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Sample records for laevis morphogenetic factor

  1. Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development.

    PubMed

    Dale, L; Howes, G; Price, B M; Smith, J C

    1992-06-01

    The mesoderm of amphibian embryos such as Xenopus laevis arises through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying equatorial and animal pole cells. Three classes of 'mesoderm-inducing factor' (MIF) that might be responsible for this interaction in vivo have been discovered. These are members of the transforming growth factor type beta (TGF-beta), fibroblast growth factor (FGF) and Wnt families. Among the most potent MIFs are the activins, members of the TGF-beta family, but RNA for activin A and B is not detectable in the Xenopus embryo until neurula and late blastula stages, respectively, and this is probably too late for the molecules to act as natural inducers. In this paper, we use the polymerase chain reaction to clone additional members of the TGF-beta family that might possess mesoderm-inducing activity. We show that transcripts encoding Xenopus bone morphogenetic protein 4 (XBMP-4) are detectable in the unfertilized egg, and that injection of XBMP-4 RNA into the animal hemisphere of Xenopus eggs causes animal caps isolated from the resulting blastulae to express mesoderm-specific markers. Surprisingly, however, XBMP-4 preferentially induces ventral mesoderm, whereas the closely related activin induces axial tissues. Furthermore, the action of XBMP-4 is 'dominant' over that of activin. In this respect, XBMP-4 differs from basic FGF, another ventral inducer, where simultaneous treatment with FGF and activin results in activin-like responses. The dominance of XBMP-4 over activin may account for the ability of injected XBMP-4 RNA to 'ventralize' whole Xenopus embryos. It is interesting, however, that blastopore formation in such embryos can occur perfectly normally. This contrasts with embryos ventralized by UV-irradiation and suggests that XBMP-4-induced ventralization occurs after the onset of gastrulation. PMID:1425340

  2. Effects of nutritional factors on the growth and heterotrophic eicosapentaenoic acid production of diatom Nitzschia laevis

    NASA Astrophysics Data System (ADS)

    Cao, Xiaohong; Li, Songyao; Wang, Chunling; Lu, Meifang

    2008-08-01

    The effects of several nutritional factors on the growth and eicosapentaenoic acid (EPA) production of diatom Nitzschia laevis were studied. 4 LDM (quadrupled concentration of the nutrient salt) was the optimal concentration of nutrient salt for the growth and EPA production of N. laevis. The growth of N. laevis was inhibited when the glucose concentration was either lower than 10 gL-1 or higher than 15 gL-1. Both sodium nitrate and urea were good nitrogen sources for the growth and EPA production, while ammonium chloride seriously decreased the dry cell weight (DW) and the EPA content. Silicate seriously influenced the growth of N. laevis. The maximum DW of 2.34 gL-1 was obtained in the presence of 150 mgL-1 Na2SiO3·9H2O. The EPA content remained almost the same when the silicate concentration was lower than 150 mgL-1; however, higher silicate concentrations resulted in a steady decrease of EPA content. Low medium salinity (⩽29) did not seem to influence the DW of N. laevis, and high salinity resulted in a decrease of DW. The highest EPA content (4.08%) and yield (110 mgL-1) were observed at the salinity of 36 and 29, respectively.

  3. Mechanisms of amphibian macrophage development: characterization of the Xenopus laevis colony-stimulating factor-1 receptor.

    PubMed

    Grayfer, Leon; Edholm, Eva-Stina; Robert, Jacques

    2014-01-01

    Macrophage-lineage cells are indispensable to vertebrate homeostasis and immunity. In turn, macrophage development is largely regulated through colony-stimulating factor-1 (CSF1) binding to its cognate receptor (CSF1R). To study amphibian monopoiesis, we identified and characterized the X. laevis CSF1R cDNA transcript. Quantitative analysis revealed that CSF1R tissue gene expression increased with X. laevis development, with greatest transcript levels detected in the adult lung, spleen and liver tissues. Notably, considerable levels of CSF1R mRNA were also detected in the regressing tails of metamorphosing animals, suggesting macrophage involvement in this process, and in the adult bone marrow; corroborating the roles for this organ in Xenopus monopoiesis. Following animal infections with the ranavirus Frog Virus 3 (FV3), both tadpole and adult X. laevis exhibited increased kidney CSF1R gene expression. Conversely, while FV3-infected tadpoles increased their spleen and liver CSF1R mRNA levels, the FV3-challenged adults did not. Notably, FV3 induced elevated bone marrow CSF1R expression, and while stimulation of tadpoles with heat-killed E. coli had no transcriptional effects, bacterial stimulation of adult frogs resulted in significantly increased spleen, liver and bone marrow CSF1R expression. We produced the X. laevis CSF1R in recombinant form (rXlCSF1R) and determined, via in vitro cross-linking studies, that two molecules of rXlCSF1R bound the dimeric rXlCSF1. Finally, administration of rXlCSF1R abrogated the rXlCSF1-induced tadpole macrophage recruitment and differentiation as well as bacterial and FV3-elicited peritoneal leukocyte accumulation. This work marks a step towards garnering greater understanding of the unique mechanisms governing amphibian macrophage biology.

  4. Activin-like factor from a Xenopus laevis cell line responsible for mesoderm induction.

    PubMed

    van den Eijnden-Van Raaij, A J; van Zoelent, E J; van Nimmen, K; Koster, C H; Snoek, G T; Durston, A J; Huylebroeck, D

    1990-06-21

    Induction of mesoderm during early amphibian embryogenesis can be mimicked in vitro by adding growth factors, including heparin-binding and type-beta transforming growth factors (TGF-beta), to isolated ectoderm explants from Xenopus laevis embryos. Although the mesoderm-inducing factor (MIF) from X. laevis XTC cells (XTC-MIF) has properties similar to TGF-beta, this factor is still unidentified. Recently, we obtained a number of homogeneous cell lines from the heterogeneous XTC population, which differ in their MIF production. Only one, XTC-GTX-11, produced MIF, although it was similar to the rest of the clones in its production of known growth factors, including TGF-beta activity. This observation, together with the identification of activin A as a potent MIF led us to study the parallel activities of MIF and activin. Here we report an analysis of activin-like activity from XTC cells and some of the XTC clones, including XTC-GTX-11. There is a clear consistent correlation between MIF activity and presence of activin activity, indicating that XTC-MIF is the Xenopus homologue of mammalian activin.

  5. Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation- regulated protein essential for development

    PubMed Central

    1996-01-01

    Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST- fusion proteins have pH-dependent actin-depolymerizing and F-actin- binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration- dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for

  6. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

    SciTech Connect

    Montesano, Roberto Sarkoezi, Rita; Schramek, Herbert

    2008-09-12

    Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

  7. Xenopus laevis as a novel model to study long bone critical-size defect repair by growth factor-mediated regeneration.

    PubMed

    Feng, Liang; Milner, Derek J; Xia, Chunguang; Nye, Holly L D; Redwood, Patrick; Cameron, Jo Ann; Stocum, David L; Fang, Nick; Jasiuk, Iwona

    2011-03-01

    We used the tarsus of an adult Xenopus laevis frog as an in vivo load-bearing model to study the regeneration of critical-size defects (CSD) in long bones. We found the CSD for this bone to be about 35% of the tarsus length. To promote regeneration, we implanted biocompatible 1,6 hexanediol diacrylate scaffolds soaked with bone morphogenetic proteins-4 and vascular endothelial growth factors. In contrast to studies that use scaffolds as templates for bone formation, we used scaffolds as a growth factor delivery vehicle to promote cartilage-to-bone regeneration. Defects in control frogs were filled with scaffolds lacking growth factors. The limbs were harvested at a series of time points ranging from 3 weeks to 6 months after implantation and evaluated using micro-computed tomography and histology. In frogs treated with growth factor-loaded scaffolds, we observed a cartilage-to-bone regeneration in the skeletal defect. Five out of eight defects were completely filled with cartilage by 6 weeks. Blood vessels had invaded the cartilage, and bone was beginning to form in ossifying centers. By 3 months, these processes were well advanced, and extensive ossification was observed in 6-month samples. In contrast, the defects in control frogs showed only formation of fibrous scar tissue. This study demonstrates the utility of a Xenopus model system for tissue engineering research and that the normal in vivo mechanism of endochondral bone development and fracture repair can be mimicked in the repair of CSD with scaffolds used as growth factor delivery mechanisms.

  8. Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells.

    PubMed

    Snoek, G T; Koster, C H; de Laat, S W; Heideveld, M; Durston, A J; van Zoelen, E J

    1990-04-01

    The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF) beta-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGF beta-like activities at an apparent molecular weight of 6-10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal explants. The relation between this activity and a recently described TGF beta-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.

  9. Crosstalk among electrical activity, trophic factors and morphogenetic proteins in the regulation of neurotransmitter phenotype specification.

    PubMed

    Borodinsky, Laura N; Belgacem, Yesser H

    2016-04-01

    Morphogenetic proteins are responsible for patterning the embryonic nervous system by enabling cell proliferation that will populate all the neural structures and by specifying neural progenitors that imprint different identities in differentiating neurons. The adoption of specific neurotransmitter phenotypes is crucial for the progression of neuronal differentiation, enabling neurons to connect with each other and with target tissues. Preliminary neurotransmitter specification originates from morphogen-driven neural progenitor specification through the combinatorial expression of transcription factors according to morphogen concentration gradients, which progressively restrict the identity that born neurons adopt. However, neurotransmitter phenotype is not immutable, instead trophic factors released from target tissues and environmental stimuli change expression of neurotransmitter-synthesizing enzymes and specific vesicular transporters modifying neuronal neurotransmitter identity. Here we review studies identifying the mechanisms of catecholaminergic, GABAergic, glutamatergic, cholinergic and serotonergic early specification and of the plasticity of these neurotransmitter phenotypes during development and in the adult nervous system. The emergence of spontaneous electrical activity in developing neurons recruits morphogenetic proteins in the process of neurotransmitter phenotype plasticity, which ultimately equips the nervous system and the whole organism with adaptability for optimal performance in a changing environment. PMID:26686293

  10. Brain-derived neurotrophic factor (BDNF) expression in normal and regenerating olfactory epithelium of Xenopus laevis.

    PubMed

    Frontera, Jimena Laura; Cervino, Ailen Soledad; Jungblut, Lucas David; Paz, Dante Agustín

    2015-03-01

    Olfactory epithelium has the capability to continuously regenerate olfactory receptor neurons throughout life. Adult neurogenesis results from proliferation and differentiation of neural stem cells, and consequently, olfactory neuroepithelium offers an excellent opportunity to study neural regeneration and the factors involved in the maintenance and regeneration of all their cell types. We analyzed the expression of BDNF in the olfactory system under normal physiological conditions as well as during a massive regeneration induced by chemical destruction of the olfactory epithelium in Xenopus laevis larvae. We described the expression and presence of BDNF in the olfactory epithelium and bulb. In normal physiological conditions, sustentacular (glial) cells and a few scattered basal (stem) cells express BDNF in the olfactory epithelium as well as the granular cells in the olfactory bulb. Moreover, during massive regeneration, we demonstrated a drastic increase in basal cells expressing BDNF as well as an increase in BDNF in the olfactory bulb and nerve. Together these results suggest an important role of BDNF in the maintenance and regeneration of the olfactory system.

  11. Williams Syndrome Transcription Factor is critical for neural crest cell function in Xenopus laevis

    PubMed Central

    Barnett, Chris; Yazgan, Oya; Kuo, Hui-Ching; Malakar, Sreepurna; Thomas, Trevor; Fitzgerald, Amanda; Harbour, Billy; Henry, Jonathan J.; Krebs, Jocelyn E.

    2012-01-01

    Williams Syndrome Transcription Factor (WSTF) is one of ~25 haplodeficient genes in patients with the complex developmental disorder Williams Syndrome (WS). WS results in visual/spatial processing defects, cognitive impairment, unique behavioral phenotypes, characteristic “elfin” facial features, low muscle tone and heart defects. WSTF exists in several chromatin remodeling complexes and has roles in transcription, replication, and repair. Chromatin remodeling is essential during embryogenesis, but WSTF’s role in vertebrate development is poorly characterized. To investigate the developmental role of WSTF, we knocked down WSTF in Xenopus laevis embryos using a morpholino that targets WSTF mRNA. BMP4 shows markedly increased and spatially aberrant expression in WSTF-deficient embryos, while SHH, MRF4, PAX2, EPHA4 and SOX2 expression are severely reduced, coupled with defects in a number of developing embryonic structures and organs. WSTF-deficient embryos display defects in anterior neural development. Induction of the neural crest, measured by expression of the neural crest-specific genes SNAIL and SLUG, is unaffected by WSTF depletion. However, at subsequent stages WSTF knockdown results in a severe defect in neural crest migration and/or maintenance. Consistent with a maintenance defect, WSTF knockdowns display a specific pattern of increased apoptosis at the tailbud stage in regions corresponding to the path of cranial neural crest migration. Our work is the first to describe a role for WSTF in proper neural crest function, and suggests that neural crest defects resulting from WSTF haploinsufficiency may be a major contributor to the pathoembryology of WS. PMID:22691402

  12. Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription.

    PubMed Central

    Bogenhagen, D F; Insdorf, N F

    1988-01-01

    The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor eluted from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA(Phe) and the displacement loop. Images PMID:2457154

  13. Several developmental and morphogenetic factors govern the evolution of stomatal patterning in land plants.

    PubMed

    Rudall, Paula J; Hilton, Jason; Bateman, Richard M

    2013-11-01

    We evaluate stomatal development in terms of its primary morphogenetic factors and place it in a phylogenetic context, including clarification of the contrasting specialist terms that are used by different sets of researchers. The genetic and structural bases for stomatal development are well conserved and increasingly well understood in extant taxa, but many phylogenetically crucial plant lineages are known only from fossils, in which it is problematic to infer development. For example, specialized lateral subsidiary cells that occur adjacent to the guard cells in some taxa can be derived either from the same cell lineage as the guard cells or from an adjacent cell file. A potentially key factor in land-plant evolution is the presence (mesogenous type) or absence (perigenous type) of at least one asymmetric division in the cell lineage leading to the guard-mother cell. However, the question whether perigenous or mesogenous development is ancestral in land plants cannot yet be answered definitively based on existing data. Establishment of 'fossil fingerprints' as developmental markers is critical for understanding the evolution of stomatal patterning. Long cell-short cell alternation in the developing leaf epidermis indicates that the stomata are derived from an asymmetric mitosis. Other potential developmental markers include nonrandom stomatal orientation and a range of variation in relative sizes of epidermal cells. Records of occasional giant stomata in fossil bennettites could indicate development of a similar type to early-divergent angiosperms.

  14. Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions.

    PubMed

    Peng, Jia; Li, Qinglei; Wigglesworth, Karen; Rangarajan, Adithya; Kattamuri, Chandramohan; Peterson, Randall T; Eppig, John J; Thompson, Thomas B; Matzuk, Martin M

    2013-02-19

    The TGF-β superfamily is the largest family of secreted proteins in mammals, and members of the TGF-β family are involved in most developmental and physiological processes. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), oocyte-secreted paralogs of the TGF-β superfamily, have been shown genetically to control ovarian physiology. Although previous studies found that GDF9 and BMP15 homodimers can modulate ovarian pathways in vitro, the functional species-specific significance of GDF9:BMP15 heterodimers remained unresolved. Therefore, we engineered and produced purified recombinant mouse and human GDF9 and BMP15 homodimers and GDF9:BMP15 heterodimers to compare their molecular characteristics and physiological functions. In mouse granulosa cell and cumulus cell expansion assays, mouse GDF9 and human BMP15 homodimers can up-regulate cumulus expansion-related genes (Ptx3, Has2, and Ptgs2) and promote cumulus expansion in vitro, whereas mouse BMP15 and human GDF9 homodimers are essentially inactive. However, we discovered that mouse GDF9:BMP15 heterodimer is ∼10- to 30-fold more biopotent than mouse GDF9 homodimer, and human GDF9:BMP15 heterodimer is ∼1,000- to 3,000-fold more bioactive than human BMP15 homodimer. We also demonstrate that the heterodimers require the kinase activities of ALK4/5/7 and BMPR2 to activate SMAD2/3 but unexpectedly need ALK6 as a coreceptor in the signaling complex in granulosa cells. Our findings that GDF9:BMP15 heterodimers are the most bioactive ligands in mice and humans compared with homodimers explain many puzzling genetic and physiological data generated during the last two decades and have important implications for improving female fertility in mammals.

  15. Maintenance of Multipotency in Human Dermal Fibroblasts Treated with Xenopus laevis Egg Extract Requires Exogenous Fibroblast Growth Factor-2

    PubMed Central

    Kole, Denis; Ambady, Sakthikumar; Page, Raymond L.

    2014-01-01

    Abstract Direct reprogramming of a differentiated somatic cell into a developmentally more plastic cell would offer an alternative to applications in regenerative medicine that currently depend on either embryonic stem cells (ESCs), adult stem cells, or induced pluripotent stem cells (iPSCs). Here we report the potential of select Xenopus laevis egg extract fractions, in combination with exogenous fibroblast growth factor-2 (FGF2), to affect life span, morphology, gene expression, protein translation, and cellular localization of OCT4 and NANOG transcription factors, and the developmental potential of human dermal fibroblasts in vitro. A gradual change in morphology is accompanied by translation of embryonic transcription factors and their nuclear localization and a life span exceeding 60 population doublings. Cells acquire the ability to follow adipogenic, neuronal, and osteogenic differentiation under appropriate induction conditions in vitro. Analysis of active extract fractions reveals that Xenopus egg protein and RNAs as well as exogenously supplemented FGF2 are required and sufficient for induction and maintenance of this phenotypic change. Factors so far identified in the active fractions include FGF2 itself, transforming growth factor-β, maskin, and nucleoplasmin. Identification of critical factors needed for reprogramming may allow for nonviral, chemically defined derivation of human-induced multipotent cells that can be maintained by exogenous FGF2. PMID:24405062

  16. Synergistic Effects of Hypoxia and Morphogenetic Factors on Early Chondrogenic Commitment of Human Embryonic Stem Cells in Embryoid Body Culture

    PubMed Central

    Yodmuang, Supansa; Marolt, Darja; Marcos-Campos, Ivan; Gadjanski, Ivana

    2015-01-01

    Derivation of articular chondrocytes from human stem cells would advance our current understanding of chondrogenesis, and accelerate development of new stem cell therapies for cartilage repair. Chondrogenic differentiation of human embryonic stem cells (hESCs) has been studied using supplemental and cell-secreted morphogenetic factors. The use of bioreactors enabled insights into the effects of physical forces and controlled oxygen tension. In this study, we investigated the interactive effects of controlled variation of oxygen tension and chondrocyte-secreted morphogenetic factors on chondrogenic differentiation of hESCs in the embryoid body format (hESC-EB). Transient hypoxic culture (2 weeks at 5 % O2 followed by 1 week at 21 % O2) of hESC-EBs in medium conditioned with primary chondrocytes up-regulated the expression of SOX9 and suppressed pluripotent markers OCT4 and NANOG. Pellets derived from these cells showed significant up-regulation of chondrogenic genes (SOX9, COL2A1, ACAN) and enhanced production of cartilaginous matrix (collagen type II and proteoglycan) as compared to the pellets from hESC-EBs cultured under normoxic conditions. Gene expression profiles corresponded to those associated with native cartilage development, with early expression of N-cadherin (indicator of cell condensation) and late expression of aggrecan (ACAN, indicator of proteoglycan production). When implanted into highly vascularized subcutaneous area in immunocompromised mice for 4 weeks, pellets remained phenotypically stable and consisted of cartilaginous extracellular matrix (ECM), without evidence of dedifferentiation or teratoma formation. Based on these results, we propose that chondrogenesis in hESC can be synergistically enhanced by a control of oxygen tension and morphogenetic factors secreted by chondrocytes. PMID:25618295

  17. Synergistic effects of hypoxia and morphogenetic factors on early chondrogenic commitment of human embryonic stem cells in embryoid body culture.

    PubMed

    Yodmuang, Supansa; Marolt, Darja; Marcos-Campos, Ivan; Gadjanski, Ivana; Vunjak-Novakovic, Gordana

    2015-04-01

    Derivation of articular chondrocytes from human stem cells would advance our current understanding of chondrogenesis, and accelerate development of new stem cell therapies for cartilage repair. Chondrogenic differentiation of human embryonic stem cells (hESCs) has been studied using supplemental and cell-secreted morphogenetic factors. The use of bioreactors enabled insights into the effects of physical forces and controlled oxygen tension. In this study, we investigated the interactive effects of controlled variation of oxygen tension and chondrocyte-secreted morphogenetic factors on chondrogenic differentiation of hESCs in the embryoid body format (hESC-EB). Transient hypoxic culture (2 weeks at 5 % O2 followed by 1 week at 21 % O2) of hESC-EBs in medium conditioned with primary chondrocytes up-regulated the expression of SOX9 and suppressed pluripotent markers OCT4 and NANOG. Pellets derived from these cells showed significant up-regulation of chondrogenic genes (SOX9, COL2A1, ACAN) and enhanced production of cartilaginous matrix (collagen type II and proteoglycan) as compared to the pellets from hESC-EBs cultured under normoxic conditions. Gene expression profiles corresponded to those associated with native cartilage development, with early expression of N-cadherin (indicator of cell condensation) and late expression of aggrecan (ACAN, indicator of proteoglycan production). When implanted into highly vascularized subcutaneous area in immunocompromised mice for 4 weeks, pellets remained phenotypically stable and consisted of cartilaginous extracellular matrix (ECM), without evidence of dedifferentiation or teratoma formation. Based on these results, we propose that chondrogenesis in hESC can be synergistically enhanced by a control of oxygen tension and morphogenetic factors secreted by chondrocytes.

  18. Natural variation in embryo mechanics: gastrulation in Xenopus laevis is highly robust to variation in tissue stiffness

    PubMed Central

    von Dassow, Michelangelo; Davidson, Lance A.

    2009-01-01

    How sensitive is morphogenesis to the mechanical properties of embryos? To estimate an upper bound on the sensitivity of early morphogenetic movements to tissue mechanical properties, we assessed natural variability in the apparent stiffness among gastrula-stage Xenopus laevis embryos. We adapted micro-aspiration methods to make repeated, non-destructive measurements of apparent tissue stiffness in whole embryos. Stiffness varied by close to a factor of 2 among embryos within a single clutch. Variation between clutches was of similar magnitude. On the other hand, the direction of change in stiffness over the course of gastrulation was the same in all embryos and in all clutches. Neither pH nor salinity – two environmental factors we predicted could affect variability in nature – affected tissue stiffness. Our results indicate that gastrulation in X. laevis is robust to at least two-fold variation in tissue stiffness. PMID:19097119

  19. Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

    SciTech Connect

    Aranguren, Xabier L.; Beerens, Manu; Vandevelde, Wouter; Dewerchin, Mieke; Carmeliet, Peter; Luttun, Aernout

    2011-06-24

    Highlights: {yields} COUP-TFII deficiency in zebrafish affects arterio-venous EC specification. {yields} COUP-TFII is indispensable for lymphatic development in zebrafish. {yields} COUP-TFII knockdown in Xenopus disrupts lymphatic EC differentiation and migration. {yields} COUP-TFII's role in EC fate decisions is evolutionary conserved. -- Abstract: Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment of venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.

  20. Bone morphogenetic protein

    SciTech Connect

    Xiao Yongtao; Xiang Lixin; Shao Jianzhong

    2007-10-26

    Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor-beta superfamily. It has been demonstrated that BMPs had been involved in the regulation of cell proliferation, survival, differentiation and apoptosis. However, their hallmark ability is that play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. In this review, we mainly concentrate on BMP structure, function, molecular signaling and potential medical application.

  1. Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma

    PubMed Central

    Yoshida, Daizo; Kim, Kyongsong; Ishii, Yudo; Tahara, Shigeyuki; Teramoto, Akira; Morita, Akio

    2015-01-01

    Gremlin is an antagonist of bone morphogenetic protein (BMP) and a major driving force in skeletal modeling in the fetal stage. Several recent reports have shown that Gremlin is also involved in angiogenesis of lung cancer and diabetic retinopathy. The purpose of this study was to investigate the role of Gremlin in tumor angiogenesis in pituitary adenoma. Double fluorescence immunohistochemistry of Gremlin and CD34 was performed in pituitary adenoma tissues obtained during transsphenoidal surgery in 45 cases (7 PRLoma, 17 GHoma, 2 ACTHoma, and 2 TSHoma). Gremlin and microvascular density (MVD) were detected by double-immunofluorescence microscopy in CD34-positive vessels from tissue microarray analysis of 60 cases of pituitary adenomas (6 PRLoma, 23 GHoma, 22 NFoma, 5 ACTHoma, and 4 TSHoma). In tissue microarray analysis, MVD was significantly correlated with an increased Gremlin level (linear regression: P < 0.005,  r2 = 0.4958). In contrast, Gremlin expression showed no correlation with tumor subtype or Knosp score. The high level of expression of Gremlin in pituitary adenoma tissue with many CD34-positive vessels and the strong coherence of these regions indicate that Gremlin is associated with angiogenesis in pituitary adenoma cells. PMID:25834571

  2. Blood cell induction in Xenopus animal cap explants: effects of fibroblast growth factor, bone morphogenetic proteins, and activin.

