorphosis is totally dependent on thyroid hormone (T3) and mimics the postembryonic period around birth in mammals. It is an excellent model to study the molecular basis of postembryonic development in vertebrate. We and others have shown that many, if not all, matrix metalloproteinases (MMPs), which cleave proteins of the extracellular matrix as well as other substrates, are induced by T3 and important for metamorphosis. MMP activity can be inhibited by tissue inhibitors of metalloproteinase (TIMPs). There are 4 TIMPs in vertebrates and their roles in postembryonic development are poorly studied.
We analyzed the TIMP2 genes in Xenopus laevis and the highly related species Xenopus tropicalis and discovered that TIMP2 is a single copy gene in Xenopus tropicalis as in mammals but is duplicated in Xenopus laevis. Furthermore, the TIMP2 locus in Xenopus tropicalis genome is different from that in human, suggesting an evolutionary reorganization of the locus. More importantly, we found that the duplicated TIMP2 genes were similarly regulated in the developing limb, remodeling intestine, resorbing tail during metamorphosis. Unexpectedly, like its MMP target genes, the TIMP2 genes were upregulated by T3 during both natural and T3-induced metamorphosis.
Our results indicate that TIMP2 is highly conserved among vertebrates and that the TIMP2 locus underwent a chromosomal reorganization during evolution. Furthermore, the unexpected upregulation of TIMP2 genes during metamorphosis suggests that proper balance of MMP activity is important for metamorphosis.
PLoS ONE 01/2012; 7(5):e36707. · 4.09 Impact Factor
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ABSTRACT: To identify molecular interaction partners of the cellular prion protein (PrP(C)), we applied a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the potassium channel tetramerization domain containing 1 (KCTD1) as a PrP(C) interacting protein. Deletion mapping showed that PrP(C) specifically binds KCTD1 through the unstructured PrP(51-136) region. We further confirmed the interaction between PrP(C) and KCDT1 protein by co-immunoprecipitation in vivo and by a biosensor assay in vitro. Interestingly, the binding of an insertion mutant PrP(8OR) to KCTD1 is higher than that of wild-type PrP(C), suggesting an important role for an unstructured region harboring octapeptide repeats in the KCTD1-PrP(C) interaction. Our results identify a novel PrP(C)-interacting protein and suggest a new approach to investigating the unidentified physiological cellular function of PrP(C).
Biochemical and Biophysical Research Communications 11/2011; 417(1):182-6. · 2.48 Impact Factor
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ABSTRACT: Alpha-subtype protein kinase C (PKCα) is closely related to cardiovascular disease. Ritonavir (RTV), which is a human immunodeficiency virus (HIV) protease inhibitor, can induce atherosclerosis in a PKC-dependent manner. However, it remains unclear how RTV acts on PKCα to induce pathological phenotypes. In this study, we obtained mouse peritoneal macrophages from adult female Kunmin mice. The results of Oil Red O staining and immunofluorescence using confocal laser scanning microscope demonstrated that RTV could induce foam cell formation and plasma membrane translocation of PKCα like phorbol-12-myristate-13-acetate (PMA, a PKC activator). Computational modeling also exhibited similar docking of RTV and PMA to PKCα and similar patterns of hydrophobic interaction and hydrogen bond formation. Further in vitro kinase activity studies revealed that RTV could elevate PKC activity. These data provided insight into the PKC-dependent induction of atherosclerosis and useful information for more in-depth toxicity research of HIV protease inhibitor (PI). In addition, western blot analysis proved RTV also up-regulate PKCα expression, which may be related to its influence on estrogen responsiveness in target cells and needs further prove.
Chemico-biological interactions 11/2011; 194(2-3):127-33. · 2.46 Impact Factor
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ABSTRACT: Thyroid hormone (TH) affects diverse biological processes and can exert its effects through both gene regulation via binding the nuclear TH receptors (TRs) and non-genomic actions via binding to cell surface and cytoplasmic proteins. The critical importance of TH in vertebrate development has long been established, ranging from the formation of human cretins to the blockage of frog metamorphosis due the TH deficiency. How TH affects vertebrate development has been difficult to study in mammals due to the complications associated with the uterus-enclosed mammalian embryos. Anuran metamorphosis offers a unique opportunity to address such an issue. Using Xenopus as a model, we and others have shown that the expression of TRs and their heterodimerization partners RXRs (9-cis retinoic acid receptors) correlates temporally with metamorphosis in different organs in two highly related species, Xenopuslaevis and Xenopus tropicalis. In vivo molecular studies have shown that TR and RXR are bound to the TH response elements (TREs) located in TH-inducible genes in developing tadpoles of both species. More importantly, transgenic studies in X. laevis have demonstrated that TR function is both necessary and sufficient for mediating the metamorphic effects of TH. Thus, the non-genomic effects of TH have little or no roles during metamorphosis and likely during vertebrate development in general.
General and Comparative Endocrinology 02/2010; 168(2):174-80. · 3.27 Impact Factor
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ABSTRACT: The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established in the so-called postembryonic developmental period in mammals when endogenous thyroid hormone (T3) levels are high.
The T3-dependent metamorphosis in anurans like Xenopus laevis resembles the mammalian postembryonic development and offers a unique opportunity to study how the adult stem cells are developed. The tadpole intestine is predominantly a monolayer of larval epithelial cells. During metamorphosis, the larval epithelial cells undergo apoptosis and, concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well-established stem cell marker in the adult mouse intestinal crypt. Here we have cloned and analyzed the spatiotemporal expression profile of LGR5 gene during frog metamorphosis. We show that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by T3 during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine.
These results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates.
PLoS ONE 01/2010; 5(10):e13605. · 4.09 Impact Factor
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ABSTRACT: The cellular prion protein (PrP(C)) is highly conserved in the evolution of mammals, and therefore, thought to have important cellular functions. Despite decades of intensive research, the physiological function of PrP(C) remains enigmatic. We carried out a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the transmembrane protein tetraspanin-7 (CD231), as a PrP(C) interacting protein. We confirmed the interaction between PrP(C) and tetraspanin-7 by yeast two-hybrid assay, immunofluorescent co-localization, and immunocoprecipitation. Our mutational studies further demonstrated that PrP(C) specifically binds tetraspanin-7 through the region corresponding to bovine PrP(154-182) containing alpha-helix 1.
Biochemical and Biophysical Research Communications 02/2008; 365(1):154-7. · 2.48 Impact Factor
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ABSTRACT: We used a bovine brain cDNA library to perform a yeast two-hybrid assay with bovine mature PrP(C) as bait. The screening result showed that alphaB-crystalline interacted with PrP(C). The interaction was further evaluated both in vivo and in vitro with different methods, such as immunofluorescent colocalization, native polyacrylamide-gel electrophoresis, and IAsys biosensor assays. The results suggested that alphaB-crystalline may have the ability to refold denatured prion proteins, and provided first evidence that alphaB-crystalline is directly associated with prion protein.
FEBS Letters 11/2005; 579(24):5419-24. · 3.54 Impact Factor