    PubMed

    Miyanaga, Y; Shiurba, R; Asashima, M

    1999-02-01

    Cultures of Xenopus blastula animal caps were used to explore the haematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expanded into egg-shaped structures, and beneath an outer layer of epidermis, a ventral mesodermal lining surrounded a fluid-filled cavity containing "blood-like cells". Immunocytochemistry identified some of these cells as early leukocytes, but erythrocytes were rare. BMP-2 or BMP-4 induced primitive erythrocytes as well as leukocytes, and a high concentration was required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induced ventral mesoderm concomitantly. High concentrations of activin A dorsalized explants, which contained infrequent leukocytes, and an optimal combination of activin A and bFGF caused differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increased the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contained a well organized cell mass in which yolk-rich cells mixed with blood cells and pigmented cells did not. BMP-2 plus bFGF also induced numerous leukocytes and fewer erythrocytes, but BMP-4 antagonized the leukopoietic effect of bFGF. The data suggest that the signalling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination.

  3. Transcriptional regulation of gilthead seabream bone morphogenetic protein (BMP) 2 gene by bone- and cartilage-related transcription factors.

    PubMed

    Marques, Cátia L; Cancela, M Leonor; Laizé, Vincent

    2016-01-15

    Bone morphogenetic protein (BMP) 2 belongs to the transforming growth factor β (TGFβ) superfamily of cytokines and growth factors. While it plays important roles in embryo morphogenesis and organogenesis, BMP2 is also critical to bone and cartilage formation. Protein structure and function have been remarkably conserved throughout evolution and BMP2 transcription has been proposed to be tightly regulated, although few data is available. In this work we report the cloning and functional analysis of gilthead seabream BMP2 promoter. As in other vertebrates, seabream BMP2 gene has a 5′ non-coding exon, a feature already present in DPP gene, the fruit fly ortholog of vertebrate BMP2 gene, and maintained throughout evolution. In silico analysis of seabream BMP2 promoter revealed several binding sites for bone and cartilage related transcription factors (TFs) and their functionality was evaluated using promoter-luciferase constructions and TF-expressing vectors. Runt-related transcription factor 3 (RUNX3) was shown to negatively regulate BMP2 transcription and combination with the core binding factor β (CBFβ) further reduced transcriptional activity of the promoter. Although to a lesser extent, myocyte enhancer factor 2C (MEF2C) had also a negative effect on the regulation of BMP2 gene transcription, when associated with SRY (sex determining region Y)-box 9 (SOX9b). Finally, v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) was able to slightly enhance BMP2 transcription. Data reported here provides new insights toward the better understanding of the transcriptional regulation of BMP2 gene in a bone and cartilage context. PMID:26456102

  4. Colony-Stimulating Factor-1-Responsive Macrophage Precursors Reside in the Amphibian (Xenopus laevis) Bone Marrow Rather than the Hematopoietic Sub-Capsular Liver

    PubMed Central

    Grayfer, Leon; Robert, Jacques

    2013-01-01

    Macrophage precursors originate from, and undergo lineage commitment within designated sites of hematopoiesis, such as the mammalian bone marrow. These cells subsequently differentiate in response to stimulation with macrophage colony-stimulating factor (CSF-1). The amphibian bone marrow, unlike that of mammals, has been overlooked as a source of leukocyte precursors in favor of the liver sub-capsular region, where hematopoiesis occurs in anurans. Here we report that the bone marrow rather than the liver periphery provides macrophage progenitors to the amphibian Xenopus laevis. We identified the amphibian CSF-1, examined its gene expression in developing and virally infected X. laevis and produce it in recombinant form (rXlCSF-1). This rXlCSF-1 did not bind or elicit proliferation/differentiation of sub-cortical liver cells. Surprisingly, a sub-population of bone marrow cells engaged this growth factor and formed rXlCSF-1-concentration-dependant colonies in semi-solid medium. Furthermore, rXlCSF-1-treated bone marrow (but not liver) cultures comprised of cells with characteristic macrophage morphology and high gene expression of the macrophage marker, colony stimulating factor-1 receptor (CSF-1R). Together, our findings indicate that in contrast to all other vertebrates studied to date, Xenopus committed macrophage precursors populations are not present in the central site of hematopoiesis, but reside in the bone marrow. PMID:23485675

  5. The two nonallelic insulin-like growth factor-I genes in Xenopus laevis are differentially regulated during development.

    PubMed

    Perfetti, R; Scott, L A; Shuldiner, A R

    1994-11-01

    To study the potential role of insulin-like growth factor-I (IGF-I) during early embryogenesis, we have used the amphibian Xenopus laevis, a versatile model of vertebrate development. Using polymerase chain reaction (PCR)-based cloning strategies, we have previously identified two different nonallelic Xenopus IGF-I genes (IGF-I' and IGF-I"). Both are expressed in similar quantities in adult liver. We now report the use of a modification of the reverse transcription-PCR method, designated RNA template-specific PCR (RS-PCR), to detect IGF-I messenger RNA (mRNA) from single oocytes and embryos. The same primer pair was used to amplify both IGF-I' and IGF-I" mRNAs. Slot blot analysis of the RS-PCR products with internal oligonucleotide probes that specifically recognize IGF-I' or IGF-I" sequences revealed that only IGF-I' mRNA was present in follicles surrounding mature (stage VI) oocytes; neither IGF-I mRNA was present in follicles surrounding less mature oocytes (stages I and IV) or within oocytes or unfertilized eggs. After fertilization, IGF-I' mRNA was first detected during early organogenesis (stages 21-23), and increased during subsequent stages of development. To localize early IGF-I' expression, a stage 27 embryo was sliced into 24-microns cross-sections; RS-PCR and slot blot analysis were performed on RNA extracts from consecutive sections. IGF-I' mRNA was expressed in all sections, but was most abundant in the body region from which the visceral organs were developing. In contrast, to the early expression of IGF-I', IGF-I" mRNA was not detected until stage 41, a period corresponding to premetamorphic growth. Reverse PCR for Xenopus GH mRNA demonstrated that the onset of GH gene expression was coincident with the onset of IGF-I" gene expression (stage 41). These data suggest that the early expression of IGF-I' may be GH independent, whereas later expression of IGF-I" is GH dependent. We conclude that the two nonallelic IGF-I genes are expressed differentially

  6. The factor VII-activating protease (FSAP) enhances the activity of bone morphogenetic protein-2 (BMP-2).

    PubMed

    Roedel, Elfie Kathrin; Schwarz, Elisabeth; Kanse, Sandip Madhav

    2013-03-01

    Factor VII-activating protease (FSAP) is a circulating protease involved in the pathogenesis of atherosclerosis, calcification, and fibrotic processes. To understand how FSAP controls the balance of local growth factors, we have investigated its effect on the regulation of bone morphogenetic proteins (BMPs). BMP-2 is produced as a large pro-form and secreted as a mature heparin-binding growth factor after intracellular processing by pro-protein convertases (PCs). In this study, we discovered that FSAP enhances the biological activity of mature BMP-2 as well as its pro-form, as shown by osteogenic differentiation of C2C12 myoblasts. These findings were complemented by knockdown of FSAP in hepatocytes, which revealed BMP-2 processing by endogenous FSAP. N-terminal sequencing indicated that pro-BMP-2 was cleaved by FSAP at the canonical PC cleavage site, giving rise to mature BMP-2 (Arg(282)↓Gln(283)), as well as in the N-terminal heparin binding region of mature BMP-2, generating a truncated mature BMP-2 peptide (Arg(289)↓Lys(290)). Similarly, mature BMP-2 was also cleaved to a truncated peptide within its N-terminal region (Arg(289)↓Lys(290)). Plasmin exhibited a similar activity, but it was weaker compared with FSAP. Thrombin, Factor VIIa, Factor Xa, and activated protein C were not effective. These results were further supported by the observation that the mutation of the heparin binding region of BMP-2 inhibited the processing by FSAP but not by PC. Thus, the proteolysis and activation of pro-BMP-2 and mature BMP-2 by FSAP can regulate cell differentiation and calcification in vasculature and may explain why polymorphisms in the gene encoding for FSAP are related to vascular diseases. PMID:23341458

  7. The Factor VII-activating Protease (FSAP) Enhances the Activity of Bone Morphogenetic Protein-2 (BMP-2)*

    PubMed Central

    Roedel, Elfie Kathrin; Schwarz, Elisabeth; Kanse, Sandip Madhav

    2013-01-01

    Factor VII-activating protease (FSAP) is a circulating protease involved in the pathogenesis of atherosclerosis, calcification, and fibrotic processes. To understand how FSAP controls the balance of local growth factors, we have investigated its effect on the regulation of bone morphogenetic proteins (BMPs). BMP-2 is produced as a large pro-form and secreted as a mature heparin-binding growth factor after intracellular processing by pro-protein convertases (PCs). In this study, we discovered that FSAP enhances the biological activity of mature BMP-2 as well as its pro-form, as shown by osteogenic differentiation of C2C12 myoblasts. These findings were complemented by knockdown of FSAP in hepatocytes, which revealed BMP-2 processing by endogenous FSAP. N-terminal sequencing indicated that pro-BMP-2 was cleaved by FSAP at the canonical PC cleavage site, giving rise to mature BMP-2 (Arg282↓Gln283), as well as in the N-terminal heparin binding region of mature BMP-2, generating a truncated mature BMP-2 peptide (Arg289↓Lys290). Similarly, mature BMP-2 was also cleaved to a truncated peptide within its N-terminal region (Arg289↓Lys290). Plasmin exhibited a similar activity, but it was weaker compared with FSAP. Thrombin, Factor VIIa, Factor Xa, and activated protein C were not effective. These results were further supported by the observation that the mutation of the heparin binding region of BMP-2 inhibited the processing by FSAP but not by PC. Thus, the proteolysis and activation of pro-BMP-2 and mature BMP-2 by FSAP can regulate cell differentiation and calcification in vasculature and may explain why polymorphisms in the gene encoding for FSAP are related to vascular diseases. PMID:23341458

  8. Control of phenotypic plasticity of smooth muscle cells by bone morphogenetic protein signaling through the myocardin-related transcription factors.

    PubMed

    Lagna, Giorgio; Ku, Manching M; Nguyen, Peter H; Neuman, Nicole A; Davis, Brandi N; Hata, Akiko

    2007-12-21

    Vascular smooth muscle cells (VSMCs), unlike other muscle cells, do not terminally differentiate. In response to injury, VSMCs change phenotype, proliferate, and migrate as part of the repair process. Dysregulation of this plasticity program contributes to the pathogenesis of several vascular disorders, such as atherosclerosis, restenosis, and hypertension. The discovery of mutations in the gene encoding BMPRII, the type II subunit of the receptor for bone morphogenetic proteins (BMPs), in patients with pulmonary arterial hypertension (PAH) provided an indication that BMP signaling may affect the homeostasis of VSMCs and their phenotype modulation. Here we report that BMP signaling potently induces SMC-specific genes in pluripotent cells and prevents dedifferentiation of arterial SMCs. The BMP-induced phenotype switch requires intact RhoA/ROCK signaling but is not blocked by inhibitors of the TGFbeta and PI3K/Akt pathways. Furthermore, nuclear localization and recruitment of the myocardin-related transcription factors (MRTF-A and MRTF-B) to a smooth muscle alpha-actin promoter is observed in response to BMP treatment. Thus, BMP signaling modulates VSMC phenotype via cross-talk with the RhoA/MRTFs pathway, and may contribute to the development of the pathological characteristics observed in patients with PAH and other obliterative vascular diseases. PMID:17947237

  9. Effects of growth differentiation factor 9 and bone morphogenetic protein 15 on the in vitro maturation of porcine oocytes.

    PubMed

    Lin, Z-L; Li, Y-H; Xu, Y-N; Wang, Q-L; Namgoong, S; Cui, X-S; Kim, N-H

    2014-04-01

    Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-β (TGF-β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

  10. Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34

    PubMed Central

    Grayfer, Leon; Robert, Jacques

    2014-01-01

    Macrophages are integral to amphibian immunity against RVs, as well as to the infection strategies of these pathogens. Although CSF-1 was considered to be the principal mediator of macrophage development, the IL-34 cytokine, which shares no sequence identity with CSF-1, is now believed to contribute to vertebrate monopoiesis. However, the respective roles of CSF-1- and IL-34-derived macrophages are still poorly understood. To delineate the contribution of these macrophage populations to amphibian immunity against the RV FV3, we identified the Xenopus laevis IL-34 and transcriptionally and functionally compared this cytokine with the previously identified X. laevis CSF-1. The X. laevis CSF-1 and IL-34 displayed strikingly nonoverlapping developmental and tissue-specific gene-expression patterns. Furthermore, only CSF-1 but not IL-34 was up-regulated in the kidneys of FV3-challenged tadpoles. Intriguingly, recombinant forms of these cytokines (rXlCSF-1, rXlIL-34) elicited morphologically distinct tadpole macrophages, and whereas rXlCSF-1 pretreatment decreased the survival of FV3-infected tadpoles, rXlIL-34 administration significantly prolonged FV3-challenged animal survival. Compared with rXlIL-34-elicited macrophages, macrophages derived by rXlCSF-1 were more phagocytic but also significantly more susceptible to in vitro FV3 infections. By contrast, rXlIL-34-derived macrophages exhibited significantly greater in vitro antiranaviral activity and displayed substantially more robust gene expression of the NADPH oxidase components (p67phox, gp91phox) and type I IFN. Moreover, FV3-challenged, rXlIL-34-derived macrophages exhibited several orders of magnitude greater up-regulation of the type I IFN gene expression. This marks the first report of the disparate roles of CSF-1 and IL-34 in vertebrate antiviral immunity. PMID:25190077

  11. Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.

    PubMed

    Grayfer, Leon; Robert, Jacques

    2014-12-01

    Macrophages are integral to amphibian immunity against RVs, as well as to the infection strategies of these pathogens. Although CSF-1 was considered to be the principal mediator of macrophage development, the IL-34 cytokine, which shares no sequence identity with CSF-1, is now believed to contribute to vertebrate monopoiesis. However, the respective roles of CSF-1- and IL-34-derived macrophages are still poorly understood. To delineate the contribution of these macrophage populations to amphibian immunity against the RV FV3, we identified the Xenopus laevis IL-34 and transcriptionally and functionally compared this cytokine with the previously identified X. laevis CSF-1. The X. laevis CSF-1 and IL-34 displayed strikingly nonoverlapping developmental and tissue-specific gene-expression patterns. Furthermore, only CSF-1 but not IL-34 was up-regulated in the kidneys of FV3-challenged tadpoles. Intriguingly, recombinant forms of these cytokines (rXlCSF-1, rXlIL-34) elicited morphologically distinct tadpole macrophages, and whereas rXlCSF-1 pretreatment decreased the survival of FV3-infected tadpoles, rXlIL-34 administration significantly prolonged FV3-challenged animal survival. Compared with rXlIL-34-elicited macrophages, macrophages derived by rXlCSF-1 were more phagocytic but also significantly more susceptible to in vitro FV3 infections. By contrast, rXlIL-34-derived macrophages exhibited significantly greater in vitro antiranaviral activity and displayed substantially more robust gene expression of the NADPH oxidase components (p67(phox), gp91(phox)) and type I IFN. Moreover, FV3-challenged, rXlIL-34-derived macrophages exhibited several orders of magnitude greater up-regulation of the type I IFN gene expression. This marks the first report of the disparate roles of CSF-1 and IL-34 in vertebrate antiviral immunity.

  12. Signalling pathways involved in the synergistic effects of human growth differentiation factor 9 and bone morphogenetic protein 15.

    PubMed

    Reader, Karen L; Mottershead, David G; Martin, Georgia A; Gilchrist, Robert B; Heath, Derek A; McNatty, Kenneth P; Juengel, Jennifer L

    2016-03-01

    Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) act synergistically to regulate granulosa cell proliferation and steroid production in several species. Several non-Sma and mothers against decapentaplegic (SMAD) signalling pathways are involved in the action of murine and ovine GDF9 and BMP15 in combination, with the pathways utilised differing between the two species. The aims of this research were to determine if human GDF9 and BMP15 also act in a synergistic manner to stimulate granulosa cell proliferation and to identify which non-SMAD signalling pathways are activated. Human GDF9 with BMP15 (GDF9+BMP15) stimulated an increase in (3)H-thymidine incorporation (P<0.001), which was greater than the increase with BMP15 alone, while GDF9 alone had no effect. The stimulation of (3)H-thymidine incorporation by GDF9+BMP15 was reduced by the addition of inhibitors to the SMAD2/3, nuclear factor-KB (NF-KB) and c-Jun N-terminal kinase (JNK) signalling pathways. Inhibitors to the SMAD1/5/8, extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK) or p38-MAPK pathways had no effect. The addition of the BMP receptor 2 (BMPR2) extracellular domain also inhibited stimulation of (3)H-thymidine incorporation by GDF9+BMP15. In conclusion, human GDF9 and BMP15 act synergistically to stimulate granulosa cell proliferation, a response that also involves species-specific non-SMAD signalling pathways.

  13. Identification of nuclear factors which interact with the 5' flanking region of the EF-1 alpha O gene in Xenopus laevis.

    PubMed

    Olesen, O F; Frydenberg, J

    1992-11-30

    The EF-1 alpha O gene of Xenopus laevis is a stage-specific gene, being transcribed in oogonia and oocytes, but not in postmeiotic germ cells and terminally differentiated cells. We found that two trans-acting factors from oocyte nuclear extract are able to interact with a DNA sequence in the 5'-upstream region of the EF-1 alpha O gene. Methylation interference experiments suggested that the two factors recognised the same DNA element. Gel retardation assays indicated that part of the protein binding site could be confined to a 21 bp sequence, located between -51 and -72, relative to the cap site. Interestingly, this region shares great homology to a negative regulatory segment in the promoter of the TFIIIA gene, another developmentally regulated gene. PMID:1446735

  14. Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor.

    PubMed

    Cooke, J; Symes, K; Smith, E J

    1989-03-01

    We have cultured explants of Xenopus blastular animal cap tissue from embryos that had received an earlier treatment with LiCl and from their untreated siblings, in various concentrations of XTC-cell-derived mesoderm-inducing factor (XTC-MIF, Smith, 1987; Smith et al. 1988). The pretreatment with lithium that we used transforms later morphogenesis in the whole embryo to give radialized body forms with anterior/dorsal levels of structure grossly over-represented. In addition, animal caps from 'Li+' embryos were allowed to develop without exposure to in vitro MIF (Li+ controls) and compared with normal uninduced control explants, and explants were made from normal early blastulae but given various initial treatments with LiCl in culture. The results confirm that the lithium ion itself will not induce mesoderm in competent, animal cap tissue of Xenopus. It does, however, enhance the responsiveness of this tissue to XTC-MIF, in a way that parallels its recently reported effect in the case of another mesoderm inducer of different character, bFGF (Slack et al. 1988). The effects observed are sufficient to imply that the altered body pattern that follows lithium treatment, in whole embryos, could be caused by modulation of the responses to an unaltered pattern of in situ inductive stimuli. We also observe evidence that appreciable inductive signals reach animal pole tissue beyond the limits of mesoderm formation in normal development. Relatively low concentrations of MIF prevent the development of an epidermis-specific marker in dissociated blastular animal cap cells (Symes et al. 1988). When such experiments are repeated in relation to the lithium pretreatment of embryos, such treatment is seen to have sensitized the cell population, so that the MIF concentration range that assures complete suppression of the marker is reduced. The results are discussed in relation to induction considered as pattern formation.

  15. Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages

    PubMed Central

    Grayfer, Leon; Robert, Jacques

    2015-01-01

    Although Mϕ represent the most primordial immune cell subsets, the mechanisms governing their functional heterogeneity remain poorly defined. However, it is well established that the CSF-1 cytokine contributes to monopoiesis and to this heterogeneity, whereas the unrelated IL-34 also binds the CSF-1R toward poorly understood immunologic roles. To delineate the molecular and evolutionary basis behind vertebrate Mϕ functional heterogeneity, we performed comprehensive transcriptional and functional studies of amphibian (Xenopus laevis) BM (in vitro) and PER (in vivo) Mϕ derived by rXlCSF-1 and rXlIL-34. Our findings indicate that these amphibian cytokines promote morphologically and functionally distinct Mϕ populations. Mϕ induced by rXlCSF-1 possess more robust iNOS gene expression, are substantially more phagocytic, display greater NO responses, and exhibit enhanced bactericidal capacities. By contrast, rXlIL-34-derived Mϕ express greater levels of Arg-1 and NADPH oxidase components and possess greater respiratory burst responses. Most notably, whereas CSF-1 Mϕ are highly susceptible to the emerging FV3 ranavirus, rXlIL-34 Mϕ exhibit potent antiviral activity against this pathogen, which is dependent on reactive oxygen production. This work marks an advance in our understanding of the possible mechanisms governing vertebrate Mϕ functional heterogeneity. PMID:26136505

  16. Cross-talk between bone morphogenetic proteins and inflammatory pathways.

    PubMed

    van der Kraan, Peter M; Davidson, Esmeralda N Blaney

    2015-11-23

    Pro-inflammatory cytokines and bone morphogenetic proteins are generally studied separately and considered to be elements of different worlds, immunology and developmental biology. Varas and colleagues report that these factors show cross-talk in rheumatoid arthritis synoviocytes. They show that pro-inflammatory cytokines not only stimulate the production of bone morphogenetic proteins but that these endogenously produced bone morphogenetic proteins interfere with the effects of pro-inflammatory cytokines on synoviocytes.

  17. Role of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Ovarian Function and Their Importance in Mammalian Female Fertility — A Review

    PubMed Central

    de Castro, Fernanda Cavallari; Cruz, Maria Helena Coelho; Leal, Claudia Lima Verde

    2016-01-01

    Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-β) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regulate absorption of amino acids, glycolysis and biosynthesis of cholesterol cumulus cells. Even though the mode of action has not been fully established, in vitro observations indicate that the factors GDF9 and BMP15 stimulate the growth of ovarian follicles and proliferation of cumulus cells through the induction of mitosis in cells and granulosa and theca expression of genes linked to follicular maturation. Thus, seeking greater understanding of the action of these growth factors on the development of oocytes, the role of GDF9 and BMP15 in ovarian function is summarized in this brief review. PMID:26954112

  18. Role of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Ovarian Function and Their Importance in Mammalian Female Fertility - A Review.

    PubMed

    de Castro, Fernanda Cavallari; Cruz, Maria Helena Coelho; Leal, Claudia Lima Verde

    2016-08-01

    Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-β) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regulate absorption of amino acids, glycolysis and biosynthesis of cholesterol cumulus cells. Even though the mode of action has not been fully established, in vitro observations indicate that the factors GDF9 and BMP15 stimulate the growth of ovarian follicles and proliferation of cumulus cells through the induction of mitosis in cells and granulosa and theca expression of genes linked to follicular maturation. Thus, seeking greater understanding of the action of these growth factors on the development of oocytes, the role of GDF9 and BMP15 in ovarian function is summarized in this brief review.

  19. Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation.

    PubMed

    Yasmin, Nighat; Bauer, Thomas; Modak, Madhura; Wagner, Karin; Schuster, Christopher; Köffel, Rene; Seyerl, Maria; Stöckl, Johannes; Elbe-Bürger, Adelheid; Graf, Daniel; Strobl, Herbert

    2013-11-18

    Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-β1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-β1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-β1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-β1-ALK5 signaling. Conversely, TGF-β1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-β1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-β1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-β1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-β1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.

  20. Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10.

    PubMed

    Susan-Resiga, Delia; Essalmani, Rachid; Hamelin, Josée; Asselin, Marie-Claude; Benjannet, Suzanne; Chamberland, Ann; Day, Robert; Szumska, Dorota; Constam, Daniel; Bhattacharya, Shoumo; Prat, Annik; Seidah, Nabil G

    2011-07-01

    Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.

  1. Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10*

    PubMed Central

    Susan-Resiga, Delia; Essalmani, Rachid; Hamelin, Josée; Asselin, Marie-Claude; Benjannet, Suzanne; Chamberland, Ann; Day, Robert; Szumska, Dorota; Constam, Daniel; Bhattacharya, Shoumo; Prat, Annik; Seidah, Nabil G.

    2011-01-01

    Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR316↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10. PMID:21550985

  2. GDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells.

    PubMed

    Ge, Gaoxiang; Hopkins, Delana R; Ho, Wen-Bin; Greenspan, Daniel S

    2005-07-01

    All transforming growth factor beta (TGF-beta) superfamily members are synthesized as precursors with prodomain sequences that are proteolytically removed by subtilisin-like proprotein convertases (SPCs). For most superfamily members, this is believed sufficient for activation. Exceptions are TGF-betas 1 to 3 and growth differentiation factor 8 (GDF8), also known as myostatin, which form noncovalent, latent complexes with their SPC-cleaved prodomains. Sequence similarities between TGF-betas 1 to 3, myostatin, and superfamily member GDF11, also known as bone morphogenetic protein 11 (BMP11), prompted us to examine whether GDF11 might be capable of forming a latent complex with its cleaved prodomain. Here we demonstrate that GDF11 forms a noncovalent latent complex with its SPC-cleaved prodomain and that this latent complex is activated via cleavage at a single specific site by members of the developmentally important BMP1/Tolloid family of metalloproteinases. Evidence is provided for a molecular model whereby formation and activation of this complex may play a general role in modulating neural differentiation. In particular, mutant GDF11 prodomains impervious to cleavage by BMP1/Tolloid proteinases are shown to be potent stimulators of neurodifferentiation, with potential for therapeutic applications.

  3. GDF11 Forms a Bone Morphogenetic Protein 1-Activated Latent Complex That Can Modulate Nerve Growth Factor-Induced Differentiation of PC12 Cells

    PubMed Central

    Ge, Gaoxiang; Hopkins, Delana R.; Ho, Wen-Bin; Greenspan, Daniel S.

    2005-01-01

    All transforming growth factor β (TGF-β) superfamily members are synthesized as precursors with prodomain sequences that are proteolytically removed by subtilisin-like proprotein convertases (SPCs). For most superfamily members, this is believed sufficient for activation. Exceptions are TGF-βs 1 to 3 and growth differentiation factor 8 (GDF8), also known as myostatin, which form noncovalent, latent complexes with their SPC-cleaved prodomains. Sequence similarities between TGF-βs 1 to 3, myostatin, and superfamily member GDF11, also known as bone morphogenetic protein 11 (BMP11), prompted us to examine whether GDF11 might be capable of forming a latent complex with its cleaved prodomain. Here we demonstrate that GDF11 forms a noncovalent latent complex with its SPC-cleaved prodomain and that this latent complex is activated via cleavage at a single specific site by members of the developmentally important BMP1/Tolloid family of metalloproteinases. Evidence is provided for a molecular model whereby formation and activation of this complex may play a general role in modulating neural differentiation. In particular, mutant GDF11 prodomains impervious to cleavage by BMP1/Tolloid proteinases are shown to be potent stimulators of neurodifferentiation, with potential for therapeutic applications. PMID:15988002

  4. Kartogenin, transforming growth factor-β1 and bone morphogenetic protein-7 coordinately enhance lubricin accumulation in bone-derived mesenchymal stem cells.

    PubMed

    Liu, Chun; Ma, Xueqin; Li, Tao; Zhang, Qiqing

    2015-09-01

    Osteoarthritis, a common joint degeneration, can cause breakdown of articular cartilage with the presence of lubricin metabolic abnormalities. Lubricin is a multi-level chondroprotective mucinous glycoprotein in articular joints. Joint defect and infection is elevated and accompanied by accelerated cartilage lesions involving degradation and loss of lubricin. However, a novel, heterocyclic compound called kartogenin (KGN) was discovered to stimulate chondrogenic differentiation of bone-derived mesenchymal stem cells (BMSCs). And the synergistic effect of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7) could provoke lubricin accumulation. This paper attempted to explore the connection between accumulation of lubricin and the effect of TGF-β1, BMP-7 and/or KGN. Hence, we investigated the expression and secretion of lubricin in BMSCs treated with different combinations of TGF-β1, BMP-7, and/or KGN. Using an in vitro BMSCs system, we observed the content of lubricin from BMSCs treated with TGF-β1, BMP-7, and KGN was the highest at both the protein level and the gene level. The accumulation of lubricin was enhanced coordinately by the increase of synthesis and decrease of degradation possibly via c-Myc and adamts5 pathway. These results further suggested that supplementation of the defect parts with lubricin by using growth factors and small molecules showed a promising potential on preventing joint deterioration in patients with acquired or genetic deficiency of lubricin in the future of regenerative medicine.

  5. Involvement of the eukaryotic initiation factor 6 and kermit2/gipc2 in Xenopus laevis pronephros formation.

    PubMed

    Tussellino, Margherita; De Marco, Nadia; Campanella, Chiara; Carotenuto, Rosa

    2012-01-01

    The translation initiation factor Eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 Serine 235 by protein kinase C (PKC). Eif6-60S is probably part of the RNA-induced silencing complex (RISC). eif6 over-expression in Xenopus embryos causes aberrant eye development. kermit2/gipc2 morphants have an eye phenotype similar to that of the eif6 overexpressors. Eye formation is regulated by insulin growth factor (IGF) signalling. eif6 interacts with the IGF receptor (IGFR) and kermit2/gipc2, which also binds to igfr. eif6 over-expression in Xenopus causes also the formation of antero-ventral oedema, suggesting a malfunction of the excretory system. Here we evaluated the pronephros phenotype. The oedema grows into the nephrocoel, expanding its boundary and is accompanied by a strong reduction of the pronephros. The three main components of the pronephros are severely impaired in eif6 over-expressors, while are not affected in eif6 morphants. Conversely, gipc2 depletion induces the oedema phenotype and reduction of the pronephros, while gipc2 overexpression does not. p110*, a constitutively active p110 subunit of the PI3 kinase partially recovers the oedema phenotype. We also determined that PKC-dependent phosphorylation of Ser235 in eif6 is not required to produce defective pronephroi. These results indicate that the levels of eif6 are highly regulated during development and instrumental for proper morphogenesis of the pronephros. Moreover, it appears that for proper pronephros development the gipc2 level should be kept within or over the physiological range and that the oedema phenotype is partly due to the inhibition of IGF signalling. PMID:22689378

  6. Characterization and early embryonic expression of a neural specific transcription factor xSOX3 in Xenopus laevis.

    PubMed

    Penzel, R; Oschwald, R; Chen, Y; Tacke, L; Grunz, H

    1997-10-01

    Using the powerful RDA-PCR-technique we could identify a novel Xenopus specific Sox-gene (xSox3) a transcription factor closely related to the sox sub-group B, which contains a HMG box. In normogenesis the xSox3 gene is expressed in the presumptive central nervous system. Furthermore a maternal component is also found in oocytes and in early cleavage stages in the animal hemisphere only. By whole-mount in situ hybridization the first zygotic transcription activities can be detected in the late blastula in the dorsal ectoderm and the dorsal and lateral part of the marginal zone. The expression reaches the highest level atthe late gastrula till the late neurula and fades after stage 30. The expression is restricted from gastrulation onwards to the presumptive brain area and the lens epithelium. Furthermore we could show that the gene is expressed in isolated Spemann organizer with adjacent neuroectoderm. The signal can be suppressed by suramin treatment, which inhibits neural development and causes a shift of dorsal to ventral mesoderm. The treatment of whole embryos with LiCl and UV results in an overexpression or an inhibition of the expression, respectively. In exogastrulae (pseudo-exogastrulae) the gene is expressed in the close vicinity to the endomesoderm only, but not in the distal most part of the ectoderm. This result indicates that it is unlikely that the gene can be activated by planar signals. The gene can also be activated in dissociated gastrula ectoderm without mesodermal or neural inducers. That means that the gene can be expressed in ectodermal cells in a cell autonomous manner. PMID:9415486

  7. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2).

    PubMed

    Maegawa, Naoki; Kawamura, Kenji; Hirose, Motohiro; Yajima, Hiroshi; Takakura, Yoshinori; Ohgushi, Hajime

    2007-01-01

    It is well known that bone marrow contains mesenchymal stromal cells (MSCs), which can show osteoblastic differentiation when cultured in osteogenic medium containing ascorbic acid, beta-glycerophosphate and dexamethasone. The differentiation results in the appearance of osteoblasts, together with the formation of bone matrix; thus, in vitro cultured bone (osteoblasts/bone matrix) could be fabricated by MSC culture. This type of cultured bone has already been used in clinical cases involving orthopaedic problems. To improve the therapeutic effect of the cultured bone, we investigated the culture conditions that contributed to extensive osteoblastic differentiation. Rat bone marrow was primarily cultured to expand the number of MSCs and further cultured in osteogenic medium for 12 days. The culture was also conducted in a medium supplemented with either bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor (FGF-2), or with sequential combinations of both supplements. Among them, the sequential supplementation of FGF-2 followed by BMP-2 showed high alkaline phosphatase activity, sufficient bone-specific osteocalcein expression and abundant bone matrix formation of the MSC culture. These data implied that the number of responding cells or immature osteoblasts was increased by the supplementation of FGF-2 in the early phase of the culture and that these cells can show osteoblastic differentiation, of which capability was augmented by BMP-2 in the late phase. The sequential supplementation of these cytokines into MSC culture might be suitable for the fabrication of ideal cultured bone for use in bone tissue engineering. PMID:18038421

  8. Tumor necrosis factor alpha represses bone morphogenetic protein (BMP) signaling by interfering with the DNA binding of Smads through the activation of NF-kappaB.

    PubMed

    Yamazaki, Masato; Fukushima, Hidefumi; Shin, Masashi; Katagiri, Takenobu; Doi, Takahiro; Takahashi, Tetsu; Jimi, Eijiro

    2009-12-18

    Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor alpha (TNFalpha) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFalpha inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFalpha had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-kappaB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-kappaB by the overexpression of the dominant negative IkappaBalpha. Although TNFalpha failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFalpha suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-kappaB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFalpha inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-kappaB.

  9. Vascular endothelial growth factor/bone morphogenetic protein-2 bone marrow combined modification of the mesenchymal stem cells to repair the avascular necrosis of the femoral head.

    PubMed

    Ma, Xiao-Wei; Cui, Da-Ping; Zhao, De-Wei

    2015-01-01

    Vascular endothelial cell growth factor (VEGF) combined with bone morphogenetic protein (BMP) was used to repair avascular necrosis of the femoral head, which can maintain the osteogenic phenotype of seed cells, and effectively secrete VEGF and BMP-2, and effectively promote blood vessel regeneration and contribute to formation and revascularization of tissue engineered bone tissues. To observe the therapeutic effect on the treatment of avascular necrosis of the femoral head by using bone marrow mesenchymal stem cells (BMSCs) modified by VEGF-165 and BMP-2 in vitro. The models were avascular necrosis of femoral head of rabbits on right leg. There groups were single core decompression group, core decompression + BMSCs group, core decompression + VEGF-165/BMP-2 transfect BMSCs group. Necrotic bone was cleared out under arthroscope. Arthroscopic observation demonstrated that necrotic bone was cleared out in each group, and fresh blood flowed out. Histomorphology determination showed that blood vessel number and new bone area in the repair region were significantly greater at various time points following transplantation in the core decompression + VEGF-165/BMP-2 transfect BMSCs group compared with single core decompression group and core decompression + BMSCs group (P < 0.05). These suggested that VEGF-165/BMP-2 gene transfection strengthened osteogenic effects of BMSCs, elevated number and quality of new bones and accelerated the repair of osteonecrosis of the femoral head.

  10. Vascular endothelial growth factor/bone morphogenetic protein-2 bone marrow combined modification of the mesenchymal stem cells to repair the avascular necrosis of the femoral head

    PubMed Central

    Ma, Xiao-Wei; Cui, Da-Ping; Zhao, De-Wei

    2015-01-01

    Vascular endothelial cell growth factor (VEGF) combined with bone morphogenetic protein (BMP) was used to repair avascular necrosis of the femoral head, which can maintain the osteogenic phenotype of seed cells, and effectively secrete VEGF and BMP-2, and effectively promote blood vessel regeneration and contribute to formation and revascularization of tissue engineered bone tissues. To observe the therapeutic effect on the treatment of avascular necrosis of the femoral head by using bone marrow mesenchymal stem cells (BMSCs) modified by VEGF-165 and BMP-2 in vitro. The models were avascular necrosis of femoral head of rabbits on right leg. There groups were single core decompression group, core decompression + BMSCs group, core decompression + VEGF-165/BMP-2 transfect BMSCs group. Necrotic bone was cleared out under arthroscope. Arthroscopic observation demonstrated that necrotic bone was cleared out in each group, and fresh blood flowed out. Histomorphology determination showed that blood vessel number and new bone area in the repair region were significantly greater at various time points following transplantation in the core decompression + VEGF-165/BMP-2 transfect BMSCs group compared with single core decompression group and core decompression + BMSCs group (P < 0.05). These suggested that VEGF-165/BMP-2 gene transfection strengthened osteogenic effects of BMSCs, elevated number and quality of new bones and accelerated the repair of osteonecrosis of the femoral head. PMID:26629044

  11. Fibroblast growth factor 17 and bone morphogenetic protein 15 enhance cumulus expansion and improve quality of in vitro-produced embryos in cattle.

    PubMed

    Machado, Mariana Fernandes; Caixeta, Ester Siqueira; Sudiman, Jaqueline; Gilchrist, Robert B; Thompson, Jeremy G; Lima, Paula Fernanda; Price, Christopher A; Buratini, José

    2015-08-01

    Bone morphogenetic protein 15 (BMP15) and members of the fibroblast growth factor (FGF) family are expressed by the oocyte and are involved in the control of cumulus cell function. We tested the hypothesis that FGF17, alone or combined with BMP15 in the maturation medium, enhances cumulus expansion, meiosis progression, embryonic development, and expression of mRNA encoding key genes regulating expansion (prostaglandin-endoperoxide synthase 2 [PTGS2], hyaluronan synthase 2 [HAS2], tumor necrosis factor-stimulated gene 6 [TNFAIP6], and pentraxin 3 [PTX3]) and markers of oocyte developmental competence (phosphofructokinase [PFKP], gremlin [GREM1], versican [VCAN], and the genomic progesterone receptor [nPR]) in cumulus cells. Fibroblast growth factor 17 and BMP15 increased the percentage of fully expanded cumulus-oocyte complexes (COCs), but there was no additive effect when both were combined. Neither FGF17 nor BMP15 altered the percentage of oocytes reaching meiosis II at the end of COC culture or cleavage and blastocyst rates after IVF. However, embryo quality, as assessed by the number of cells in the inner cell mass, was improved by the combination of FGF17 with BMP15. Fibroblast growth factor 17 alone did not alter gene expression in cumulus cells at the end of IVM, whereas BMP15 increased PTGS2 and PTX3 mRNA levels. The combination of FGF17 and BMP15 increased nPR mRNA abundance in cumulus cells but did not change the expression of other markers of developmental competence. This study provides novel evidence that FGF17 enhances cumulus expansion in bovine COCs submitted to IVM and that the supplementation of the IVM medium with FGF17 and BMP15 may improve embryo quality.

  12. Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring.

    PubMed

    Abadjieva, Desislava; Kistanova, Elena

    2016-01-01

    Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers' oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris.

  13. Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): cellular localization, developmental profiles, and response to unilateral ovariectomy.

    PubMed

    García-López, Ángel; Sánchez-Amaya, María Isabel; Halm, Silke; Astola, Antonio; Prat, Francisco

    2011-12-01

    Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass.

  14. Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring

    PubMed Central

    2016-01-01

    Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers’ oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris. PMID:26928288

  15. Low melting point amphiphilic microspheres for delivery of bone morphogenetic protein-6 and transforming growth factor-β3 in a hydrogel matrix.

    PubMed

    Sukarto, Abby; Amsden, Brian G

    2012-02-28

    Low melting-point poly(1,3-trimethylene carbonate-co-ε-caprolactone)-b-poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate-co-ε-caprolactone), P(TMC-CL)(2)-PEG, was employed to fabricate microspheres for sustained growth factor delivery in a photocrosslinked N-methacrylate glycol chitosan hydrogel matrix. The P(TMC-CL)(2)-PEG had a melting range such that it was solid at 10°C, yet liquid with a low degree of crystallinity at 37°C. The in vitro degradation of P(TMC-CL)(2)-PEG microspheres was slow, regardless of the triblock copolymer molecular weight and so did not influence protein release. The size of protein loaded P(TMC-CL)(2)-PEG microspheres manufactured using a low-temperature electrospray technique was between 65 and 85μm. Initial formulation work was done with the model protein lysozyme, co-lyophilized with trehalose and encapsulated as approximately 2μm particles within P(TMC-CL)(2)-PEG microspheres. This work indicated a sustained release could be achieved with high trehalose content (90% w/w) in the particles. Under these conditions, the release rate of bone morphogenetic protein-6 was more sustained than that of the excipient bovine serum albumin (BSA) and closely followed that of lysozyme. On the other hand, transforming growth factor-β3 and the stabilizing agent BSA generated similar release profiles. This difference in release was proposed to be linked to the protein isoelectric point, with positively charged proteins possibly being more strongly adsorbed to the P(TMC-CL)(2)-PEG. Both growth factors were released in highly bioactive form, indicating the potential of the release approach. PMID:22037107

  16. Chondrogenesis of periodontal ligament stem cells by transforming growth factor-β3 and bone morphogenetic protein-6 in a normal healthy impacted third molar.

    PubMed

    Choi, Sunyoung; Cho, Tae-Jun; Kwon, Soon-Keun; Lee, Gene; Cho, Jaejin

    2013-03-01

    The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 µg·L⁻¹ TGF-β3 or 100 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-β3 and 220% by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-β3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

  17. Expression of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and BMP receptors in the ovaries of goats.

    PubMed

    Silva, J R V; van den Hurk, R; van Tol, H T A; Roelen, B A J; Figueiredo, J R

    2005-01-01

    The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. In goats, relatively little information is available on the local factors that regulate this process. We studied the presence and distribution of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and BMP receptors types 2 (BMPR2), 1A (BMPR1A), and 1B (BMPR1B) in goat ovaries to find evidence for their possible roles in folliculogenesis. Ovaries of cyclic goats were collected and fixed in paraformaldehyde for immunohistochemical localization of GDF9 and BMP15 proteins or used to collect follicles and luteal tissue to study the mRNA expression of GDF9, BMP15, and BMP receptors using reverse transcriptase polymerase chain reaction (RT-PCR). GDF9 and BMP15 proteins were found in oocytes of all types of follicles and granulosa cells of primary, secondary, and antral but not primordial follicles. The mRNAs for GDF9, BMP15, BMPR2, BMPR1A, and BMPR1B were detected in primordial, primary, and secondary follicles as well as in oocyte and granulosa cells of antral follicles. Transcripts for BMPR2, BMPR1A, BMPR1B, and GDF9, and GDF9 protein were furthermore found in corpora lutea. It is concluded that the mRNAs and proteins of GDF9 and BMP15 and the mRNAs of BMP receptors are expressed in goat ovarian follicles at all stages of their development, and that they form a complex intrafollicular regulatory system during folliculogenesis. Expression of all BMP receptor mRNAs and GDF9 mRNA and protein in luteal tissue additionally points to a role of GDF9 in corpus luteum function.

  18. Evolutionary origin of bone morphogenetic protein 15 and growth and differentiation factor 9 and differential selective pressure between mono- and polyovulating species.

    PubMed

    Monestier, Olivier; Servin, Bertrand; Auclair, Sylvain; Bourquard, Thomas; Poupon, Anne; Pascal, Géraldine; Fabre, Stéphane

    2014-10-01

    Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are TGFbeta-like oocyte-derived growth factors involved in ovarian folliculogenesis as critical regulators of many granulosa cell processes and ovulation rate. Ovarian phenotypic effect caused by alterations in BMP15 and GDF9 genes appears to differ between species and may be relevant to their mono- or polyovulating status. Through phylogenetic analysis we recently showed that these two paralogous genes are strongly divergent and in rapid evolution as compared to other members of the TGFbeta superfamily. Here, we evaluate the amino acid substitution rates of a set of proteins implicated in the ovarian function, including BMP15 and GDF9, with special attention to the mono- or polyovulating status of the species. Among a panel of mono- and polyovulating mammals, we demonstrate a better conservation of some areas in BMP15 and GDF9 within mono-ovulating species. Homology modeling of BMP15 and GDF9 homodimer and heterodimer 3-D structures was suggestive that these areas may be involved in dimer formation and stability. A phylogenetic study of BMP15/GDF9-related proteins reveals that these two genes diverged from the same ancestral gene along with BMP3 and GDF10, two other paralogous genes. A substitution rate analysis based on this phylogenetic tree leads to the hypothesis of an acquisition of BMP15/GDF9-specific functions in ovarian folliculogenesis in mammals. We propose that high variations observed in specific areas of BMP15 and GDF9 in polyovulating species change the equilibrium between homodimers and heterodimers, modifying the biological activity and thus allowing polyovulation to occur.

  19. [Species specificity of morphogenetic factors of Acetabularia, localized in the cytoplasmic zone adjacent to the cell membrane].

    PubMed

    Naumova, G A; Naumova, L P; Puchkova, L I; Savchenko, S M; Sandakhchiev, L S

    1976-01-01

    The species specificity of the factors controlling the cap development was established in the experiments with the transplantation of both the intact and centrifuged in the basal direction apical regions of Acetabularia meditteranea on nuclear basal regions of A. crenulata. These factors are found at the stage of 72 hrs of regeneration primarily in the cytoplasmic zone adjacent to the cell membrane which is not displaced during centrifugation. Using direct measurements and radiochemical method, we have shown that the accumulation of proteins proceeded in this zone due, mainly, to their transition from the cytoplasmic zone displaced during centrifugation.

  20. A new growth factor controlled drug release system to promote healing of bone fractures: nanospheres of recombinant human bone morphogenetic-2 and polylactic acid.

    PubMed

    Chen, Lin; Liu, Lei; Li, Cai; Tan, Yinghui; Zhang, Gang

    2011-04-01

    To prepare a new drug control release system, which can markedly promote the healing of bone fractures. Optimized water-in-oil-in-water multiple emulsion evaporation method, prepared nanospheres of recombinant human bone morphogenetic-2 and polylactic acid (rhBMP-2-PLA-Ns). Its physical character was determined by the enzyme linked immunosorbent assay method. Its bioactivity was measured with the microculture tetrazolium test immunohistochemical analyses, alizarin red staining and western blot analysis. rhBMP-2-PLA-Ns exhibited an even and uniform spherical appearance without adhesion, with a particle size distribution between 35 and 65 nm, and a mean size of 45 nm. The drug loading volume and encapsulation efficiency reached ([124.73 +/- 0.41] x 10(-3))% and (90.54 +/- 1.32)%, respectively. The drug release in vitro persisted for 14 days, with a mean concentration of 73.44 +/- 5.38 ng/ml, and corresponded to the Higuichi equation (r = 0.9962). The microculture tetrazolium test showed that 4 days later, the optical density value ranking was rhBMP-2-PLA-N group > rhBMP-2 group > blank control group. Fluorescence immunocytochemical analysis showed that 10 days later the fluorescent density of the rhBMP-2-PLA-N group was significantly higher than the other two groups. Western blot analysis confirmed that the amount of vascular endothelial growth factor in the rhBMP-2-PLA-N group was the greatest. This study showed that rhBMP-2-PLA-Ns have excellent biological activity, can promote proliferation, differentiation and mineralization of osteoblasts. The drug release time is suitable for fracture healing and is an ideal delivery system for fracture healing. PMID:21776677

  1. Insulin-Like Growth Factor-1 and Bone Morphogenetic Protein-2 Jointly Mediate Prostaglandin E2-Induced Adipogenic Differentiation of Rat Tendon Stem Cells

    PubMed Central

    Liu, Junpeng; Chen, Lei; zhou, You; Liu, Xiangzhou; Tang, Kanglai

    2014-01-01

    Tendinopathy is characterized histopathologically by lipid accumulation and tissue calcification. Adipogenic and osteogenic differentiation of tendon stem cells (TSCs) are believed to play key roles in these processes. The major inflammatory mediator prostaglandin E2 (PGE2) has been shown to induce osteogenic differentiation of TSCs via bone morphogenetic protein-2 (BMP-2), and BMP-2 has also been implicated in adipogenic differentiation of stem cells. We therefore examined the mechanisms responsible for PGE2-induced adipogenesis in rat TSCs in vitro. Insulin-like growth factor-1 (IGF-1) mRNA and protein were significantly up-regulated in PGE2-stimulated TSCs, measured by quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Incubation with specific inhibitors of cAMP, cAMP-dependent protein kinase A (PKA), and CCAAT/enhancer binding protein-δ (CEBPδ) demonstrated that IGF-1 up-regulation occurred via a cAMP/PKA/CEBPδ pathway. Furthermore, neither IGF-1 nor BMP-2 alone was able to mediate adipogenic differentiation of TSCs, but IGF-1 together with BMP-2 significantly increased adipogenesis, indicated by Oil Red O staining. Moreover, knock-down of endogenous IGF-1 and BMP2 abolished PGE2-induced adipogenic differentiation. Phosphorylation of CREB and Smad by IGF-1 and BMP-2, respectively, were required for induction of the adipogenesis-related peroxisome proliferator-activated receptor γ2 (PPARγ2) gene and for adipogenic differentiation. In conclusion, IGF-1 and BMP-2 together mediate PGE2-induced adipogenic differentiation of TSCs in vitro via a CREB- and Smad-dependent mechanism. This improved understanding of the mechanisms responsible for tendinopathies may help the development of more effective therapies. PMID:24416413

  2. Nanoplex-Mediated Co-delivery of Fibroblast Growth Factor and Bone Morphogenetic Protein Genes Promotes Osteogenesis in Human Adipocyte-Derived Mesenchymal Stem Cells

    PubMed Central

    Atluri, Keerthi; Seabold, Denise; Hong, Liu; Elangovan, Satheesh; Salem, Aliasger K.

    2015-01-01

    This study highlights the importance of transfection mediated coordinated bone morphogenetic protein 2 (BMP-2) and fibroblast growth factor 2 (FGF-2) signaling in promoting osteogenesis. We employed plasmids independently encoding BMP-2 and FGF-2 complexed with polyethylenimine (PEI) to transfect human adipose derived mesenchymal stem cells (hADMSCs) in vitro. The nanoplexes were characterized for size, surface charge, in vitro cytotoxicity and transfection ability in hADMSCs. A significant enhancement in BMP-2 protein secretion was observed on day 7 post-transfection of hADMSCs with PEI nanoplexes loaded with both pFGF-2 and pBMP-2 (PEI/(pFGF-2 + pBMP-2)) versus transfection with PEI nanoplexes of either pFGF-2 alone or pBMP-2 alone. Osteogenic differentiation of transfected hADMSCs was determined by measuring osteocalcin and Runx-2 gene expression using real time polymerase chain reactions. A significant increase in the expression of Runx-2 and osteocalcin was observed on day 3 and day 7 post-transfection, respectively, by cells transfected with PEI/(pFGF-2 + pBMP-2) compared to cells transfected with nanoplexes containing pFGF-2 or pBMP-2 alone. Alizarin Red staining and atomic absorption spectroscopy revealed elevated levels of calcium deposition in hADMSC cultures on day 14 and day 30 post-transfection with PEI/(pFGF-2 + pBMP-2) compared to other treatments. We have shown that co-delivery of pFGF-2 and pBMP-2 results in a significant enhancement in osteogenic protein synthesis, osteogenic marker expression and subsequent mineralization. This research points to a new clinically translatable strategy for achieving efficient bone regeneration. PMID:26121311

  3. Low-dose bone morphogenetic protein-2/stromal cell-derived factor-1β cotherapy induces bone regeneration in critical-size rat calvarial defects.

    PubMed

    Herberg, Samuel; Susin, Cristiano; Pelaez, Manuel; Howie, R Nicole; Moreno de Freitas, Rubens; Lee, Jaebum; Cray, James J; Johnson, Maribeth H; Elsalanty, Mohammed E; Hamrick, Mark W; Isales, Carlos M; Wikesjö, Ulf M E; Hill, William D

    2014-05-01

    Increasing evidence suggests that stromal cell-derived factor-1 (SDF-1/CXCL12) is involved in bone formation, though underlying molecular mechanisms remain to be fully elucidated. Also, contributions of SDF-1β, the second most abundant splice variant, as an osteogenic mediator remain obscure. We have shown that SDF-1β enhances osteogenesis by regulating bone morphogenetic protein-2 (BMP-2) signaling in vitro. Here we investigate the dose-dependent contribution of SDF-1β to suboptimal BMP-2-induced local bone formation; that is, a dose that alone would be too low to significantly induce bone formation. We utilized a critical-size rat calvarial defect model and tested the hypotheses that SDF-1β potentiates BMP-2 osteoinduction and that blocking SDF-1 signaling reduces the osteogenic potential of BMP-2 in vivo. In preliminary studies, radiographic analysis at 4 weeks postsurgery revealed a dose-dependent relationship in BMP-2-induced new bone formation. We then found that codelivery of SDF-1β potentiates suboptimal BMP-2 (0.5 μg) osteoinduction in a dose-dependent order, reaching comparable levels to the optimal BMP-2 dose (5.0 μg) without apparent adverse effects. Blocking the CXC chemokine receptor 4 (CXCR4)/SDF-1 signaling axis using AMD3100 attenuated the osteoinductive potential of the optimal BMP-2 dose, confirmed by qualitative histologic analysis. In conclusion, SDF-1β provides potent synergistic effects that support BMP-induced local bone formation and thus appears a suitable candidate for optimization of bone augmentation using significantly lower amounts of BMP-2 in spine, orthopedic, and craniofacial settings.

  4. Functional differentiation of uterine stromal cells involves cross-regulation between bone morphogenetic protein 2 and Kruppel-like factor (KLF) family members KLF9 and KLF13.

    PubMed

    Pabona, John Mark P; Zeng, Zhaoyang; Simmen, Frank A; Simmen, Rosalia C M

    2010-07-01

    The inability of the uterine epithelium to enter a state of receptivity for the embryo to implant is a significant underlying cause of early pregnancy loss. We previously showed that mice null for the progesterone receptor (PGR)-interacting protein Krüppel-like factor (KLF) 9 are subfertile and exhibit reduced uterine progesterone sensitivity. KLF9 expression is high in predecidual stroma, undetectable in decidua, and enhanced in uteri of mice with conditional ablation of bone morphogenetic protein 2 (BMP2). Given the individual importance of KLF9 and BMP2 for implantation success, we hypothesized that the establishment of uterine receptivity involves KLF9 and BMP2 functional cross-regulation. To address this, we used early pregnant wild-type and Klf9 null mice and KLF9 small interfering RNA-transfected human endometrial stromal cells (HESCs) induced to differentiate under standard conditions. Loss of KLF9 in mice and HESCs enhanced BMP2 expression, whereas recombinant BMP2 treatment of HESCs attenuated KLF9 mRNA levels. IGFBP1 and KLF9-related KLF13 expression were positively associated with BMP2 and inversely associated with KLF9. Prolonged, but not short-term, knockdown of KLF9 in HESCs reduced IGFBP1 expression. Mouse uterine Igfbp1 expression was similarly reduced with Klf9 ablation. PGR-A and PGR-B expression were positively associated with KLF9 in predecidual HESCs but not decidualizing HESCs. KLF13 knockdown attenuated BMP2 and PGR-B and abrogated BMP2-mediated inhibition of KLF9 expression. Results support cross-regulation among BMP2, KLF9, and KLF13 to maintain progesterone sensitivity in stromal cells undergoing differentiation and suggest that loss of this regulatory network compromises establishment of uterine receptivity and implantation success.

  5. Comparison of expression patterns of fibroblast growth factor 8, bone morphogenetic protein 4 and sonic hedgehog in jaw development of the house shrew, Suncus murinus.

    PubMed

    Ogi, Hidenao; Tabata, Makoto J; Yamanaka, Atsushi; Yasui, Kinya; Uemura, Masanori

    2002-01-01

    To elucidate the mechanism underlying jaw development in mammals, we used a new laboratory animal, Suncus murinus (house shrew, an insectivore) as the subject for the investigation, because Suncus has all types of teeth (incisor, canine, premolar and molar) in its upper and lower jaws and is thought to be a good model animal having a general mammalian tooth pattern. At the start, by use of degenerate primers we cloned Suncus homologues of fibroblast growth factor 8 (sFgf8), bone morphogenetic protein 4 (sBmp4) and sonic hedgehog (sShh) genes from cDNA library derived from whole Suncus embryos at day 12 (E12). Thereafter, we examined the expression patterns of these genes in the jaw development of Suncus E11-16 embryos (for mouse E9.5-12 embryos). sFgf8 and sBmp4 were expressed in E11 but not in E15 and onward during orofacial development. sShh was expressed from E11 onward, and its expression was increased in the orofacial area. The expression pattern of sFgf8 in the maxillary and mandibular arches of E14 coincided with the area of the presumptive tooth arch. However, sShh and sBmp4 were expressed only in the outer area (= buccal/labial side) of presumptive tooth arch. Thus, these 3 genes showed specific expression pattern in jaw development of Suncus, and their distributions did not overlap each other except in a few regions. These findings suggest that sFgf8, sBmp4 and sShh have a specific function respectively during jaw development in Suncus murinus.

  6. Oocytes in sheep homozygous for a mutation in bone morphogenetic protein receptor 1B express lower mRNA levels of bone morphogenetic protein 15 but not growth differentiation factor 9.

    PubMed

    Crawford, Janet L; Heath, Derek A; Reader, Karen L; Quirke, Laurel D; Hudson, Norma L; Juengel, Jennifer L; McNatty, Kenneth P

    2011-07-01

    The aim of this study was to test the hypothesis that the high ovulation rate in ewes (BB) homozygous for a mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene is linked to lower BMP15 and/or GDF9 mRNA in oocytes compared with those in wild-type (++) ewes. Cumulus cell-oocyte complexes (COC) and granulosa cells (GC) were recovered from ≥1 mm diameter follicles of BB and ++ ewes during a prostaglandin-induced follicular phase. Expression levels of GDF9 and BMP15 were measured by multiplex qPCR from individual COC. The gonadotropin-induced cAMP responses of the GC from each non-atretic follicle were measured following treatment with FSH or human chorionic gonadotropin. In a separate validation experiment, GDF9 and BMP15 expression was present only in oocytes and not in cumulus cells. There was no effect of follicular diameter on oocyte-derived GDF9 or BMP15 mRNA levels. The mean expression levels of BMP15, but not GDF9, were significantly lower in all non-atretic follicles, including the subsets containing either FSH- or LH-responsive GC in BB, compared with ++, ewes. No genotype effects were noted for FSH-induced cAMP production by GC either with respect to dose of, or number of follicles responding to, FSH. However, ovaries from BB ewes contained significantly more follicles responsive to LH, with respect to cAMP production in GC. We propose that these findings are consistent with the hypothesis that the higher ovulation rate in BB sheep is due, at least in part, to lower oocyte-derived BMP15 mRNA levels together with the earlier onset of LH-responsiveness in GC.

  7. Platelet-derived growth factor inhibits bone regeneration induced by osteogenin, a bone morphogenetic protein, in rat craniotomy defects.

    PubMed Central

    Marden, L J; Fan, R S; Pierce, G F; Reddi, A H; Hollinger, J O

    1993-01-01

    Platelet-derived growth factor (PDGF) is a potent moderator of soft tissue repair through induction of the inflammatory phase of repair and subsequent enhanced collagen deposition. We examined the effect of recombinant BB homodimer PDGF (rPDGF-BB) applied to rat craniotomy defects, treated with and without bovine osteogenin (OG), to see if bone regeneration would be stimulated. Implants containing 0, 20, 60, or 200 micrograms rPDGF-BB, reconstituted with insoluble rat collagenous bone matrix containing 0, 30, or 150 micrograms OG, were placed into 8-mm craniotomies. After 11 d, 21 of the 144 rats presented subcutaneous masses superior to the defect sites. The masses, comprised of serosanguinous fluid encapsulated by fibrous connective tissue, were larger and occurred more frequently in rats treated with 200 micrograms rPDGF-BB, and were absent in rats not treated with rPDGF-BB. The masses underwent resorption within 28 d after surgery. OG (2-256 micrograms) caused a dose-dependent increase in radiopacity and a marked regeneration of calcified tissue in a dose-dependent fashion within defect sites. However, OG-induced bone regeneration was inhibited 17-53% in the presence of rPDGF-BB. These results suggest that rPDGF-BB inhibited OG-induced bone regeneration and stimulated a soft tissue repair wound phenotype and response. Images PMID:8254045

  8. In Vitro and In Vivo Synergistic Interactions Between the Runx2/Cbfa1 Transcription Factor and Bone Morphogenetic Protein-2 in Stimulating Osteoblast Differentiation

    PubMed Central

    YANG, SHUYING; WEI, DAOYAN; WANG, DIAN; PHIMPHILAI, MATTABHORN; KREBSBACH, PAUL H; FRANCESCHI, RENNY T

    2013-01-01

    Bone regeneration requires interactions between a number of factors including bone morphogenetic proteins (BMPs), growth factors, and transcriptional regulators such as Runx2/Cbfa1 (Runx2). Because each component may provide a unique contribution to the overall osteogenic response, we hypothesized that bone formation may be enhanced by using combinations of complimentary factors. As an initial test of this concept, interactions between BMP2 and Runx2 were examined using adenovirus-based expression vectors (AdCMV-Runx2, AdCMV-BMP2) in the pluripotent C3H10T1/2 cell line. Cells transduced with AdCMV-Runx2 strongly expressed osteoblast markers, such as alkaline phosphatase and osteocalcin, but formed only a weakly mineralized extracellular matrix in vitro, whereas cells transduced with AdCMV-BMP2 exhibited higher levels of mineralization, but only expressed low levels of Runx2 and osteocalcin mRNA. Significantly, when cells were transduced with optimal titers of both viruses, osteoblast differentiation was stimulated to levels that were 10-fold greater than those seen with either AdCMV-Runx2 or AdCMV-BMP2 alone. To measure in vivo osteogenic activity, virally transduced cells were subcutaneously implanted into immunodeficient mice. Cells transduced with control virus produced only fibrous tissue while those with AdCMV-Runx2 produced limited amounts of both cartilage and bone. In contrast, cells transduced with either AdCMV-BMP2 alone or AdCMV-BMP2 plus AdCMV-Cbfa1 generated large ossicles containing cartilage, bone, and a marrow cavity. However, ossification in the AdCMV-BMP2 plus AdCMV-Cbfa1 group was more extensive in that both mineral content and fractional bone area were greater than that seen in the AdCMV-BMP2 group. Thus, the increased osteoblast differentiation observed with combined adenovirus treatment in vitro is also manifested by increased bone formation in vivo. These results suggest that Runx2 and BMP2 have distinct, but complementary, roles in

  9. Combinatorial Analysis of Growth Factors Reveals the Contribution of Bone Morphogenetic Proteins to Chondrogenic Differentiation of Human Periosteal Cells.

    PubMed

    Mendes, Luis Filipe; Tam, Wai Long; Chai, Yoke Chin; Geris, Liesbet; Luyten, Frank P; Roberts, Scott J

    2016-05-01

    Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-β1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (μMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of μMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-β1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control μMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated μMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches. PMID:27018617

  10. Histologic and Histomorphometric Comparison of Bone Regeneration Between Bone Morphogenetic Protein-2 and Platelet-Derived Growth Factor-BB in Experimental Groups.

    PubMed

    Guven, Gokhan; Gultekin, B Alper; Guven, Gamze Senol; Guzel, Elif; Furat, Selenay; Ersanli, Selim

    2016-05-01

    Efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human platelet-derived growth factor-BB (rhPDGF-BB) delivered via absorbable collagen sponge (ACS) on bone formation was evaluated in guinea pig tibias. Three-millimeter-circular bone tibia defects were created in 24 guinea pigs assigned randomly to 4 groups according to the following defect filling materials: ACS only, rhBMP-2+ACS, rhPDGF-BB+ACS, or empty. New bone formation was evaluated histologically and histomorphometrically at 15 (early healing) and 45 days (late healing). Mean new bone per total defect area ratio was 0.73, 0.57, 0.43, and 0.42 in rhBMP-2+ACS, rhPDGF-BB+ACS, ACS only, and empty groups at early healing, respectively. During early healing, significantly more new bone formation was observed in rhBMP-2+ACS and rhPDGF-BB+ACS groups than in the control groups. New bone formation was significantly higher with rhBMP-2+ACS than with rhPDGF-BB+ACS. Mean new bone per total defect area ratio was 0.81, 0.86, 0.74, and 0.75 in the rhBMP-2+ACS, rhPDGF-BB+ACS, ACS only, and empty groups at late healing, respectively. During late healing, new bone formation was significantly higher in the rhPDGF-BB+ACS group relative to both control groups, but the results did not differ significantly from those in the rhBMP-2+ACS group. New bone formation in the rhBMP-2+ACS group did not change significantly between the healing periods. In the rhPDGF-BB+ACS group, however, new bone formation was significantly higher in the late healing period. Both growth factors accelerated new bone formation in the early healing period. Although rhBMP-2 was more effective in the early healing period, the effects of rhPDGF-BB were longer lasting. PMID:27092911

  11. Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

    PubMed Central

    Koivuniemi, Raili; Mäkelä, Johanna; Hokkanen, Marie-Estelle; Bruelle, Céline; Ho, Tho Huu; Ola, Roxana; Korhonen, Laura; Schröder, Jim; Kataoka, Hiroaki; Lindholm, Dan

    2013-01-01

    Background Neural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain. Methodology/Principal Findings In this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner. Conclusions This study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of

  12. Tetramethylpyrazine inhibits agiontensin II-induced nuclear factor-kappaB activation and bone morphogenetic protein-2 downregulation in rat vascular smooth muscle cells.

    PubMed

    Ren, Xin-Yu; Ruan, Qiu-Rong; Zhu, Da-He; Zhu, Min; Qu, Zhi-Ling; Lu, Jun

    2007-06-25

    Tetramethylpyrazine (TMP), an effective component of traditional Chinese medicine Chuanxiong, is commonly used to resolve embolism. Its possible therapeutic effect against atherosclerosis has received considerable attention recently. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. The mechanisms of TMP in the proliferation of VSMCs induced by Ang II remain to be defined. The present study was aimed to study the effect of TMP on Ang II-induced VSMC proliferation through detection of nuclear factor-kappaB (NF-kappaB) activity and bone morphogenetic protein-2 (BMP-2) expression. Primary cultured rat aortic smooth muscle cells were divided into the control group, Ang II group, Ang II + TMP group and TMP group. Cells in each group were harvested at different time points (15, 30 and 60 min for detection of NF-kappaB activity; 6, 12 and 24 h for measurement of BMP-2 expression). NF-kappaB activation was identified as nuclear staining by immunohistochemistry. BMP-2 expression was observed through Western blot, immunohistochemistry and in situ hybridization. The results showed that: (1) Ang II stimulated the activation of NF-kappaB. Translocation of NF-kappaB p65 subunit from cytoplasm to nucleus appeared as early as 15 min, peaked at 30 min (P<0.01) and declined after 1 h. (2) TMP inhibited Ang II-induced NF-kappaB activation (P<0.01). (3) Ang II increased BMP-2 expression at 6 h but declined it significantly at 12 and 24 h (P<0.01). (4) BMP-2 expression was also kept at high level at 6 h in Ang II + TMP group but maintained at the normal level at 12 and 24 h. (5) There was no significant difference in NF-kappaB activation and BMP-2 expression between the control group and TMP group. These results indicate that TMP inhibits Ang II-induced VSMC proliferation through repression of NF-kappaB activation and BMP-2 reduction, and BMP-2 expression is independent of the NF-kappaB pathway. In

  13. Co-delivery and controlled release of stromal cell-derived factor-1α chemically conjugated on collagen scaffolds enhances bone morphogenetic protein-2-driven osteogenesis in rats

    PubMed Central

    SUN, HAIPENG; WANG, JINMING; DENG, FEILONG; LIU, YUN; ZHUANG, XIUMEI; XU, JIAYUN; LI, LONG

    2016-01-01

    There has been considerable focus in investigations on the delivery systems and clinical applications of bone morphogenetic protein-2 (BMP-2) for novel bone formation. However, current delivery systems require high levels of BMP-2 to exert a biological function. There are several concerns in using of high levels of BMP-2, including safety and the high cost of treatment. Therefore, the development of strategies to decrease the levels of BMP-2 required in these delivery systems is required. In our previous studies, a controlled-release system was developed, which used Traut's reagent and the cross-linker, 4-(N-maleimi-domethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), to chemically conjugate BMP-2 directly on collagen discs. In the current study, retention efficiency and release kinetics of stromal cell-derived factor-1α (SDF-1α) cross-linked on collagen scaffolds were detected. In addition, the osteogenic activity of SDF-1α and suboptimal doses of BMP-2 cross-linked on collagen discs following subcutaneous implantation in rats were evaluated. Independent two-tailed t-tests and one-way analysis of variance were used for analysis. In the present study, the controlled release of SDF-1α chemically conjugated on collagen scaffolds was demonstrated. By optimizing the concentrations of Traut's reagent and the Sulfo-SMCC cross-linker, a significantly higher level of SDF-1α was covalently retained on the collagen scaffold, compared with that retained using a physical adsorption method. Mesenchymal stem cell homing indicated that the biological function of the SDF-1α cross-linked on the collagen scaffolds remained intact. In rats, co-treatment with SDF-1α and a suboptimal dose of BMP-2 cross-linked on collagen scaffolds using this chemically conjugated method induced higher levels of ectopic bone formation, compared with the physical adsorption method. No ectopic bone formation was observed following treatment with a

  14. A Xenopus laevis gene encoding EF-1 alpha S, the somatic form of elongation factor 1 alpha: sequence, structure, and identification of regulatory elements required for embryonic transcription.

    PubMed

    Johnson, A D; Krieg, P A

    1995-01-01

    Transcription of the Xenopus laevis EF-1 alpha S gene commences at the mid-blastula stage of embryonic development and then continues constitutively in all somatic tissues. The EF-1 alpha S promoter is extremely active in the early Xenopus embryo where EF-1 alpha S transcripts account for as much as 40% of all new polyadenylated transcripts. We have isolated the Xenopus EF-1 alpha S gene and used microinjection techniques to identify promoter elements responsible for embryonic transcription. These in vivo expression studies have identified an enhancer fragment, located approximately 4.4 kb upstream of the transcription start site, that is required for maximum expression from the EF-1 alpha S promoter. The enhancer fragment contains both an octamer and a G/C box sequence, but mutation studies indicate that the octamer plays no significant role in regulation of EF-1 alpha S expression in the embryo. The presence of a G/C element in the enhancer and of multiple G/C boxes in the proximal promoter region suggests that the G/C box binding protein, Sp1, plays a major role in the developmental regulation of EF-1 alpha S promoter activity. PMID:8565334

  15. Morphogenetic Aspects of Murein Structure and Biosynthesis

    PubMed Central

    Schwarz, Uli; Leutgeb, Werner

    1971-01-01

    The shape of Escherichia coli is fixed by the form of the sacculus. This sacculus is a macromolecule made up from the polymer murein. In an investigation of the possible factors determining the shape of the sacculus, we attempted to resolve between two fundamental alternatives. (i) Is the shape of the sacculus automatically fixed by its chemical composition? or (ii) does a special morphogenetic system exist which determines the shape of the sacculus? An analysis of sacculi from cells grown in poor and rich media and harvested at different stages of growth was made. Significant variations in the composition of murein were found, whereas the general shape of the cells remained unchanged. This finding stands opposed to the assumption of a strict correlation between chemistry and shape of the sacculus. The second alternative was investigated by attempting to change artificially the shape of the sacculus by modifying the form of the hypothetical morphogenetic system. Rod-shaped cells were converted into spherical spheroplasts which were subsequently allowed to reform a new spherical sacculus. In chemical composition this spherical sacculus was found to be indistinguishable from the rod-shaped sacculus. This finding is taken as evidence for the existence of a distinct morphogenetic apparatus in the cell wall whose form is reflected by the shape of the sacculus. Images PMID:4929868

  16. Differential expression dynamics of Growth differentiation factor9 (GDF9) and Bone morphogenetic factor15 (BMP15) mRNA transcripts during in vitro maturation of buffalo (Bubalus bubalis) cumulus-oocyte complexes.

    PubMed

    Kathirvel, Muralidharan; Soundian, Eswari; Kumanan, Vijayarani

    2013-12-01

    The present study has evaluated the association of growth differentiation factor9 (GDF9) and bone morphogenetic protein15 (BMP15) mRNA expression in cumulus-oocyte complexes (COCs) of buffalo ovary during in vitro maturation (IVM). GDF9 and BMP15 are expressed specifically in mammalian oocytes and also participate in cumulus-oocyte crosstalk. Quantitative real-time polymerase chain reaction (qRT-PCR) technique was applied to investigate the relative abundance (RA) of GDF9 and BMP15 mRNA transcripts throughout the IVM process. Relative mRNA expression pattern of these specific genes were assessed in oocytes and cumulus cells at 0, 6, 12 and 24 h of in vitro culture. Our results revealed that RA of GDF9 during different hours of IVM showed significant reduction between 0 h and 24 h of maturation in oocytes and BMP15 transcript increased significantly (P<0.05) between 6 h and 12 h and decreased again between 12 h and 24. In cumulus cells, GDF9 remained stable during IVM upto 12 h of maturation and decreased significantly between 12 h and 24 h of maturation. Conversely, significant reduction of BMP15 was observed between 0 h and 6 h, stayed stable upto 12 h and became undetectable at 24 h of maturation. In conclusion, these two genes were differentially expressed during the period of oocyte maturation process and notably, BMP15 expression pattern is associated specifically with the period of cumulus cell expansion.

  17. The classic: Bone morphogenetic protein.

    PubMed

    Urist, Marshall R; Strates, Basil S

    2009-12-01

    This Classic Article is a reprint of the original work by Marshall R. Urist and Basil S. Strates, Bone Morphogenetic Protein. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1069-2; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1971 by Sage Publications Inc. Journals and is reprinted with permission from Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res. 1971;50:1392-1406.

  18. The classic: Bone morphogenetic protein.

    PubMed

    Urist, Marshall R; Strates, Basil S

    2009-12-01

    This Classic Article is a reprint of the original work by Marshall R. Urist and Basil S. Strates, Bone Morphogenetic Protein. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1069-2; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1971 by Sage Publications Inc. Journals and is reprinted with permission from Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res. 1971;50:1392-1406. PMID:19727989

  19. Exogenous bone morphogenetic protein-7 reduces hepatic fibrosis in Schistosoma japonicum-infected mice via transforming growth factor-β/Smad signaling

    PubMed Central

    Chen, Bo-Lin; Peng, Jie; Li, Qing-Fu; Yang, Min; Wang, Yuan; Chen, Wei

    2013-01-01

    AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum)-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson’s staining. Transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95 ± 6.66 vs 2.02 ± 0.76; week 15: 12.84 ± 4.36 vs 1.74 ± 0.80; P < 0.05), but significantly lower than that in group B (week 9: 22.95 ± 6.66 vs 34.43 ± 6.96; week 15: 12.84 ± 4.36 vs 18.90 ± 5.07; P < 0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-β1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24 ± 5.73 vs 0.33 ± 0.20; week 15: 12.42 ± 4.88 vs 0.34 ± 0.27; TGF-β1: week 9: 37.00 ± 13.74 vs 3.73 ± 2.14; week 15: 16.71 ± 9.80 vs 3.08 ± 2.35; pSmad2/3: week 9: 12.92 ± 4.81 vs 0.83 ± 0.48; week 15: 7.87 ± 4

  20. Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor-β (TGF-β) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro.

    PubMed

    Yamasaki, Atsushi; Kasai, Atsushi; Toi, Akihiro; Kurita, Maki; Kimoto, Saki; Hayata-Takano, Atsuko; Nakazawa, Takanobu; Nagayasu, Kazuki; Shintani, Norihito; Hashimoto, Ryota; Ito, Akira; Meltzer, Herbert Y; Ago, Yukio; Waschek, James A; Onaka, Yusuke; Matsuda, Toshio; Baba, Akemichi; Hashimoto, Hitoshi

    2015-02-01

    The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock-in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet-1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate-based screening and identified BMP type I receptor kinase inhibitors LDN-193189 and DMH1 as activators of luciferase. LDN-193189 induced ES cells to express the genes encoding Pet-1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF-β receptor inhibitor SB-431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN-193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF-β target gene Lefty, whereas the opposite effect was observed with SB-431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF-β receptor signaling are implicated in serotonergic differentiation. Candidate-based screening for serotonergic induction using a rapid assay in mouse embryonic stem cells revealed that the bone morphogenetic protein (BMP) type I receptor kinase inhibitors selectively induce serotonergic differentiation, whereas the TGF-β receptor inhibitor SB-431542 inhibits the differentiation. These results suggest that inhibition of BMP type I receptors and concomitant activation of transforming growth factor-β (TGF-β) receptor signaling are involved in the early trajectory of serotonergic

  1. Single nucleotide polymorphism of bone morphogenetic protein 4 gene: A risk factor of non-syndromic cleft lip with or without palate

    PubMed Central

    Savitha, Sathyaprasad; Sharma, S. M.; Veena, Shetty; Rekha, R.

    2015-01-01

    Background: The bone morphogenetic protein (BMP) signalling pathway is crucial in a number of developmental processes and is critical in the formation of variety of craniofacial elements including cranial neural crest, facial primordium, tooth, lip and palate. It is an important mediator in regulation of lip and palate fusion, cartilage and bone formation. Aim: To study the role of mutation of BMP4 genes in the aetiology of non-syndromic cleft lip with or without palate (NSCL ± P) and identify it directly from human analyses. Materials and Methods: A case-control study was done to evaluate whether BMP4T538C polymorphism, resulting in an amino acid change of Val=Ala (V152A) in the polypeptide, is associated with NSCL ± P in an Indian paediatric population. Genotypes of 100 patients with NSCL ± P and 100 controls (in whom absence of CL ± P was confirmed in three generations) were detected using a polymerase chain reaction-restriction fragment length polymorphism strategy. Logistic regression was performed to evaluate allele and genotype association with NSCLP. Results: Results showed significant association between homozygous CC genotype with CL ± P (odds ratio [OR]-5.59 and 95% confidence interval [CI] = 2.85-10.99). The 538C allele carriers showed an increased risk of NSCL ± P as compared with 538 T allele (OR - 4.2% CI = 2.75-6.41). Conclusion: This study suggests an association between SNP of BMP4 gene among carriers of the C allele and increased risk for NSCLP in an Indian Population. Further studies on this aspect can scale large heights in preventive strategies for NSCLP that may soon become a reality. PMID:26424979

  2. Role of piperonyl butoxide in the toxicity of chlorpyrifos to Ceriodaphnia dubia and Xenopus laevis.

    PubMed

    El-Merhibi, A; Kumar, A; Smeaton, T

    2004-02-01

    The use of chemical inhibitors/inducers is one of the strategies employed to determine whether a particular metabolic pathway is involved in the metabolism of a xenobiotic. The objective of this study was to assess the role of piperonyl butoxide (PBO) on the toxicity of an organophosphorus insecticide, chlorpyrifos (CPF) to two species, Ceriodaphnia dubia (waterflea) and Xenopus laevis (South African clawed frog). Chlorpyrifos was highly toxic to C. dubia (48-h LC50: 0.05 microg/L) in comparison with X. laevis (96-h LC50: 2410 microg/L). Piperonyl butoxide at 200 microg/L reduced the toxicity of chlorpyrifos to C. dubia by a factor of 6. Piperonyl butoxide at 3000 microg/L also reduced the toxicity of CPF to X. laevis with respect to mortality and malformations. Acetylcholinesterase (AChE) activity was used as a biomarker to further assess the role of PBO in chlorpyrifos toxicity. X. laevis exposed to CPF and PBO exhibited a biphasic response in terms of AChE activity with an initial increase in the AChE activity followed by a drastic decrease. The results from the present study indicate that C. dubia and X. laevis have the capability to metabolize chlorpyrifos via cytochromes P450 mediated reactions. The results also indicate that the use of the biomarker AChE is useful in determining metabolic processes of organophosphorus insecticides, which require metabolic activation.

  3. Circus movements and blebbing locomotion in dissociated embryonic cells of an amphibian, Xenopus laevis.

    PubMed

    Johnson, K E

    1976-12-01

    Circus movements, which involve the circumferential rotation of a hyaline cytoplasmic protrusion, occur in cells obtained by EDTA dissociation of gastrula-stage Xenopus laevis embryos. Only a few dissociated blastula-stage cells show circus movements, more early gastrula-stage cells show them, and nearly all late gastrula-stage cells show them. Circus movements cease in cells prior to mitosis and begin again in daughter cells after mitosis is completed. In early gastrulae, only 17% of prospective endodermal cells show circus movements while 79% of prospective mesodern, archenteric roof, and posterior neural ectoderm do so. Isolated cells as well as groups of cells in vitro are often propelled by circus movements. There is an obvious antagonism between cell contact and circus movements. The morphogenetic significance of circus movements and blebbing locomotion is discussed.

  4. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    PubMed Central

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  5. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures.

    PubMed

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A; Harris, William A

    2013-04-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  6. The ratio of growth differentiation factor 9: bone morphogenetic protein 15 mRNA expression is tightly co-regulated and differs between species over a wide range of ovulation rates.

    PubMed

    Crawford, Janet L; McNatty, Kenneth P

    2012-01-01

    Recent evidence suggests that the species-specific ovulation-rate phenotypes may be influenced by differences in the expression levels of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) mRNA and protein. The aim of this study was to compare GDF9 and BMP15 mRNA levels in individual denuded oocytes (DO) from a range of single (i.e. cow, red deer), single-to-triple (i.e. sheep) and high (i.e. pig, mouse, rat) ovulation-rate species. Compared to all other species studied, GDF9 mRNA levels were lower in DO of cows and deer, whilst BMP15 levels were highest in DO of pigs. There was no detectable expression of either GDF9 or BMP15 mRNA in CC from any species. The ratio of GDF9:BMP15 mRNA expression was highly correlated (R(2)>0.80) within each species but differed markedly between species (P<0.01). Thus, we conclude that the ratio of GDF9:BMP15 mRNA is species-specific across a wide range of ovulation-rate phenotypes.

  7. Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein-transgenic mice exhibiting a diversity in intracellular transforming growth factor-β and bone morphogenetic protein signaling.

    PubMed

    Sawada, Shunsuke; Chosa, Naoyuki; Takizawa, Naoki; Yokota, Jun; Igarashi, Yasuyuki; Tomoda, Koichi; Kondo, Hisatomo; Yaegashi, Takashi; Ishisaki, Akira

    2016-03-01

    Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG‑2, ‑3, and ‑5 from the bone marrow of green fluorescent protein (GFP)‑transgenic mice. These cell lines clearly expressed mouse MSC markers Sca‑1 and CD44, and SG‑2 and ‑5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)‑β superfamily. By contrast, SG‑3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG‑3 cells, which underwent osteogenesis in response to BMP, while TGF‑β receptor II was most highly expressed in SG‑3 and ‑5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF‑β1 in SG‑2 cells but not in SG‑3 or ‑5 cells. Furthermore, TGF‑β1 clearly induced the expression of Smad‑interacting transcription factor CCAAT/enhancer binding protein‑β in SG‑2 but not in SG‑3 or ‑5 cells. These results demonstrated the establishment of TGF‑β‑responsive SG‑2 MSCs, BMP‑responsive SG‑3 MSCs and TGF‑β/BMP‑unresponsive SG‑5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF‑β superfamily affects the proliferation and differentiation status of MSCs in vivo. PMID:26781600

  8. Molecular characterisation of growth differentiation factor 9 (gdf9) and bone morphogenetic protein 15 (bmp15) and their patterns of gene expression during the ovarian reproductive cycle in the European sea bass.

    PubMed

    Halm, S; Ibañez, A J; Tyler, C R; Prat, F

    2008-09-10

    Members of the transforming growth factor-beta superfamily, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have crucial roles in primary follicle growth in mammals. To initiate investigations into their significance in teleost oogenesis, we set out to clone and characterise the cDNAs of gdf9 and bmp15 and analysed their patterns of gene expression during the ovarian reproductive cycle in the European sea bass (Dicentrachus labrax). Sea bass gdf9 and bmp15 cDNAs were 2200 and 2049 bp long, coding for 438 and 459 amino acids (aas), respectively, and were most similar to zebrafish gdf9 and bmp15 (64.4 and 56.1%, respectively). By Northern analysis, sea bass gdf9 and bmp15 mRNA transcripts were detected in the ovary only of the tissues analysed and their sizes were 2.2 and 2.1 kb, respectively. Dot-blot analysis revealed high levels of gdf9 and bmp15 expression in the ovary during primary oocyte growth and previtellogenesis (July to October), with a significant decline at the onset of vitellogenesis (November) and remaining low until the beginning of new oocyte growth (April/May). There was a highly significant positive correlation (r=0.939) between gdf9 and bmp15 gene expression in individual samples. The high levels of gdf9 and bmp15 mRNA transcripts in the ovary, especially during the previtellogenic growth period suggest an important role for these factors in early primary oocyte growth in the European sea bass.

  9. XENOPUS LAEVIS: A CULTURING AND REARING PROTOCOL

    EPA Science Inventory

    Xenopus laevis are used extensively here at MED-Duluth as a model for assessing development toxicity to xenobiotics. As a result, a culturing system has been developed that provides eggs and tadpoles of consistent high quality for use by researchers at the facility. The methods ...

  10. Xenopus laevis in Developmental and Molecular Biology.

    ERIC Educational Resources Information Center

    Dawid, Igor B.; Sargent, Thomas D.

    1988-01-01

    Discusses the advantages of Xenopus laevis as an experimental animal in the study of embryogenesis in vertebrates. Summarizes the contributions of this system to the analysis of ribosomal and 5S RNA genes, and the diverse and highly productive applications of the oocyte injection technology. (RT)

  11. Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes.

    PubMed

    Caixeta, Ester S; Sutton-McDowall, Melanie L; Gilchrist, Robert B; Thompson, Jeremy G; Price, Christopher A; Machado, Mariana F; Lima, Paula F; Buratini, José

    2013-07-01

    Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.

  12. Functional Differentiation of Uterine Stromal Cells Involves Cross-regulation between Bone Morphogenetic Protein 2 and Kruppel-like Factor (KLF) Family Members KLF9 and KLF13

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The inability of the uterine epithelium to enter a state of receptivity for the embryo to implant is a significant underlying cause of early pregnancy loss. We previously showed that mice null for the Progesterone Receptor (PGR)-interacting protein Kruppel-like Factor (KLF) 9 are subfertile and exhi...

  13. Structure of bone morphogenetic protein 9 procomplex

    PubMed Central

    Mi, Li-Zhi; Brown, Christopher T.; Gao, Yijie; Tian, Yuan; Le, Viet Q.; Walz, Thomas; Springer, Timothy A.

    2015-01-01

    Bone morphogenetic proteins (BMPs) belong to the TGF-β family, whose 33 members regulate multiple aspects of morphogenesis. TGF-β family members are secreted as procomplexes containing a small growth factor dimer associated with two larger prodomains. As isolated procomplexes, some members are latent, whereas most are active; what determines these differences is unknown. Here, studies on pro-BMP structures and binding to receptors lead to insights into mechanisms that regulate latency in the TGF-β family and into the functions of their highly divergent prodomains. The observed open-armed, nonlatent conformation of pro-BMP9 and pro-BMP7 contrasts with the cross-armed, latent conformation of pro-TGF-β1. Despite markedly different arm orientations in pro-BMP and pro-TGF-β, the arm domain of the prodomain can similarly associate with the growth factor, whereas prodomain elements N- and C-terminal to the arm associate differently with the growth factor and may compete with one another to regulate latency and stepwise displacement by type I and II receptors. Sequence conservation suggests that pro-BMP9 can adopt both cross-armed and open-armed conformations. We propose that interactors in the matrix stabilize a cross-armed pro-BMP conformation and regulate transition between cross-armed, latent and open-armed, nonlatent pro-BMP conformations. PMID:25751889

  14. Expansion of murine periosteal progenitor cells with fibroblast growth factor 2 reveals an intrinsic endochondral ossification program mediated by bone morphogenetic protein 2.

    PubMed

    van Gastel, Nick; Stegen, Steve; Stockmans, Ingrid; Moermans, Karen; Schrooten, Jan; Graf, Daniel; Luyten, Frank P; Carmeliet, Geert

    2014-09-01

    The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects. PMID:24989687

  15. cnrip1 is a regulator of eye and neural development in Xenopus laevis.

    PubMed

    Zheng, Xiaona; Suzuki, Toshiyasu; Takahashi, Chika; Nishida, Eisuke; Kusakabe, Morioh

    2015-04-01

    Cannabinoid receptor interacting protein 1 (CNRIP1), which has been originally identified as the binding partner of cannabinoid receptor 1 (CNR1), is evolutionarily conserved throughout vertebrates, but its physiological function has been unknown. Here, we identify a developmental role of CNRIP1 using Xenopus laevis embryos. During early embryogenesis, expression of Xenopus laevis cnrip1 is highly restricted to the animal region of gastrulae where neural and eye induction occur, and afterward it is seen in neural and other tissues with a temporally and spatially regulated pattern. Morpholino-mediated knockdown experiments indicate that cnrip1 has an essential role in early eye and neural development by regulating the onset of expression of key transcription factor genes, sox2, otx2, pax6 and rax. Also, over-expression experiments suggest that cnrip1 has a potential to expand sox2, otx2, pax6 and rax expression. These results suggest an instructive role of Xenopus laevis cnrip1 in early eye and neural development. Furthermore, Xenopus laevis cnr1 knockdown leads to eye defects, which are partly similar to, but milder than, those caused by cnrip1 knockdown, suggesting a possible functional similarity between CNRIP1 and CNR1. This study is the first characterization of an in vivo role of CNRIP1 in the context of whole organisms.

  16. Vocal competition in male Xenopus laevis frogs.

    PubMed

    Tobias, Martha L; Corke, Anna; Korsh, Jeremy; Yin, David; Kelley, Darcy B

    2010-11-01

    Male Xenopus laevis frogs produce underwater advertisement calls that attract gravid females and suppress calling by male competitors. Here we explore whether groups of males establish vocal ranks and whether auditory cues alone suffice for vocal suppression. Tests of male-male pairs within assigned groups reveal linear vocal dominance relations, in which each male has a defined rank. Both the duration over which males interact, as well as the number of competitive opportunities, affect linearity. Linear dominance across the group is stable for about 2 weeks; rank is dynamic. Males engage in physical interactions (clasping) while paired but clasping and vocal rank are not correlated. Playbacks of advertisement calls suppress calling and calls from high- and low-ranking males are equally effective. Thus, auditory cues alone suffice to suppress vocal behavior. Playback intensities equivalent to a nearby male advertising effectively suppress calling while low-intensity playbacks are either ineffective or stimulate vocal behavior. X. laevis advertisement calls are biphasic, composed of alternating fast and slow click trills. Approximately half the males tested are more vocally suppressed by all slow than by all fast trills; thus, these males can distinguish between the two phases. The fully aquatic family Pipidae diverged from terrestrial ancestors approximately 170 mya. Vocal suppression in the X. laevis mating system may represent the translation of an ancient anuran social strategy to underwater life. PMID:21442049

  17. Hepatoregenerative role of bone morphogenetic protein-9

    PubMed Central

    Sosa, Ivan; Cvijanovic, Olga; Celic, Tanja; Cuculic, Drazen; Crncevic-Orlic, Zeljka; Vukelic, Lucian; Cvek, Sanja Zoricic; Dudaric, Luka; Bosnar, Alan; Bobinac, Dragica

    2011-01-01

    Summary Bone morphogenetic protein-9 (BMP-9) is a member of the transforming growth factor beta (TGF-β) superfamily of cytokines, which regulate cell growth and differentiation during embryogenesis. Apart of that, the hypoglycemic potential of BMP-9 is of great interest. It has been confirmed that BMP-9, like insulin, improves glycemia in diabetic mice and regulates directional glucose metabolism in hepatocytes; therefore it is proposed to be a candidate hepatic insulin-sensitizing substance (HISS). In liver fibrosis, due to the portocaval shunt, insulin bypasses the organ and the liver undergoes atrophy. Parenteral administration of insulin reverses atrophy by stimulating mitogenic activity of the hepatocytes. Because BMP-9 has a signaling pathway similar to other BMPs and insulin, it is to be expected that BMP-9 has a certain regenerative role in the liver, supporting the above-mentioned is evidence of BMP-9 expression in Dissè’s spaces and BMP-7’s mitogenic activity in mucosal cells. However, further studies are needed to confirm the possible regenerative role of BMP-9. PMID:22129908

  18. Skin wound healing in different aged Xenopus laevis.

    PubMed

    Bertolotti, Evelina; Malagoli, Davide; Franchini, Antonella

    2013-08-01

    Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8- and 15-month-old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re-epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti-inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti-tumor necrosis factor (TNF)-α, iNOS, transforming growth factor (TGF)-β1, and matrix metalloproteinase (MMP)-9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti-α-SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar-like pattern. The quantitative PCR analysis demonstrated an up-regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar-like tissue formation.

  19. Induction of superficial zone protein (SZP)/lubricin/PRG 4 in muscle-derived mesenchymal stem/progenitor cells by transforming growth factor-β1 and bone morphogenetic protein-7

    PubMed Central

    2012-01-01

    Introduction Articular cartilage (AC) is an avascular tissue with precise polarity and organization. The three distinct zones are: surface, middle and deep. The production and accumulation of the superficial zone protein (SZP), also known as lubricin, by the surface zone is a characteristic feature of AC. To date, there is a wealth of evidence showing differentiation of AC from mesenchymal stem cells. Most studies that described chondrogenic differentiation did not focus on AC with characteristic surface marker SZP/lubricin. The present investigation was initiated to determine the induction of SZP/lubricin in skeletal muscle-derived mesenchymal stem/progenitor cells (MDMSCs) by transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7). Methods MDMSCs were cultured as a monolayer at a density of 1 × 105 cells/well in 12-well tissue culture plates. Cell cultures were treated for 3, 7 and 10 days with TGF-β1 and BMP-7. The medium was analyzed for SZP. The cells were used to isolate RNA for RT-PCR assays for SZP expression. Results The SZP/lubricin increased in a time-dependent manner on Days 3, 7 and 10 in the medium. As early as Day 3, there was a three-fold increase in response to 3 ng/ml of TGF-β1 and 300 ng/ml of BMP-7. This was confirmed by immunochemical localization of SZP as early as Day 3 after treatment with TGF-β1. The expression of SZP mRNA was enhanced by TGF-β1. Conclusions The present investigation demonstrated the efficient and reproducible induction of SZP/lubricin accumulation by TGF-β1 and BMP-7 in skeletal MDMSCs. Optimization of the experimental conditions may permit the utility of MDMSCs in generating surface zone-like cells with phenotypic markers of AC and, therefore, constitute a promising cell source for tissue engineering approaches of superficial zone cartilage. PMID:22490392

  20. Profiling bone morphogenetic proteins and transforming growth factor-βs by hTGF-β3 pre-treated coral-derived macroporous bioreactors: the power of one.

    PubMed

    Ripamonti, Ugo; Dix-Peek, Thérèse; Parak, Ruqayya; Milner, Brenda; Duarte, Raquel

    2015-05-01

    To study the expression profile of bone morphogenetic proteins and transforming growth factor-βs (BMPs and TGFβs), coral-derived calcium carbonate-based macroporous bioreactors with limited conversion to hydroxyapatite (7% HA/CC) were pre-loaded with and without 250 μg hTGF-β3 and implanted in the rectus abdominis of 3 non-human primates Papio ursinus euthanized on day 60. To investigate the required dose of hNoggin, a BMPs antagonist that controls the induction of bone formation, 7% HA/CC were pre-loaded with 150 μg hNoggin, with 125 μg hTGF-β3/150 μg hNoggin, with or without 125 μg hTGF-β3 and implanted in the r. abdominis of 3 additional animals euthanized on day 90. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) evaluated the expression' profile of BMP-2, BMP-3, BMP-4, BMP-6, BMP-7 and TGF-β1, -β2, and -β3 in tissue generating bioreactors as well as in the adjacent r. abdominis muscle. On day 60, 250 μg hTGF-β3 induced bone formation at the periphery of the implanted bioreactors only. On day 90, 125 μg hTGF-β3/treated bioreactors showed the induction of bone formation throughout the macroporous spaces. Untreated bioreactors induced bone, 4.11% vs. 2.00% on days 60 and 90, respectively. In hTGF-β3/treated bioreactors, BMP-2 and BMP-3 were up-regulated at both time periods, both in the homogenized constructs and in the adjacent r. abdominis muscle whilst BMP-4 in the homogenized construct only. In untreated 7% HA/CC constructs, BMP-2 was up-regulated in the macroporous construct only. On day 60, 250 μg hTGF-β3/treated and untreated macroporous constructs showed up-regulation of TGF-β1 with a six fold increase vs. TGF-β1 expression in adjacent muscle of untreated constructs. TGF-β2 was down regulated in both untreated and 250 μg hTGF-β3/treated bioreactors. On day 60, 250 μg hTGF-β3/treated bioreactors showed TGF-β3 expression in untreated, treated and adjacent muscle tissues. On day 90, BMP-2 was up

  1. Effects of bioactive glass S53P4 or beta-tricalcium phosphate and bone morphogenetic protein-2 and bone morphogenetic protein-7 on osteogenic differentiation of human adipose stem cells

    PubMed Central

    Patrikoski, Mimmi; Juntunen, Miia; Kujala, Kasperi; Kääriäinen, Minna; Kuokkanen, Hannu; Sándor, George K; Vapaavuori, Outi; Suuronen, Riitta; Mannerström, Bettina; von Rechenberg, Brigitte; Miettinen, Susanna

    2012-01-01

    The effects of bioactive glass S53P4 or beta-tricalcium phosphate; and bone morphogenetic proteins bone morphogenetic protein-2, bone morphogenetic protein-7, or bone morphogenetic protein-2 + 7 on osteogenic differentiation of human adipose stem cells were compared in control medium, osteogenic medium, and bone morphogenetic protein-supplemented osteogenic medium to assess suitability for bone tissue engineering. Cell amount was evaluated with qDNA measurements; osteogenic differentiation using marker gene expression, alkaline phosphate activity, and angiogenic potential was measured by vascular endothelial growth factor expression. As compared to beta-tricalcium phosphate, cell amount was significantly greater for bioactive glass in control medium after 7 days and in osteogenic medium after 14 days, and alkaline phosphate activity was always significantly greater for bioactive glass in control medium. However, alkaline phosphate activity increased for beta-tricalcium phosphate and decreased for bioactive glass granules in osteogenic medium. For both biomaterials, bone morphogenetic protein supplementation decreased cell amount and osteogenic differentiation of human adipose stem cells, and vascular endothelial growth factor expressions correlated with cell amounts. Effects of culture medium on human adipose stem cells are biomaterial dependent; bioactive glass in control medium enhanced osteogenic differentiation most effectively. PMID:23316275

  2. The Bone Morphogenetic Protein 1/Tolloid-like Metalloproteinases

    PubMed Central

    Hopkins, Delana R.; Keles, Sunduz; Greenspan, Daniel S.

    2009-01-01

    A decade ago, bone morphogenetic protein 1 (BMP1) was shown to provide the activity necessary for proteolytic removal of the C-propeptides of procollagens I–III: precursors of the major fibrillar collagens. Subsequent studies have shown BMP1 to be the prototype of a small group of extracellular metalloproteinases that play manifold roles in regulating formation of the extracellular matrix (ECM). Soon after initial cloning of BMP1, genetic studies showed the related Drosophila proteinase Tolloid (TLD) to be necessary for formation of the dorsal-ventral axis in early embryogenesis. It is now clear that the BMP1/TLD-like proteinases, conserved in species ranging from Drosophila to humans, act in dorsal-ventral patterning via activation of transforming growth factor β (TGFβ)-like proteins BMP2, BMP4 (vertebrates) and decapentaplegic (arthropods). More recently, it has become apparent that the BMP1/TLD-like proteinases are key activators of a broader subset of the TGFβ superfamily of proteins, with implications that these proteinases may be key in orchestrating formation of ECM with growth factor activation and BMP signaling in morphogenetic processes. PMID:17560775

  3. Effect of water hardness on oocyte quality and embryo development in the African clawed frog (Xenopus laevis).

    PubMed

    Godfrey, Earl W; Sanders, George E

    2004-04-01

    Husbandry and health of the African clawed frog, Xenopus laevis, greatly influences the quality of oocytes produced. One factor affecting oocyte quality is the water conditions in which females are maintained. Dechlorination and adequate salt concentration are known to affect oocytes, but water hardness has not been considered an important factor in Xenopus husbandry by the research community. We found that, when females were kept in soft water or water with marine salts alone, even when it was cooled to 17 to 18 degrees C, the quality of oocytes decreased; only 20 to 25% of resulting embryos developed to tailbud stages. Survival and normal development of embryos increased significantly within one month of addition to the laboratory housing water of salts that mimic conditions in African Rift Valley lakes. These salts greatly increased water hardness; development of embryos to tailbud stages remained high (50 to 70% on average) for more than a year after their addition to the water housing females. Water from South African ponds where X. laevis are collected, and from wells used by the major suppliers of X. laevis, also was moderately to very hard. Our results suggest that X. laevis is naturally adapted to hard water, and indicate that increasing general hardness during laboratory housing is more important for oocyte quality and embryo development than is increasing carbonate hardness (alkalinity) in the water used to house females.

  4. Photoconversion for tracking the dynamics of cell movement in Xenopus laevis embryos.

    PubMed

    Chernet, Brook T; Adams, Dany S; Levin, Michael

    2012-06-01

    Xenopus laevis is an ideal model system for investigating dynamic morphogenetic processes during embryogenesis, regeneration, and homeostasis. Our understanding of these events has been greatly facilitated by lineage labeling, that is, marking a cell or a group of cells and all their descendants using vital dyes, fluorescent molecules, or transplantation techniques. Unfortunately, these strategies are limited in their spatiotemporal resolution: They do not allow long-term dynamic in vivo imaging, are generally invasive, and labeling is restricted to cells on the surface. Genetically encoded fluorescent proteins (FPs), on the other hand, provide excellent alternative methods to traditional lineage labeling, enabling labeling with high spatiotemporal resolution and tracking of cellular and subcellular structures to study patterning events. Over the past decade, FPs have evolved to allow fine control of their spectral properties (in a defined region of interest) for greater labeling specificity. One example is EosFP, which is a protein cloned from the scleractinian coral Lobophyllia hemprichii that can be photoconverted from green to red fluorescence state with near-ultraviolet (UV) light irradiation. Here, we describe EosFP-photoconversion of Xenopus embryos to track cells during developmental and regenerative processes using a metal-halide- or xenon-arc-based fluorescent microscope system, which provides a simpler, less expensive alternative to photoconversion using laser microscopy. PMID:22661444

  5. Expression analysis of epb41l4a during Xenopus laevis embryogenesis

    PubMed Central

    Guo, Yanchun; Christine, Kathleen S.; Conlon, Frank; Gessert, Susanne

    2013-01-01

    Epbl41l4a (erythrocyte protein band 4.1-like 4a, also named Nbl4) is a member of the band 4.1/Nbl4 (novel band 4.1-like protein 4) group of the FERM (4.1, ezrin, radixin, moesin) protein superfamily. Proteins encoded by this gene family are involved in many cellular processes such as organization of epithelial cells and signal transduction. On a molecular level, band 4.1/Nbl4 proteins have been shown to link membrane-associated proteins and lipids to the actin cytoskeleton. Epbl41l4a has also recently been identified as a target gene of the Wnt/β-catenin pathway. Here, we describe for the first time the spatiotemporal expression of epbl41l4a using Xenopus laevis as a model system. We observed a strong and specific expression of epb41l4a in the developing somites, in particular during segmentation as well as in the nasal and cranial placodes, pronephros, and neural tube. Thus, epbl41l4a is expressed in tissues undergoing morphogenetic movements, suggesting a functional role of epbl41l4a during these processes. PMID:21556855

  6. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

  7. Thyroid hormone signaling in the Xenopus laevis embryo is functional and susceptible to endocrine disruption.

    PubMed

    Fini, J B; Le Mével, S; Palmier, K; Darras, V M; Punzon, I; Richardson, S J; Clerget-Froidevaux, M S; Demeneix, B A

    2012-10-01

    Thyroid hormone (TH) is essential for vertebrate brain development. Most research on TH and neuronal development focuses on late development, mainly the perinatal period in mammals. However, in human infants neuromotor development correlates best with maternal TH levels in the first trimester of pregnancy, suggesting that TH signaling could affect early brain development. Studying TH signaling in early embryogenesis in mammals is experimentally challenging. In contrast, free-living embryos, such as Xenopus laevis, permit physiological experimentation independent of maternal factors. We detailed key elements of TH signaling: ligands, receptors (TR), and deiodinases during early X. laevis development, before embryonic thyroid gland formation. Dynamic profiles for all components were found. Between developmental stages 37 and 41 (~48 h after hatching, coincident with a phase of continuing neurogenesis) significant increases in T(3) levels as well as in mRNA encoding deiodinases and TR occurred. Exposure of embryos at this developmental stage for 24 h to either a TH antagonist, NH-3, or to tetrabromobisphenol A, a flame retardant and known TH disruptor, differentially modulated the expression of a number of TH target genes implicated in neural stem cell function or neural differentiation. Moreover, 24-h exposure to either NH-3 or tetrabromobisphenol A diminished cell proliferation in the brain. Thus, these data show first, that TH signaling exerts regulatory roles in early X. laevis neurogenesis and second, that this period represents a potential window for endocrine disruption. PMID:22968643

  8. Xenopus laevis - A success story in biological research in Space

    NASA Astrophysics Data System (ADS)

    Horn, E.

    A feature of sensory, neuronal and motor systems is the existence of a critical period during their development. Environmental modifications, in particular stimulus depri-vation, during this period of life affects development in a long-term manner. For gravity sensory systems, space flights offer the only opportunity for deprivation conditions. Studies in the amphibian Xenopus laevis presented the most complete picture. The presentation demonstrates the importance of Xenopus laevis as an ex-perimental model animal in the past and even for future research in Space. Studies are presented which range from fertilization in Space and anatomical studies during early development under weightlessness up to post-flight studies on the anatomy of the peripheral sense organ, the spinal motor activity and behavior. Gravity depriva-tion induces anatomical as well as behavioral and neurophysiological modifications, which are normalized either during flight (thickening of the blastocoel roof) or after reentry in 1g-conditions (swimming and reflex behavior, spinal motor activity). The physiological changes can be explained by mechanisms of physiological adaptation. However, the studies also revealed stages which were insensitive to gravity depriva-tion; they point to the existence of a critical period. Observations on morphological mal-formations are described which are reversible after termination of microgravity and which are linked to a depression of vestibular reflex behavior. They might be caused by a competition between dorsalization and ventralization inducing growth factors. This observation offers the possibility for a genetic approach in finding ba-sics for microgravity effects on the development of Xenopus, and in a general frame, on the development of vertebrates including men. At the present stage of research, it remains open whether adaptive processes during exposure to altered gravity or the existence of a critical period in vestibular development are responsible for

  9. Chromatin immunoprecipitation and deep sequencing in Xenopus tropicalis and Xenopus laevis

    PubMed Central

    Wills, Andrea E.; Gupta, Rakhi; Chuong, Edward; Baker, Julie C.

    2014-01-01

    Chromatin immunoprecipitation and deep sequencing (ChIP-SEQ) represents a powerful tool for identifying the genomic targets of transcription factors, chromatin remodeling factors, and histone modifications. The frogs Xenopus laevis and Xenopus tropicalis have historically been outstanding model systems for embryology and cell biology, with emerging utility as highly accessible embryos for genome-wide studies. Here we focus on the particular strengths and limitations of Xenopus cell biology and genomics as they apply to ChIP-SEQ, and outline a methodology for ChIP-SEQ in both species, providing detailed strategies for sample preparation, antibody selection, quality control, sequencing library preparation, and basic analysis. PMID:24064036

  10. Plasticity of lung development in the amphibian, Xenopus laevis

    PubMed Central

    Rose, Christopher S.; James, Brandon

    2013-01-01

    Summary Contrary to previous studies, we found that Xenopus laevis tadpoles raised in normoxic water without access to air can routinely complete metamorphosis with lungs that are either severely stunted and uninflated or absent altogether. This is the first demonstration that lung development in a tetrapod can be inhibited by environmental factors and that a tetrapod that relies significantly on lung respiration under unstressed conditions can be raised to forego this function without adverse effects. This study compared lung development in untreated, air-deprived (AD) and air-restored (AR) tadpoles and frogs using whole mounts, histology, BrdU labeling of cell division and antibody staining of smooth muscle actin. We also examined the relationship of swimming and breathing behaviors to lung recovery in AR animals. Inhibition and recovery of lung development occurred at the stage of lung inflation. Lung recovery in AR tadpoles occurred at a predictable and rapid rate and correlated with changes in swimming and breathing behavior. It thus presents a new experimental model for investigating the role of mechanical forces in lung development. Lung recovery in AR frogs was unpredictable and did not correlate with behavioral changes. Its low frequency of occurrence could be attributed to developmental, physical and behavioral changes, the effects of which increase with size and age. Plasticity of lung inflation at tadpole stages and loss of plasticity at postmetamorphic stages offer new insights into the role of developmental plasticity in amphibian lung loss and life history evolution. PMID:24337117

  11. Xenopus laevis a success story of biological research in space

    NASA Astrophysics Data System (ADS)

    Horn, Eberhard R.

    2006-01-01

    The clawed toad Xenopus laevis is a common experimental animal used in many disciplines of life sciences, such as integrative, developmental and molecular biology or experimental medicine. Since 30 years, Xenopus is used in biological research in space. Important milestones were the years 1975, when Xenopus embryos flew for the first time on the Russian space station Salut-4 and 1994, when Xenopus eggs were successfully fertilized for the first time in space during the Japanese Spacelab mission STS-47 and developed in microgravity to vital tadpoles. Most Xenopus studies were related to embryogenesis and development. Observations during and after altered gravity revealed changes such as the thickening of the blastocoel roof, the dorsalization of the tail, and modifications of vestibular reflexes, fictive and freely swimming. Many changes were reversible even during microgravity exposure. Studies about the vestibuloocular reflex or synapse formation revealed an age-related sensitivity to altered gravity. Xenopus offers useful tools for studies about microgravity effects on living systems. Its oocyte is a suitable model to study ion channel function in space; the dorsalization model can be used to analyse growth factor sensibilities. Hardware for life support of adults, tadpoles and embryos (cf. SUPPLY unit in combination with miniaquaria) as well as for controlled experiments in space are prerequisites for an extension of research with Xenopus. The application aspect is based on the fact that fundamental research per se brings benefit to man.

  12. Protein pattern of Xenopus laevis embryos grown in simulated microgravity.

    PubMed

    Tedeschi, Gabriella; Pagliato, Lara; Negroni, Manuela; Montorfano, Gigliola; Corsetto, Paola; Nonnis, Simona; Negri, Armando; Rizzo, Angela Maria

    2011-03-01

    Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.

  13. Early development of the thymus in Xenopus laevis

    PubMed Central

    Lee, Young-Hoon; Williams, Allison; Hong, Chang-Soo; You, Youngjae; Senoo, Makoto; Saint-Jeannet, Jean-Pierre

    2012-01-01

    Background Although Xenopus laevis has been a model of choice for comparative and developmental studies of the immune system, little is known about organogenesis of the thymus, a primary lymphoid organ in vertebrates. Here we examined the expression of three transcription factors that have been functionally associated with pharyngeal gland development, gcm2, hoxa3 and foxn1, and evaluated the neural crest contribution to thymus development. Results In most species Hoxa3 is expressed in the third pharyngeal pouch endoderm where it directs thymus formation. In Xenopus, the thymus primordium is derived from the second pharyngeal pouch endoderm, which is hoxa3-negative, suggesting that a different mechanism regulates thymus formation in frogs. Unlike other species foxn1 is not detected in the epithelium of the pharyngeal pouch in Xenopus, rather, its expression is initiated as thymic epithelial cell starts to differentiate and express MHC class II molecules. Using transplantation experiments we show that while neural crest cells populate the thymus primordia, they are not required for the specification and initial development of this organ or for T cell differentiation in frogs. Conclusions These studies provide novel information on early thymus development in Xenopus, and highlight a number of features that distinguish Xenopus from other organisms. PMID:23172757

  14. Facial Transplants in Xenopus laevis Embryos

    PubMed Central

    Sive, Hazel

    2014-01-01

    Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals. PMID:24748020

  15. Bone Morphogenetic Protein (BMP) signaling in development and human diseases

    PubMed Central

    Wang, Richard N.; Green, Jordan; Wang, Zhongliang; Deng, Youlin; Qiao, Min; Peabody, Michael; Zhang, Qian; Ye, Jixing; Yan, Zhengjian; Denduluri, Sahitya; Idowu, Olumuyiwa; Li, Melissa; Shen, Christine; Hu, Alan; Haydon, Rex C.; Kang, Richard; Mok, James; Lee, Michael J.; Luu, Hue L.; Shi, Lewis L.

    2014-01-01

    Bone Morphogenetic Proteins (BMPs) are a group of signaling molecules that belongs to the Transforming Growth Factor-β (TGF-β) superfamily of proteins. Initially discovered for their ability to induce bone formation, BMPs are now known to play crucial roles in all organ systems. BMPs are important in embryogenesis and development, and also in maintenance of adult tissue homeostasis. Mouse knockout models of various components of the BMP signaling pathway result in embryonic lethality or marked defects, highlighting the essential functions of BMPs. In this review, we first outline the basic aspects of BMP signaling and then focus on genetically manipulated mouse knockout models that have helped elucidate the role of BMPs in development. A significant portion of this review is devoted to the prominent human pathologies associated with dysregulated BMP signaling. PMID:25401122

  16. Role of bone morphogenetic proteins in adrenal physiology and disease.

    PubMed

    Johnsen, Inga K; Beuschlein, Felix

    2010-04-01

    Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of ligands that impact on a multitude of biological processes including cell type specification, differentiation and organogenesis. Furthermore, a large body of evidence points towards important BMP-dependent mechanisms in tumorigenesis. In accordance with their diverse actions, BMPs have been demonstrated to serve as auto-, para- and endocrine modulators also in a number of hormonal systems. In this review, we highlight novel aspects of BMP-dependent regulatory networks that pertain to adrenal physiology and disease, which have been uncovered during recent years. These aspects include the role of BMP-dependent mechanism during adrenal development, modulating effects on catecholamine synthesis and steroidogenesis and dysregulation of BMP signalling in adrenal tumorigenesis. Furthermore, we summarize potential therapeutic approaches that are based on reconstitution of BMP signalling in adrenocortical tumour cells. PMID:20133384

  17. Fine-tuned shuttles for bone morphogenetic proteins.

    PubMed

    Wharton, Kristi A; Serpe, Mihaela

    2013-08-01

    Bone morphogenetic proteins (BMPs) are potent secreted signaling factors that trigger phosphorylation of Smad transcriptional regulators through receptor complex binding at the cell-surface. Resulting changes in target gene expression impact critical cellular responses during development and tissue homeostasis. BMP activity is tightly regulated in time and space by secreted modulators that control BMP extracellular distribution and availability for receptor binding. Such extracellular regulation is key for BMPs to function as morphogens and/or in the formation of morphogen activity gradients. Here, we review shuttling systems utilized to control the distribution of BMP ligands in tissue of various geometries, developing under different temporal constraints. We discuss the biological advantages for employing specific strategies for BMP shuttling and roles of varied ligand forms.

  18. Responses of Squalius cephalus intestinal mucous cells to Pomphorhynchus laevis.

    PubMed

    Bosi, Giampaolo; Sayyaf Dezfuli, Bahram

    2015-04-01

    Intestinal mucous cell numbers and their glycoconjugate composition were investigated by histochemical methods in uninfected chub, Squalius cephalus, and in conspecifics naturally parasitised with the acanthocephalan Pomphorhynchus laevis. A sub-population of 42 chub from the River Tiber (Perugia, Italy) were sampled and screened for ecto and endoparasites. No parasites were found in gills and in other visceral organs of chub and P. laevis appeared to be the only enteric worm encountered. In all infected chub (twenty-eight out of 42) this acanthocephalan was encountered mainly in the mid-gut. In situ, an excessive yellowish mucus or catarrh was observed around each acanthocephalan. Hyperplasia and hypertrophy of the mucous cells were only evident near the site of P. laevis attachment where the total number of mucous cells and the number of those containing acidic, particularly non-sulphated mucins, or mixed glycoconjugates were significantly higher. In intestinal regions of infected fish far away from the point of parasite attachment, there were no statistical differences in the density of mucous cells in comparison to uninfected fish. Interestingly, in parasitised chub, the length of intestinal folds was significantly larger close to the sites at which P. laevis attach when compared to the length of the intestinal folds located further away from the acanthocephalans and/or in uninfected intestines. The effect of P. laevis on intestinal mucous cells of S. cephalus was compared to other parasite-host systems and the role of enhanced mucus production in parasitized intestines was discussed. PMID:25486440

  19. Homeolog-specific targeted mutagenesis in Xenopus laevis using TALENs.

    PubMed

    Nakade, Shota; Sakuma, Tetsushi; Sakane, Yuto; Hara, Yoshihiro; Kurabayashi, Atsushi; Kashiwagi, Keiko; Kashiwagi, Akihiko; Yamamoto, Takashi; Obara, Masanobu

    2015-10-01

    Transcription activator-like effector nucleases (TALENs) have previously been used for targeted genome editing in various organisms including Xenopus laevis. However, because of genomic polyploidization, X. laevis usually possess homeologous genes (homeologs) with quite similar sequences that make the analysis of gene function difficult. In the present study, we show methodological examples of targeted gene modification of X. laevis homeologs. The X. laevis cytoglobin gene (cygb) consists of two homeologs (xlcygba and xlcygbb), and molecular phylogenetic analysis suggested that they have potentially different functions. Thus, there is a need to establish a method of homeolog-specific gene disruption to clarify gene functions in detail. Here, we show successful examples of homeolog-specific and simultaneous gene disruption for xlcygba and xlcygbb. We found that selective digestion can be performed with at least three mismatches in TALEN target sites in both homeologs. This report paves the way for the functional analyses of X. laevis homeologs, even those containing nearly identical sequences. PMID:25920501

  20. Tebuconazole disrupts steroidogenesis in Xenopus laevis.

    PubMed

    Poulsen, Rikke; Luong, Xuan; Hansen, Martin; Styrishave, Bjarne; Hayes, Tyrone

    2015-11-01

    A 27-day controlled exposure study of adult male African clawed frogs (Xenopus laevis) was conducted to examine the mechanism by which tebuconazole may disrupt steroidogenesis. The fungicide was measured by LC-MS/MS in tank water and in target tissues (adipose, kidney, liver, and brain), and we observed tissue-specific bioconcentration with BCF up to 238. Up to 10 different steroid hormones were quantified in gonads using LC-MS/MS and in plasma using GC-MS/MS and a radioimmunoassay was performed for further measurement of androgens. In order to assess whether effects increased with exposure or animals adapted to the xenobiotic, blood samples were collected 12 days into the study and at termination (day 27). After 12 days of exposure to 100 and 500μgL(-1) tebuconazole, plasma levels of testosterone (T) and dihydrotestosterone (DHT) were increased, while plasma 17β-estradiol (E2) concentrations were greatly reduced. Exposure to 0.1μgL(-1), on the other hand, resulted in decreased levels of T and DHT, with no effects observed for E2. After 27 days of exposure, effects were no longer observed in circulating androgen levels while the suppressive effect on E2 persisted in the two high-exposure groups (100 and 500μgL(-1)). Furthermore, tebuconazole increased gonadal concentrations of T and DHT as well as expression of the enzyme CYP17 (500μgL(-1), 27 days). These results suggest that tebuconazole exposure may supress the action of CYP17 at the lowest exposure (0.1μgL(-1)), while CYP19 suppression dominates at higher exposure concentrations (increased androgens and decreased E2). Increased androgen levels in plasma half-way into the study and in gonads at termination may thus be explained by compensatory mechanisms, mediated through increased enzymatic expression, as prolonged exposure had no effect on circulating androgen levels. PMID:26432166

  1. Functional domains of the Xenopus laevis 5S gene promoter.

    PubMed Central

    Pieler, T; Oei, S L; Hamm, J; Engelke, U; Erdmann, V A

    1985-01-01

    To study the fine structure of the Xenopus laevis somatic 5S gene internal control region, we have created 15 different transversions using mutagenic oligonucleotide primers. The effects of these mutations on 5S DNA transcription in vitro as well as on stable complex formation with transcription factor TF III A and TF III C in crude nuclear extracts were analyzed. Mutations in the common class III 5' promoter element (nucleotides 50-61 in the 5S gene) interfere with transcription activity and stable complex formation whenever they contradict the tDNA box A consensus sequence. The second promoter element is defined by a major sequence block (nucleotides 80-89, box C) and two additional internal residues (70 and 71) at a distance of roughly one helical turn from both the major 3' and 5' control sequences; these two 3' elements contain the primary TF III A binding domain. The remaining nucleotides (62-69 and 71-79) when mutated do not interfere with transcription activity or factor binding and thus they constitute two spacer elements within a symmetrically structured 5S gene promoter. An increase in the relative spacing of box A and box C by insertion of 3 bp between nucleotides 66 and 67 leads to a drastic reduction in transcription activity and the ability to form a stable complex with TF III A and/or TF III C. Thus, accurate spacing is essential for the proper orientation of TF III A on 5S DNA and/or TF III C binding. Images Fig. 1. Fig. 3. Fig. 4. PMID:3004969

  2. Plasticity in the melanotrope neuroendocrine interface of Xenopus laevis.

    PubMed

    Jenks, Bruce G; Kidane, Adhanet H; Scheenen, Wim J J M; Roubos, Eric W

    2007-01-01

    Melanotrope cells of the amphibian pituitary pars intermedia produce alpha-melanophore-stimulating hormone (alpha-MSH), a peptide which causes skin darkening during adaptation to a dark background. The secretory activity of the melanotrope of the South African clawed toad Xenopus laevis is regulated by multiple factors, both classical neurotransmitters and neuropeptides from the brain. This review concerns the plasticity displayed in this intermediate lobe neuroendocrine interface during physiological adaptation to the environment. The plasticity includes dramatic morphological plasticity in both pre- and post-synaptic elements of the interface. Inhibitory neurons in the suprachiasmatic nucleus, designated suprachiasmatic melanotrope-inhibiting neurons (SMINs), possess more and larger synapses on the melanotrope cells in white than in black-background adapted animals; in the latter animals the melanotropes are larger and produce more proopiomelanocortin (POMC), the precursor of alpha-MSH. On a white background, pre-synaptic SMIN plasticity is reflected by a higher expression of inhibitory neuropeptide Y (NPY) and is closely associated with postsynaptic melanotrope plasticity, namely a higher expression of the NPY Y1 receptor. Interestingly, melanotrope cells in such animals also display higher expression of the receptors for thyrotropin-releasing hormone (TRH) and urocortin 1, two neuropeptides that stimulate alpha-MSH secretion. Possibly, in white-adapted animals melanotropes are sensitized to neuropeptide stimulation so that, when the toad moves to a black background, they can immediately initiate alpha-MSH secretion to achieve rapid adaptation to the new background condition. The melanotrope cell also produces brain-derived neurotrophic factor (BDNF), which is co-sequestered with alpha-MSH in secretory granules within the cells. The neurotrophin seems to control melanotrope cell plasticity in an autocrine way and we speculate that it may also control presynaptic

  3. Localization of two IQGAPs in cultured cells and early embryos of Xenopus laevis.

    PubMed

    Yamashiro, Sawako; Noguchi, Tatsuhiko; Mabuchi, Issei

    2003-05-01

    Mammalian IQGAP1 is considered to modulate organization of the actin cytoskeleton under regulation of signaling proteins Cdc42 or Rac and calmodulin [Bashour et al., 1997: J Cell Biol 137:1555-1566; Hart et al., 1996: EMBO J 15:2997-3005] and also to be involved in cadherin-based cell adhesion [Kuroda et al., 1998: Science 281:832-835]. However, its function in the cell has not been clear. In order to clarify the function of IQGAP, we investigated IQGAP in Xenopus laevis cells. We isolated two Xenopus cDNAs encoding homologues of mammalian IQGAP, XIQGAP1, and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. Immunofluorescent localization of XIQGAPs in Xenopus tissue cultured cells (XTC cells) and in developing embryos was examined. In XTC cells, XIQGAP1 was colocalized with F-actin at cell-to-cell contact sites, membrane ruffles in lamellipodia, and filopodia. During development of embryos, XIQGAP1 was concentrated in the borders of all embryonic cells. An intense staining for XIQGAP1 was found in regions undergoing active morphogenetic movements, such as the blastopore lip of gastrulae, and the neural plate, the notochord, and the somite of neurulae. These results suggest that XIQGAP1 is involved in both cell-to-cell adhesion and cell migration during Xenopus embryogenesis and in cultured cells. On the other hand, the localization of XIQGAP2 in XTC cells was distinct from that of XIQGAP1 although it was also seen in lamellipodia, filopodia, and borders between cells. In addition to these regions, strong nuclear staining was observed in both XTC cells and embryonic cells.

  4. Dehydration triggers differential microRNA expression in Xenopus laevis brain.

    PubMed

    Luu, Bryan E; Storey, Kenneth B

    2015-11-15

    African clawed frogs, Xenopus laevis, although primarily aquatic, have a high tolerance for dehydration, being capable of withstanding the loss of up to 32-35% of total water body water. Recent studies have shown that microRNAs play a role in the response to dehydration by the liver, kidney and ventral skin of X. laevis. MicroRNAs act by modulating the expression of mRNA transcripts, thereby affecting diverse biochemical pathways. In this study, 43 microRNAs were assessed in frog brains comparing control and dehydrated (31.2±0.83% of total body water lost) conditions. MicroRNAs of interest were measured using a modified protocol which employs polyadenylation of microRNAs prior to reverse transcription and qPCR. Twelve microRNAs that showed a significant decrease in expression (to 41-77% of control levels) in brains from dehydrated frogs (xla-miR-15a, -150, -181a, -191, -211, -218, -219b, -30c, -30e, -31, -34a, and -34b) were identified. Genomic analysis showed that the sequences of these dehydration-responsive microRNAs were highly conserved as compared with the comparable microRNAs of mice (91-100%). Suppression of these microRNAs implies that translation of the mRNA transcripts under their control could be enhanced in response to dehydration. Bioinformatic analysis using the DIANA miRPath program (v.2.0) predicted the top two KEGG pathways that these microRNAs collectively regulate: 1. Axon guidance, and 2. Long-term potentiation. Previous studies indicated that suppression of these microRNAs promotes neuroprotective pathways by increasing the expression of brain-derived neurotrophic factor and activating anti-apoptotic pathways. This suggests that similar actions may be triggered in X. laevis brains as a protective response to dehydration. PMID:26169019

  5. Morphogenetic functions of extraembryonic membranes in insects.

    PubMed

    Schmidt-Ott, Urs; Kwan, Chun Wai

    2016-02-01

    Morphogenetic functions of the amnioserosa, the serosa, the amnion, and the yolk sac are reviewed on the basis of recent studies in flies (Drosophila, Megaselia), beetles (Tribolium), and hemipteran bugs (Oncopeltus). Three hypotheses are presented. First, it is suggested that the amnioserosa of Drosophila and the dorsal amnion of other fly species function in a similar manner. Second, it is proposed that in many species with an amniotic cavity, the amnion determines the site of serosa rupture, which, through interactions between the serosa and the amnion, enables the embryo to break free from the amniotic cavity and to close its backside. Finally, it is concluded that the yolk sac is likely an important player in insect morphogenesis. PMID:27436557

  6. RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

    PubMed

    Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K; Menssen, Ruth; Wolf, Dieter H; Hollemann, Thomas

    2015-01-01

    In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development. PMID:25793641

  7. RMND5 from Xenopus laevis Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

    PubMed Central

    Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K.; Menssen, Ruth; Wolf, Dieter H.; Hollemann, Thomas

    2015-01-01

    In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development. PMID:25793641

  8. Novel Approaches to Bone Grafting: Porosity, Bone Morphogenetic Proteins, Stem Cells, and the Periosteum

    PubMed Central

    Petrochenko, Peter; Narayan, Roger J.

    2011-01-01

    The disadvantages involving the use of a patient’s own bone as graft material have led surgeons to search for alternative materials. In this review, several characteristics of a successful bone graft material are discussed. In addition, novel synthetic materials and natural bone graft materials are being considered. Various factors can determine the success of a bone graft substitute. For example, design considerations such as porosity, pore shape, and interconnection play significant roles in determining graft performance. The effective delivery of bone morphogenetic proteins and the ability to restore vascularization also play significant roles in determining the success of a bone graft material. Among current approaches, shorter bone morphogenetic protein sequences, more efficient delivery methods, and periosteal graft supplements have shown significant promise for use in autograft substitutes or autograft extenders. PMID:21488823

  9. Biochemical response to exposure to six textile dyes in early developmental stages of Xenopus laevis.

    PubMed

    Güngördü, Abbas; Birhanli, Ayse; Ozmen, Murat

    2013-01-01

    The present study was undertaken to determine the toxic effect of a lethal concentration of six different commercially used textile dyes on the 46th stage of Xenopus laevis tadpoles. The tadpoles were exposed to Astrazon Red FBL, Astrazon Blue FGRL, Remazol Red RR, Remazol Turquoise Blue G-A, Cibacron Red FN-3G, and Cibacron Blue FN-R for 168 h in static test conditions, and thus, 168-h median lethal concentrations (LC(50)s) of each dye were determined to be 0.35, 0.13, 112, 7, 359, and 15.8 mg/L, respectively. Also, to evaluate the sublethal effects of each dye, tadpoles were exposed to different concentrations of dyes (with respect to 168-h LC(50)s) for 24 h. The alteration of selected enzyme activities was tested. For this aim, glutathione S-transferase (GST), carboxylesterase, and lactate dehydrogenase (LDH) were assayed. After dye exposure, the GST induction or inhibition and LDH induction indicated some possible mechanisms of oxidative stress and deterioration in aerobic respiration processes induced by the tested dyes. Findings of the study suggest that selected biomarker enzymes are useful in understanding the toxic mechanisms of these dyes in X. laevis tadpoles as early warning indicators. Therefore, these selected biomarkers may evaluate the effect of environmental factors, such as textile dye effluents and other industrial pollutants, on amphibians in biomonitoring studies.

  10. Biochemical response to exposure to six textile dyes in early developmental stages of Xenopus laevis.

    PubMed

    Güngördü, Abbas; Birhanli, Ayse; Ozmen, Murat

    2013-01-01

    The present study was undertaken to determine the toxic effect of a lethal concentration of six different commercially used textile dyes on the 46th stage of Xenopus laevis tadpoles. The tadpoles were exposed to Astrazon Red FBL, Astrazon Blue FGRL, Remazol Red RR, Remazol Turquoise Blue G-A, Cibacron Red FN-3G, and Cibacron Blue FN-R for 168 h in static test conditions, and thus, 168-h median lethal concentrations (LC(50)s) of each dye were determined to be 0.35, 0.13, 112, 7, 359, and 15.8 mg/L, respectively. Also, to evaluate the sublethal effects of each dye, tadpoles were exposed to different concentrations of dyes (with respect to 168-h LC(50)s) for 24 h. The alteration of selected enzyme activities was tested. For this aim, glutathione S-transferase (GST), carboxylesterase, and lactate dehydrogenase (LDH) were assayed. After dye exposure, the GST induction or inhibition and LDH induction indicated some possible mechanisms of oxidative stress and deterioration in aerobic respiration processes induced by the tested dyes. Findings of the study suggest that selected biomarker enzymes are useful in understanding the toxic mechanisms of these dyes in X. laevis tadpoles as early warning indicators. Therefore, these selected biomarkers may evaluate the effect of environmental factors, such as textile dye effluents and other industrial pollutants, on amphibians in biomonitoring studies. PMID:22802115

  11. Renal branching morphogenesis: morphogenetic and signaling mechanisms.

    PubMed

    Blake, Joshua; Rosenblum, Norman D

    2014-12-01

    The human kidney is composed of an arborized network of collecting ducts, calyces and urinary pelvis that facilitate urine excretion and regulate urine composition. The renal collecting system is formed in utero, completed by the 34th week of gestation in humans, and dictates final nephron complement. The renal collecting system arises from the ureteric bud, a derivative of the intermediate-mesoderm derived nephric duct that responds to inductive signals from adjacent tissues via a process termed ureteric induction. The ureteric bud subsequently undergoes a series of iterative branching and remodeling events in a process called renal branching morphogenesis. Altered signaling that disrupts patterning of the nephric duct, ureteric induction, or renal branching morphogenesis leads to varied malformations of the renal collecting system collectively known as congenital anomalies of the kidney and urinary tract (CAKUT) and is the most frequently detected congenital renal aberration in infants. Here, we describe critical morphogenetic and cellular events that govern nephric duct specification, ureteric bud induction, renal branching morphogenesis, and cessation of renal branching morphogenesis. We also highlight salient molecular signaling pathways that govern these processes, and the investigative techniques used to interrogate them. PMID:25080023

  12. Polarities of the centriolar structure: morphogenetic consequences.

    PubMed

    Beisson, J; Jerka-Dziadosz, M

    1999-01-01

    Centrioles and basal bodies are two versions of the same conserved eukaryotic organelle and share two remarkable properties: nine-fold symmetry of their microtubular shaft and capacity to generate a new organelle in a fixed geometrical relationship to the mother organelle. It can thus be postulated that what is true for basal bodies is likely to be true also for centrioles. While the functions of centrioles are difficult to dissect, the functions of basal bodies are easier to approach. Over more than two decades, studies on protists have led to the notion that ciliary and flagellar basal bodies display polarities, not only a proximo-distal polarity, like in centrioles, but also a circumferential polarity accorded to the polarities of the cell and of its cytoskeleton. The major cytological and genetical data, mainly of Chlamydomonas, Paramecium and Tetrahymena, which support the notion that the microtubule triplets of basal bodies are non-equivalent, are reviewed. The morphogenetic implications of this circumferential anisotropy, perpetuated through the process of basal body duplication itself, are discussed. The question is raised of the possibility that centrioles also display a circumferential polarity, like basal bodies, and whether at least certain of their functions depend on such asymmetries.

  13. Atomic force microscopy of living and fixed Xenopus laevis embryos.

    PubMed

    Efremov, Yu M; Pukhlyakova, E A; Bagrov, D V; Shaitan, K V

    2011-12-01

    Xenopus laevis embryos are a rather simple and at the same time a very interesting animal model, which is widely used for research in developmental biology. Intensive coordinated cell movements take place during the multi-cellular organism development. Little is known of the cellular, molecular and biomechanical mechanisms of these movements. The conceptual framework for analysis of cell interactions within integrated populations is poorly developed. We have used atomic force microscopy (AFM) to observe the surface of fixed X. laevis embryos at different stages of their development. We have developed a new sample preparation protocol for these observations. The obtained images were compared with scanning electronic microscopy (SEM) data. Cell rearrangement during morphogenesis in vivo was also visualized by AFM. In the current paper we discuss facilities and challenges of using this technique for further embryo researching.

  14. Photoreceptor development in premetamorphic and metamorphic Xenopus laevis.

    PubMed

    Parker, Ryan O; Mccarragher, Brent; Crouch, Rosalie; Darden, Alix G

    2010-03-01

    Transgenic Xenopus laevis are commonly used to study gene expression in photoreceptors, but only red rods and red cones are known to exist in the pre-metamorphic stages commonly used in transgenic studies. Using RT-PCR, this study shows that violet cones develop in early pre-metamorphic stages (Stage 35) with the red rods and red cones. Green rod development began in Stage 53 with the onset of metamorphosis.

  15. Sustained release emphasizing recombinant human bone morphogenetic protein-2.

    PubMed

    Hollinger; Uludag; Winn

    1998-05-01

    Bone homeostasis is a dynamic process involving a myriad of cells and substrates modulated by regulatory signals such as hormones, growth and differentiating factors. When this environment is damaged, the regenerative sequalae follows a programmed pattern, and the capacity for successful recovery is often dependent on the extent of the injury. Many bony deficits that are excessively traumatic will not result in complete recovery and require therapeutic intervention(s) such as autografting or grafting from banked bone. However, for numerous reasons, an unacceptably high rate of failure is associated with these conventional therapies. Thus, alternative approaches are under investigation. A class of osteogenic regulatory molecules, the bone morphogenetic proteins (BMPs), have been isolated, cloned and characterized as potent supplements to augment bone regeneration. Optimizing a therapeutic application for BMPs may be dependent upon localized sustained release which in kind relies on a safe and well characterized carrier system. This review will discuss the current status of BMPs in bone regeneration and specifically will present the potential for a clinical therapeutic role of recombinant human BMP-2 sustained release carrier systems. PMID:10837631

  16. DRAGON, a bone morphogenetic protein co-receptor.

    PubMed

    Samad, Tarek A; Rebbapragada, Anuradha; Bell, Esther; Zhang, Ying; Sidis, Yisrael; Jeong, Sung-Jin; Campagna, Jason A; Perusini, Stephen; Fabrizio, David A; Schneyer, Alan L; Lin, Herbert Y; Brivanlou, Ali H; Attisano, Liliana; Woolf, Clifford J

    2005-04-01

    Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)beta superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFbeta ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFbeta signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFbeta ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling.

  17. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation*

    PubMed Central

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O.; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  18. Enhanced bone morphogenetic protein-2 performance on hydroxyapatite ceramic surfaces.

    PubMed

    Schuessele, A; Mayr, H; Tessmar, J; Goepferich, A

    2009-09-15

    The immobilization of biomolecules on biomaterial surfaces allows for the control of their localization and retention. In numerous studies, proteins have been simply adsorbed to enhance the biological performance of various materials in vivo. We investigated the potential of surface modification techniques on hydroxyapatite (HA) ceramic discs in an in vitro approach. A novel method for protein immobilization was evaluated using the aminobisphosphonates pamidronate and alendronate, which are strong Ca chelating agents, and was compared with the established silanization technique. Lysozyme and bone morphogenetic protein-2 (BMP-2) were used to assess the suitability of the two surface modification methods with regard to the enzymatic activity of lysozyme and to the capacity of BMP-2 to stimulate the osteoblastic differentiation of C2C12 mouse myoblasts. After immobilization, a 2.5-fold increase in enzymatic activity of lysozyme was observed compared with the control. The alkaline phosphatase activity per cell stimulated by immobilized BMP-2 was 2.5-fold higher [9 x 10(-6) I.U.] than the growth factor on unmodified surfaces [2-4 x 10(-6) I.U.]. With regard to the increase in protein activity, both procedures lead to equivalent results. Thus, the bisphosphonate-based surface modification represents a safe and easy alternative for the attachment of proteins to HA surfaces. PMID:18655137

  19. Expression of bone morphogenetic proteins of human neoplastic epithelial cells.

    PubMed

    Hatakeyama, S; Gao, Y H; Ohara-Nemoto, Y; Kataoka, H; Satoh, M

    1997-07-01

    Bone morphogenetic proteins (BMPs) are crucial factors of osteogenesis. We investigated the expressions of BMP subtypes in human salivary adenocarcinoma cell line (HSG-S8), tongue squamous cell (HSC-4) and gingival squamous cell (Ca9-22) carcinoma cell lines, gastric poorly differentiated adenocarcinoma cell (MNK45) and signet ring cell (KATOIII) carcinoma cell lines, rectal adenocarcinoma (RCM-1, RCM-2, and RCM-3), and thyroid (8505C) and bladder (T24) carcinoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR disclosed that BMP-1 was expressed in all cell lines examined, and BMP-2 was amplified in almost all cells except MKN45. Two squamous cell carcinomas, HSC-4 and Ca9-22, and KATOIII expressed only BMP-1 and BMP-2. MKN45 did not express BMP-2, but expressed BMP-7 and weakly BMP-4 and BMP-5. In addition to the expression BMP-7, and HSG-S8 expressed BMP-6. These findings indicated that the neoplastic epithelial cells possessed a rather great potency to express BMP mRNAs. On the other hand, among these carcinoma cells, HSG-S8 solely induced bone in nude mouse tumors, and HSC-4 and KATOIII contained many calcified masses in tumors while the rest did not induce either. PMID:9247707

  20. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    NASA Astrophysics Data System (ADS)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  1. Promotion of Bone Morphogenetic Protein Signaling by Tetraspanins and Glycosphingolipids

    PubMed Central

    Szymczak, Lindsey C.; Aydin, Taner; Yun, Sijung; Constas, Katharine; Schaeffer, Arielle; Ranjan, Sinthu; Kubba, Saad; Alam, Emad; McMahon, Devin E.; He, Jingpeng; Shwartz, Neta; Tian, Chenxi; Plavskin, Yevgeniy; Lindy, Amanda; Dad, Nimra Amir; Sheth, Sunny; Amin, Nirav M.; Zimmerman, Stephanie; Liu, Dennis; Schwarz, Erich M.; Smith, Harold; Krause, Michael W.; Liu, Jun

    2015-01-01

    Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like “Sma/Mab” signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development. PMID:25978409

  2. Morphogenetic action through flux-limited spreading.

    PubMed

    Verbeni, M; Sánchez, O; Mollica, E; Siegl-Cachedenier, I; Carleton, A; Guerrero, I; Ruiz i Altaba, A; Soler, J

    2013-12-01

    A central question in biology is how secreted morphogens act to induce different cellular responses within a group of cells in a concentration-dependent manner. Modeling morphogenetic output in multicellular systems has so far employed linear diffusion, which is the normal type of diffusion associated with Brownian processes. However, there is evidence that at least some morphogens, such as Hedgehog (Hh) molecules, may not freely diffuse. Moreover, the mathematical analysis of such models necessarily implies unrealistic instantaneous spreading of morphogen molecules, which are derived from the assumptions of Brownian motion in its continuous formulation. A strict mathematical model considering Fick's diffusion law predicts morphogen exposure of the whole tissue at the same time. Such a strict model thus does not describe true biological patterns, even if similar and attractive patterns appear as results of applying such simple model. To eliminate non-biological behaviors from diffusion models we introduce flux-limited spreading (FLS), which implies a restricted velocity for morphogen propagation and a nonlinear mechanism of transport. Using FLS and focusing on intercellular Hh-Gli signaling, we model a morphogen gradient and highlight the propagation velocity of morphogen particles as a new key biological parameter. This model is then applied to the formation and action of the Sonic Hh (Shh) gradient in the vertebrate embryonic neural tube using our experimental data on Hh spreading in heterologous systems together with published data. Unlike linear diffusion models, FLS modeling predicts concentration fronts and the evolution of gradient dynamics and responses over time. In addition to spreading restrictions by extracellular binding partners, we suggest that the constraints imposed by direct bridges of information transfer such as nanotubes or cytonemes underlie FLS. Indeed, we detect and measure morphogen particle velocity in such cell extensions in different

  3. A model of morphogenetic pattern formation.

    PubMed

    Cummings, F W

    1990-06-21

    A model for the morphogenetic movement of surfaces composed of cellular monolayers is proposed. The cells are presumed joined at their lateral surfaces. An otherwise unspecified substance called a "morphogen" is introduced which is the agent of change in the individual cell (or cell-like region). The distribution of these cellular deformations define a surface (the middle surface, through the middle of the cell heights) via equations given for the Gauss and Mean curvatures of the surface defined at each point. The Gauss curvature as a function of the morphogen level determines the metric of the surface "g(u, v)" in conformal co-ordinates u, v. A unique equation for the morphogen distribution over the survace is presented which has the property of size invariance, that is, the model "regulates" without need of further arguments. The two resulting coupled equations for the metric and the morphogen, eqns (4) and (2), both non-linear equations, are to be solved self-consistently, once the individual cell deformation as a function of morphogen is given. The surface geometry determines the morphogen distribution, and the morphogen distribution in turn affects the surface geometry. Extension of the model to two or more morphogens is straightforward, and the key property of "regulation" or size invariance of the model is retained. Numerical integration of the two coupled equations is carried out in the case of axial symmetry, and the results presented by the case that individual cells deform by changing the ratio of their apical to basal areas, as well as their heights. Gastrulation in small regulating holoblastic eggs (e.g. starfish, sea urchin and amphioxus) is discussed in light of the present model.

  4. A quantitative adverse outcome pathway model for thyroid axis disruption in Xenopus laevis tadpoles

    EPA Science Inventory

    The development of Xenopus laevis tadpoles is tightly controlled by the thyroid hormones tetraiodothyronine (T4) and triiodothyronine (T3). Toxicity testing efforts have shown that several compounds interfere with development in X. laevis tadpoles by disrupting the thyroid axis a...

  5. Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication

    PubMed Central

    Uno, Y; Nishida, C; Takagi, C; Ueno, N; Matsuda, Y

    2013-01-01

    It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD. PMID:23820579

  6. Expanding the Morphogenetic Repertoire: Perspectives from the Drosophila Egg

    PubMed Central

    Bilder, David; Haigo, Saori L.

    2012-01-01

    SUMMARY Tissue and organ architectures are incredibly diverse, yet our knowledge of the morphogenetic behaviors that generate them is relatively limited. Recent studies have revealed unexpected mechanisms that drive axis elongation in the Drosophila egg, including an unconventional planar polarity signaling pathway, a distinctive type of morphogenetic movement termed ‘global tissue rotation’, a molecular corset-like role of extracellular matrix, and oscillating basal cellular contractions. We review here what is known about Drosophila egg elongation, compare it to other instances of morphogenesis, and highlight several issues of general developmental relevance. PMID:22264728

  7. Turning Bone Morphogenetic Protein 2 (BMP2) On and Off in Mesenchymal Cells†

    PubMed Central

    Rogers, Melissa B.; Shah, Tapan A.; Shaikh, Nadia N.

    2016-01-01

    The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. This article is protected by copyright. All rights reserved PMID:25776852

  8. Bacteriophage φC31 Integrase Mediated Transgenesis in Xenopus laevis for Protein Expression at Endogenous Levels

    NASA Astrophysics Data System (ADS)

    Allen, Bryan G.; Weeks, Daniel L.

    Bacteriophage φC31 inserts its genome into that of its host bacterium via the integrase enzyme which catalyzes recombination between a phage attachment site (attP) and a bacterial attachment site (attB). Integrase requires no accessory factors, has a high efficiency of recombination, and does not need perfect sequence fidelity for recognition and recombination between these attachment sites. These imperfect attachment sites, or pseudo-attachment sites, are present in many organisms and have been used to insert transgenes in a variety of species. Here we describe the φC31 integrase approach to make transgenic Xenopus laevis embryos.

  9. Distribution of two species of sea snakes, Aipysurus laevis and Emydocephalus annulatus, in the southern Great Barrier Reef: metapopulation dynamics, marine protected areas and conservation

    NASA Astrophysics Data System (ADS)

    Lukoschek, V.; Heatwole, H.; Grech, A.; Burns, G.; Marsh, H.

    2007-06-01

    Aipysurus laevis and Emydocephalus annulatus typically occur in spatially discrete populations, characteristic of metapopulations; however, little is known about the factors influencing the spatial and temporal stability of populations or whether specific conservation strategies, such as networks of marine protected areas, will ensure the persistence of species. Classification tree analyses of 35 years of distribution data (90 reefs, surveyed 1-11 times) in the southern Great Barrier Reef (GBR) revealed that longitude was a major factor determining the status of A. laevis on reefs (present = 38, absent = 38 and changed = 14). Reef exposure and reef area were also important; however, these factors did not specifically account for the population fluctuations and the recent local extinctions of A. laevis in this region. There were no relationships between the status of E. annulatus (present = 16, absent = 68 and changed = 6) and spatial or physical variables. Moreover, prior protection status of reefs did not account for the distribution of either species. Biotic factors, such as habitat and prey availability and the distribution of predators, which may account for the observed patterns of distribution, are discussed. The potential for inter-population exchange among sea snake populations is poorly understood, as is the degree of protection that will be afforded to sea snakes by the recently implemented network of No-take areas in the GBR. Data from this study provide a baseline for evaluating the responses of A. laevis and E. annulatus populations to changes in biotic factors and the degree of protection afforded on reefs within an ecosystem network of No-take marine protected areas in the southern GBR.

  10. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

    PubMed Central

    Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  11. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity.

    PubMed

    Alsamarah, Abdelaziz; LaCuran, Alecander E; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  12. Regulation of bone morphogenetic proteins in early embryonic development

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  13. Induction of neural crest in Xenopus by transcription factor AP2alpha.

    PubMed

    Luo, Ting; Lee, Young-Hoon; Saint-Jeannet, Jean-Pierre; Sargent, Thomas D

    2003-01-21

    We report experiments with Xenopus laevis, using both intact embryos and ectodermal explants, showing that the transcription factor AP2alpha is positively regulated by bone morphogenetic protein (BMP) and Wnt signaling, and that this activation is an essential step in the induction of neural crest (NC). Ectopic expression of AP2alpha is sufficient to activate high-level expression of NC-specific genes such as Slug and Sox9, which can occur as isolated domains within the neural plate as well as by expansion of endogenous NC territories. AP2alpha also has the property of inducing NC in isolated ectoderm in which Wnt signaling is provided but BMP signaling is minimized by overexpression of chordin. Like other NC regulatory factors, activation of AP2alpha requires some attenuation of endogenous BMP signaling; however, this process occurs at a lower threshold for AP2alpha. Furthermore, AP2alpha expression domains are larger than for other NC factors. Loss-of-function experiments with antisense AP2alpha morpholino oligonucleotides result in severe reduction in the NC territory. These results support a central role for AP2alpha in NC induction. We propose a model in which AP2alpha expression, along with inactivation of NC inhibitory factors such as Dlx3, establish a feedback loop comprising AP2alpha, Sox9, and Slug, leading to and maintaining NC specification. PMID:12511599

  14. Collagenoma in an African Clawed Frog (Xenopus laevis)

    PubMed Central

    Johnston, Jessica M; Philips, Blythe H; Carty, Anthony J; Klein, Peter S; Brice, Angela K

    2016-01-01

    A 3-y-old female Xenopus laevis was reported for a gray mass on the abdomen. The frog was used for egg collection and was otherwise experimentally naïve. On physical exam, the frog was bright and active and had a firm, gray, lobulated mass (1.5 cm × 0.5 cm × 0.5 cm) in the cutaneous tissue of the left lateral abdomen. An excisional biopsy was performed under anesthesia, and the entire mass was removed and processed for histopathology. Microscopically, the dermis was greatly expanded by connective tissue with a marked decrease in the number of glands, and occasional degenerative glands were present. When stained with Masson trichrome, the excessive connective tissue stained blue, indicating that it was composed of collagen. With Verhoeff–van Gieson staining, the connective tissue stained bright red with an absence of black-staining material, demonstrating the presence of collagen and ruling out elastic fibers. In light of the morphology of the mass and the results of the special stains, the mass was diagnosed as a collagenoma. To our knowledge, this report is the first description of a collagenoma in X. laevis. PMID:26884406

  15. Susceptibility of early life stages of Xenopus laevis to cadmium

    SciTech Connect

    Herkovits, J.; Perez-Coll, C.S.; Cardellini, P.; Pavanati, C.

    1997-02-01

    The susceptibility of Xenopus laevis to cadmium during different stages of development was evaluated by exposing embryos to cadmium concentrations ranging from 0.1 to 10 mg Cd{sup 2+}/L for 24, 48, and 72 h and assessing lethality and malformations. Susceptibility increased from the two blastomeres stage (stage 2) to stage 40, in which the 24-h LC100 was 1.13 mg Cd{sup 2+}/L, and resistance increased from this stage onward. Malformations occurred at all developmental stages evaluated, the most common being reduced size, incurvated axis, underdeveloped or abnormally developed fin, microcephaly, and microphtalmy. Scanning electron microscopy revealed changes in the ectodermal surface ranging from slightly vaulted cells to a severe reduction in the number of ciliated cells as the concentration of cadmium increased. The intraspecific variation evaluated in embryos (from four sets of parents) at seven developmental stages, expressed as the coefficient of variation of the LC100, ranged from 10 to 112% and reflects the capacity of Xenopus laevis to adapt to changing environmental conditions at different embryonic stages.

  16. The Direct Interaction between Two Morphogenetic Proteins Is Essential for Spore Coat Formation in Bacillus subtilis

    PubMed Central

    Isticato, Rachele; Sirec, Teja; Vecchione, Stefano; Crispino, Anna; Saggese, Anella; Baccigalupi, Loredana; Notomista, Eugenio; Driks, Adam; Ricca, Ezio

    2015-01-01

    In Bacillus subtilis the protective layers that surround the mature spore are formed by over seventy different proteins. Some of those proteins have a regulatory role on the assembly of other coat proteins and are referred to as morphogenetic factors. CotE is a major morphogenetic factor, known to form a ring around the forming spore and organize the deposition of the outer surface layers. CotH is a CotE-dependent protein known to control the assembly of at least nine other coat proteins. We report that CotH also controls the assembly of CotE and that this mutual dependency is due to a direct interaction between the two proteins. The C-terminal end of CotE is essential for this direct interaction and CotH cannot bind to mutant CotE deleted of six or nine C-terminal amino acids. However, addition of a negatively charged amino acid to those deleted versions of CotE rescues the interaction. PMID:26484546

  17. 180 degrees rotation of ciliary rows and its morphogenetic implications in Tetrahymena pyriformis.

    PubMed

    Ng, S F; Frankel, J

    1977-03-01

    With quasi-surgical techniques, longitudinal somatic ciliary rows in Tetrahymena pyriformis have been rotated 180 degrees. New structures formed in the rotated ciliary rows during growth and reproduction are disposed 180 degrees opposite to their normal positions or orientations, confirming the earlier findings of Beisson and Sonneborn on Paramecium. However, during cell fission the rotated ciliary rows exhibit abnormality in orientation along the fission zone; the configuration of these rows near the anterior end of the posterior product of fission is consequently affected. Rotated ciliary rows have been employed as a tool in the analysis of morphogenetic problems: (a) The contractile vacuole pore is normally located on the left side of a ciliary row; but it is on the right of inverted rows. Hence, the morphogenetic properties of the two sides of the ciliary row associated with the contractile vacuole pore are different and this difference is the sole determinative factor as to the side of the ciliary row on which the contractile vacuole pore is located. (b) The process that generates the rotated ciliary rows frequently also brings about the implantation of an extra band of longitudinal microtubules at a specific site on the cell surface. This extra structure is inheritable, which opens up opportunities for the study of microtubular assembly in vivo. PMID:403524

  18. Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening.

    PubMed

    Lindsay, Leann L; Hedrick, Jerry L

    2004-11-12

    The egg envelope of most animal eggs is modified following fertilization, resulting in the prevention of polyspermy and hardening of the egg envelope. In frogs and mammals a prominent feature of envelope modification is N-terminal proteolysis of the envelope glycoprotein ZPA. We have purified the ZPA protease from Xenopus laevis eggs and characterized it as a zinc metalloprotease. Proteolysis of isolated egg envelopes by the isolated protease resulted in envelope hardening. The N-terminal peptide fragment of ZPA remained disulfide bond linked to the ZPA glycoprotein moiety following proteolysis. We propose a mechanism for egg envelope hardening involving ZPA proteolysis by an egg metalloprotease as a triggering event followed by induction of global conformational changes in egg envelope glycoproteins. PMID:15474476

  19. Toward defining the phosphoproteome of Xenopus laevis embryos

    PubMed Central

    McGivern, Jered V.; Swaney, Danielle L.; Coon, Joshua J.; Sheets, Michael D.

    2010-01-01

    Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context. PMID:19384857

  20. Xenopus laevis alpha and beta thyroid hormone receptors.

    PubMed Central

    Yaoita, Y; Shi, Y B; Brown, D D

    1990-01-01

    The Xenopus laevis genome encodes two genes for the alpha (TR alpha) and two genes for the beta (TR beta) thyroid hormone receptors. The two TR alpha genes closely resemble their rat, human, and chicken counterparts. No alternatively spliced TR alpha cDNA clones were found in the 5' untranslated region (5' UTR). In contrast, complex alternative splicing of TR beta mRNA occurs within the 5' UTR as well as possible alternative transcriptional start sites. As many as eight exons encoding mainly the 5' UTR are alternatively spliced, giving rise to at least two amino termini for each of the two TR beta proteins. The 5' UTR of transcripts from both TR alpha and TR beta genes contain multiple AUG sequences with short open reading frames suggesting translational control mechanisms might play a role in expression of TR genes. Images PMID:2402492

  1. Expanding the genetic code in Xenopus laevis oocytes.

    PubMed

    Ye, Shixin; Riou, Morgane; Carvalho, Stéphanie; Paoletti, Pierre

    2013-01-21

    Heterologous expression of ligand-gated ion channels (LGICs) in Xenopus laevis oocytes combined with site-directed mutagenesis has been demonstrated to be a powerful approach to study structure-function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl-tRNA synthetase (aaRS)/suppressor tRNA(CUA) pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N-terminal domain of N-methyl-D-aspartate receptors (NMDARs) serve as photo-crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes. PMID:23292655

  2. Linker scanner mutagenesis of the Xenopus laevis ribosomal gene promoter.

    PubMed Central

    Reeder, R H; Pennock, D; McStay, B; Roan, J; Tolentino, E; Walker, P

    1987-01-01

    We have assayed a series of linker scanner mutants which cover the Xenopus laevis ribosomal gene promoter at approximately ten base pair intervals. All of these mutations adversely affect promoter activity with the exception of one mutation which stimulates activity. Thus, none are neutral. We show that most of the mutations can be partially rescued by ligating a block of enhancer elements upstream of the promoter. In addition, we have made extracts from liver nuclei which produce DNaseI protection footprints over the promoter. Analysis of both strands reveals a prominent footprinting domain from about -5 to -30. However, lesser changes in the digestion pattern are detected over most of the promoter. Previously published analyses have suggested that this promoter might be composed of three functional domains. The experiments presented here suggest that either 1) the three putative domains are so closely arranged that the boundaries are difficult to discern, or 2) the situation is more complex. Images PMID:3658698

  3. The structure of the mitochondrial cloud of Xenopus laevis oocytes.

    PubMed

    Billett, F S; Adam, E

    1976-12-01

    The ultrastructure of the mitochondrial cloud (Balbiani body) of the pre-vitellogenic oocytes of Xenopus laevis has been examined using transmission and stereoscan electron microscopy. Examination of conventional thin sections confirm previous observations which suggest that the cloud consists essentially of many thousands mitochondria and numerous small vesicles; larger clouds, in oocytes greater than 200 mum in diameter, contain relatively more vesicles. Using a standard electron microscope at 100 kV very long and coursing arrays of mitochondrial profiles can be detected. The presence of very long mitochondrial elements has been confirmed using a high voltage microscope operating at 500-1000 kV. Stereoscan preparations, isolated from pre-vitellogenic oocytes, lend some support to the view that the mitochondrial cloud amy consist of a mass of long filamentous mitochondria and the possibility that there are large continuous regions of mitochondrial material cannot be ruled out.

  4. Cytoplasmic effect on gene function in Xenopus laevis.

    PubMed

    Yu, H J; Shi, C P; Niu, M C

    1987-05-01

    The pigmentation gene of Xenopus laevis is dominant and that of albino aP mutant recessive. Heterologous haploid hybrids are produced by UV inactivation of the egg nuclei during second polar body formation in the mutant sperm-fertilized Xenopus eggs. During development of these hybrids, melanin appeared in the eye and melanophores in the skin at stages comparable to those of the wild type, but much earlier than in the albino mutant. The number and intensity of pigment cells are intermediate between the black Xenopus and albino mutant. While a number of pigment cells remain in the hybrids, those in the albino eventually degenerate. Therefore, the development and maintenance of pigmentation in heterologous hybrids are contributed by Xenopus cytoplasm. Tadpole tail-tips were squashed and stained for chromosome counting. The results show that Xenopus and mutants are diploid (36 chromosomes) and heterologous haploid hybrids have 18 chromosomes. PMID:3672098

  5. Gut specific expression using mammalian promoters in transgenic Xenopus laevis.

    PubMed

    Beck, C W; Slack, J M

    1999-11-01

    The recent development of transgenic methods for the frog Xenopus laevis provides the opportunity to study later developmental events, such as organogenesis, at the molecular level. Our studies have focused on the development of the tadpole gut, where tissue specific promoters have yet to be identified. We have used mammalian promoters, for the genes elastase, pancreatic duodenal homeobox-1, transthyretin, and intestinal fatty acid binding protein to drive green fluorescent protein expression in live tadpoles. All of these were shown to drive appropriate tissue specific expression, suggesting that the molecular mechanisms organising the gut are similar in amphibians and mammals. Furthermore, expression from the elastase promoter is initiated in the pancreatic buds before morphological definition becomes possible, making it a powerful tool for the study of pancreatic determination. PMID:10534620

  6. Squalane as a possible carrier of bone morphogenetic protein.

    PubMed

    Kawakami, T; Uji, H; Antoh, M; Hasegawa, H; Kise, T; Eda, S

    1993-07-01

    Gelatin capsules containing squalane partially purified bone morphogenetic protein (BMP) complex were placed on the perimuscular membrane of rats. Two kinds of control, gelatin capsules containing only BMP and those bearing squalane only, were used. The embedded areas were histopathologically examined at 3 and 6 wk after the operation. The observations revealed that the squalane/BMP complex elicited wide heterotopic bone formation with bone marrow tissue, suggesting that squalane is a possible carrier of BMP for clinical applications.

  7. Identification of novel microRNAs in Xenopus laevis metaphase II arrested eggs

    PubMed Central

    Ambady, Sakthikumar; Wu, Zheyang; Dominko, Tanja

    2012-01-01

    Using a combination of deep sequencing and bioinformatics approach, we for the first time identify miRNAs and their relative abundance in mature, metaphase II arrested eggs in X. laevis. We characterize 115 miRNAs that have been described either in X. tropicalis (85), X. laevis (9) or other vertebrate species (21) that also map to known Xenopus pre-miRNAs and to the X. tropicalis genome. Additionally, 72 new X. laevis putative candidate miRNAs are identified based on mapping to X. tropicalis genome within regions that have the propensity to form hairpin loops. These data expand on the availability of genetic information in X. laevis and identifies target miRNAs for future functional studies. PMID:22223599

  8. THYROID AXIS INHIBITION IN XENOPUS LAEVIS: DEVELOPMENT OF AN AMPHIBIAN-BASED SCREENING ASSAY

    EPA Science Inventory

    In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with relying on tail resorption as a measu...

  9. Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

    PubMed Central

    Ruberti, I; Fragapane, P; Pierandrei-Amaldi, P; Beccari, E; Amaldi, F; Bozzoni, I

    1982-01-01

    Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved. Images PMID:6296782

  10. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    SciTech Connect

    Yonezawa, Takayuki; Lee, Ji-Won; Hibino, Ayaka; Asai, Midori; Hojo, Hironori; Cha, Byung-Yoon; Teruya, Toshiaki; Nagai, Kazuo; Chung, Ung-Il; Yagasaki, Kazumi; and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a {beta}-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related {beta}-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of

  11. Bone morphogenetic protein signaling and growth suppression in colon cancer

    PubMed Central

    Beck, Stayce E.; Jung, Barbara H.; Fiorino, Antonio; Gomez, Jessica; Del Rosario, Eunice; Cabrera, Betty L.; Huang, Sherry C.; Chow, Jimmy Y. C.; Carethers, John M.

    2014-01-01

    Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily, which utilize BMP receptors and intracellular SMADs to transduce their signals to regulate cell differentiation, proliferation, and apoptosis. Because mutations in BMP receptor type IA (BMPRIA) and SMAD4 are found in the germline of patients with the colon cancer predisposition syndrome juvenile polyposis, and because the contribution of BMP in colon cancers is largely unknown, we examined colon cancer cells and tissues for evidence of BMP signaling and determined its growth effects. We determined the presence and functionality of BMPR1A by examining BMP-induced phosphorylation and nuclear translocation of SMAD1; transcriptional activity via a BMP-specific luciferase reporter; and growth characteristics by cell cycle analysis, cell growth, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolic as-says. These assays were also performed after transfection with a dominant negative (DN) BMPR1A construct. In SMAD4-null SW480 cells, we examined BMP effects on cellular wound assays as well as BMP-induced transcription in the presence of transfected SMAD4. We also determined the expression of BMPR1A, BMP ligands, and phospho-SMAD1 in primary human colon cancer specimens. We found intact BMP signaling and modest growth suppression in HCT116 and two derivative cell lines and, surprisingly, growth suppression in SMAD4-null SW480 cells. BMP-induced SMAD signaling and BMPR1A-mediated growth suppression were reversed with DN BMPR1A transfection. BMP2 slowed wound closure, and transfection of SMAD4 into SW480 cells did not change BMP-specific transcriptional activity over controls due to receptor stimulation by endogenously produced ligand. We found no cell cycle alterations with BMP treatment in the HCT116 and derivative cell lines, but there was an increased G1 fraction in SW480 cells that was not due to increased p21 transcription. In human colon cancer

  12. Comparison of morphogenetic networks of filamentous fungi and yeast.

    PubMed

    Wendland, J

    2001-11-01

    Fungi generally display either of two growth modes, yeast-like or filamentous, whereas dimorphic fungi, upon environmental stimuli, are able to switch between the yeast-like and the filamentous growth mode. Signal transduction pathways have been elucidated in the budding yeast Saccharomyces cerevisiae, establishing a morphogenetic network that links cell-cycle events with cellular morphogenesis. Recent molecular genetic studies in several filamentous fungal model systems revealed key components required for distinct steps from fungal spore germination to the maintenance of polar hyphal growth, mycelium formation, and nuclear division. This allows a mechanistic comparison of yeast-like and hyphal growth and the establishment of a core model morphogenetic network for filamentous growth including signaling via the cAMP pathway, Rho modules, and cell cycle kinases. Appreciating similarities between morphogenetic networks of the unicellular yeasts and the multicellular filamentous fungi will open new research directions, help in isolating the central network components, and ultimately pave the way to elucidate the central differences (of many) that distinguish, e.g., the growth mode of filamentous fungi from that of their yeast-like relatives, the role of cAMP signaling, and nuclear division. PMID:11686673

  13. The first record of the slender sunfish Ranzania laevis from the Red Sea.

    PubMed

    Abu El-Regal, M A; El-Moselhy, K

    2013-11-01

    A female specimen of the slender sunfish Ranzania laevis of 600 mm total length was recorded for the first time from the Red Sea after being stranded on a shallow sandy bay at Hurghada beach (27° 06' 16″ N; 33° 50' 01″ E) on 13 May 2012. Ranzania laevis is believed to have migrated from the Indian Ocean as the nearest area where it was found is coastal waters of Oman.

  14. Biochemical and Hematologic Reference Intervals for Aged Xenopus laevis in a Research Colony

    PubMed Central

    Chang, Angela G; Hu, Jing; Lake, Elizabeth; Bouley, Donna M; Johns, Jennifer L

    2015-01-01

    Xenopus laevis, the African clawed frog, is commonly used in developmental and toxicology research studies. Little information is available on aged X. laevis; however, with the complete mapping of the genome and the availability of transgenic animal models, the number of aged animals in research colonies is increasing. The goals of this study were to obtain biochemical and hematologic parameters to establish reference intervals for aged X. laevis and to compare results with those from young adult X. laevis. Blood samples were collected from laboratory reared, female frogs (n = 52) between the ages of 10 and 14 y. Reference intervals were generated for 30 biochemistry analytes and full hematologic analysis; these data were compared with prior results for young X. laevis from the same vendor. Parameters that were significantly higher in aged compared with young frogs included calcium, calcium:phosphorus ratio, total protein, albumin, HDL, amylase, potassium, CO2, and uric acid. Parameters found to be significantly lower in aged frogs included glucose, AST, ALT, cholesterol, BUN, BUN:creatinine ratio, phosphorus, triglycerides, LDL, lipase, sodium, chloride, sodium:potassium ratio, and anion gap. Hematology data did not differ between young and old frogs. These findings indicate that chemistry reference intervals for young X. laevis may be inappropriate for use with aged frogs. PMID:26424243

  15. Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes.

    PubMed Central

    Bogan, B W; Lamar, R T

    1996-01-01

    The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium. PMID:8633857

  16. Small molecule inhibitor of the bone morphogenetic protein pathway DMH1 reduces ovarian cancer cell growth.

    PubMed

    Hover, Laura D; Young, Christian D; Bhola, Neil E; Wilson, Andrew J; Khabele, Dineo; Hong, Charles C; Moses, Harold L; Owens, Philip

    2015-11-01

    The bone morphogenetic protein (BMP) pathway belonging to the Transforming Growth Factor beta (TGFβ) family of secreted cytokines/growth factors is an important regulator of cancer. BMP ligands have been shown to play both tumor suppressive and promoting roles in human cancers. We have found that BMP ligands are amplified in human ovarian cancers and that BMP receptor expression correlates with poor progression-free-survival (PFS). Furthermore, active BMP signaling has been observed in human ovarian cancer tissue. We also determined that ovarian cancer cell lines have active BMP signaling in a cell autonomous fashion. Inhibition of BMP signaling with a small molecule receptor kinase antagonist is effective at reducing ovarian tumor sphere growth. Furthermore, BMP inhibition can enhance sensitivity to Cisplatin treatment and regulates gene expression involved in platinum resistance in ovarian cancer. Overall, these studies suggest targeting the BMP pathway as a novel source to enhance chemo-sensitivity in ovarian cancer.

  17. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  18. A Tunable Silk Hydrogel Device for Studying Limb Regeneration in Adult Xenopus Laevis.

    PubMed

    Golding, Anne; Guay, Justin A; Herrera-Rincon, Celia; Levin, Michael; Kaplan, David L

    2016-01-01

    In certain amphibian models limb regeneration can be promoted or inhibited by the local wound bed environment. This research introduces a device that can be utilized as an experimental tool to characterize the conditions that promotes limb regeneration in the adult frog (Xenopus laevis) model. In particular, this device was designed to manipulate the local wound environment via a hydrogel insert. Initial characterization of the hydrogel insert revealed that this interaction had a significant influence on mechanical forces to the animal, due to the contraction of the hydrogel. The material and mechanical properties of the hydrogel insert were a factor in the device design in relation to the comfort of the animal and the ability to effectively manipulate the amputation site. The tunable features of the hydrogel were important in determining the pro-regenerative effects in limb regeneration, which was measured by cartilage spike formation and quantified by micro-computed tomography. The hydrogel insert was a factor in the observed morphological outcomes following amputation. Future work will focus on characterizing and optimizing the device's observed capability to manipulate biological pathways that are essential for limb regeneration. However, the present work provides a framework for the role of a hydrogel in the device and a path forward for more systematic studies. PMID:27257960

  19. A Tunable Silk Hydrogel Device for Studying Limb Regeneration in Adult Xenopus Laevis

    PubMed Central

    Golding, Anne; Levin, Michael; Kaplan, David L.

    2016-01-01

    In certain amphibian models limb regeneration can be promoted or inhibited by the local wound bed environment. This research introduces a device that can be utilized as an experimental tool to characterize the conditions that promotes limb regeneration in the adult frog (Xenopus laevis) model. In particular, this device was designed to manipulate the local wound environment via a hydrogel insert. Initial characterization of the hydrogel insert revealed that this interaction had a significant influence on mechanical forces to the animal, due to the contraction of the hydrogel. The material and mechanical properties of the hydrogel insert were a factor in the device design in relation to the comfort of the animal and the ability to effectively manipulate the amputation site. The tunable features of the hydrogel were important in determining the pro-regenerative effects in limb regeneration, which was measured by cartilage spike formation and quantified by micro-computed tomography. The hydrogel insert was a factor in the observed morphological outcomes following amputation. Future work will focus on characterizing and optimizing the device’s observed capability to manipulate biological pathways that are essential for limb regeneration. However, the present work provides a framework for the role of a hydrogel in the device and a path forward for more systematic studies. PMID:27257960

  20. The Effect of Plasma Exposure on Tail Regeneration of Tadpoles Xenopus Laevis

    NASA Astrophysics Data System (ADS)

    June, Joyce; Rivie, Adonis; Ezuduemoih, Raphael; Menon, Jaishri; Martus, Kevin

    2014-03-01

    Wound healing requires a balanced combination of nutrients and growth factors for healing and tissue regeneration. The effect of plasma exposure on tail regeneration of tadpoles, Xenopus laevis is investigated. The exposure of the wound to the helium plasma immediately followed the amputation of 40% of the tail. Amputation of the tail initiates regeneration of spinal cord, muscle, notochord, skin and connective tissues. By 24 h, the wound was covered by wound epithelium and blastema was formed by day 5. There was increased angiogenesis in plasma exposed tail regenerate compared to the control following 5 d post amputation. Observed was an increase in NO production in the regenerate of plasma exposed tadpoles was derived from increased activity of nNOS and iNOS. Western blot analysis for vascular endothelial growth factor showed stronger bands for the protein in amputated tadpoles of both the groups. Analysis of the composition and characteristics of the plasma using optical emission spectroscopy indicates excited state species consisting of N2, N2+,and OH is present in the plasma. This study was supported, in part, by the NSF Grant 1040108.

  1. Regulation of the activin-inhibin-follistatin system by bone morphogenetic proteins in the zebrafish ovary.

    PubMed

    Li, Cheuk Wun; Ge, Wei

    2013-09-01

    In the zebrafish, the dynamic expression of the activin-inhibin-follistatin system during folliculogenesis and its exclusive localization (except follistatin) in follicle cells suggests that the system plays important roles in follicle development and that its expression is subject to tight controls, probably by external factors including those derived from the oocyte. We have previously identified zebrafish bone morphogenetic proteins (BMPs) as oocyte factors that may act on follicle cells; however, the targets of BMPs in the follicle cells remain unknown. Considering their spatiotemporal expression in the follicle, we hypothesized that members of the activin-inhibin-follistatin system in follicle cells could be potential target genes of BMPs. In the present study, we developed a novel coculture system to co-incubate zebrafish bone morphogenetic protein 2b or 4 (zfBMP2b/4)-producing Chinese hamster ovary (CHO) cells with zebrafish follicle cells. During incubation, the zfBMPs secreted from the CHO cells would act directly on the follicle cells in a paracrine manner. Our results showed that all activin beta subunits (inhbaa, inhbab, and inhbb) were down-regulated by both zfBMP2b and zfBMP4, while follistatin (fst, an activin-binding protein) and inhibin alpha (inha, an activin antagonist) were significantly up-regulated. The specificity of bone morphogenetic protein (BMP) actions was confirmed by short interfering RNA knockdown of zfBMP4 expression in the CHO cells. The robust response of inha to zfBMPs, together with our previous observation that inha expression surged at the full-grown stage prior to oocyte maturation, led us to hypothesize that the full-grown oocyte may signal upper levels of the hypothalamic-pituitary-gonadal axis its readiness to mature by releasing BMPs, which in turn stimulate inhibin production. As an ovarian hormone and activin antagonist, inhibin may suppress the action of activin in the pituitary to reduce follicle-stimulating hormone

  2. Effect of methoxychlor on various life stages of Xenopus laevis.

    PubMed

    Fort, Douglas J; Guiney, Patrick D; Weeks, John A; Thomas, John H; Rogers, Robert L; Noll, Andra M; Spaulding, Clinton D

    2004-10-01

    The toxicological effects of the organochlorine pesticide methoxychlor were evaluated at various life stages of the South African clawed frog, Xenopus laevis, in an effort to determine stage-specific sensitivity. A battery of four separate assays, including a short-term (4-day) early embryo-larval assay (FETAX) (NF stages 8-46 [Nieuwkoop and Faber, 1994]), 30-day hind limb development assay (NF stages 8-54), 18-day metamorphic climax assay (NF stages 58-66), and 30-day adult reproduction assay were performed. Test concentrations for the FETAX, hind limb development, metamorphic climax, and reproductive assays ranged from 0.0001-1.0 mg/l, 0.0001-0.1 mg/l, 0.0001-0.1 mg/l, and 0.001-0.1 mg/l, respectively. Results from the short-term embryo-larval assay indicated that increased embryo-lethality, malformation, and growth inhibition were not induced at /=0.01 mg/l delayed hind limb digit differentiation. Follicular hyperplasia of the thyroid glands was noted in specimens exposed to 0.1 mg/l methoxychlor. Results from the 18-day metamorphic climax assay indicated that methoxychlor inhibited the rate of tail resorption in a concentration-dependent manner. Whole body tissue triiodothyronine (T(3)) profiles showed a reduced and delayed surge during climax compared to controls. For the reproductive assessment, adult female X. laevis were super-ovulated and both female and male were then exposed to varying concentrations of methoxychlor. A concentration-dependent reduction in ovary weight and the number of viable oocytes was observed. In exposed male specimens, a concentration-dependent reduction in testis weight and sperm count was found. Methoxychlor was found to accumulate in the ovary, and to a lesser extent in the testis. Based on breeding studies in which exposed females were bred with control males and exposed males bred with control

  3. Dissection, culture, and analysis of Xenopus laevis embryonic retinal tissue.

    PubMed

    McDonough, Molly J; Allen, Chelsea E; Ng-Sui-Hing, Ng-Kwet-Leok A; Rabe, Brian A; Lewis, Brittany B; Saha, Margaret S

    2012-01-01

    The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation(1-16). The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates (12,14-18). While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells (7,19-23). For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues (8,19-22,24-33). Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level (5,8,21,24,27-30,33-39). Xenopus laevis, a classic model system for the study of early neural development (19,27,29,31-32,40-42), serves as a particularly suitable system for retinal primary cell culture (10,38,43-45). Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction (25,38,43). In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding

  4. Recombinant human bone morphogenetic protein 2 in lateral ridge augmentation.

    PubMed

    Mehanna, Robert; Koo, Samuel; Kim, David M

    2013-01-01

    This case report describes the augmentation of severe lateral ridge defects in the maxilla and mandible using recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS). The surgical technique used tenting screws and a membrane to maintain space for the ACS. After 7 months of healing, the ridge width increased from 1 to 2 mm to 6 to 9 mm, thus allowing successful placement of dental implants. De novo bone formation through use of the surgical technique for space maintenance of rhBMP-2/ACS was demonstrated without the need for additional particulate bone grafting. PMID:23342352

  5. CHAPERONE-MEDIATED CHROMATIN ASSEMBLY AND TRANSCRIPTION REGULATION IN XENOPUS LAEVIS

    PubMed Central

    Onikubo, Takashi; Shechter, David

    2016-01-01

    Chromatin is the complex of DNA and histone proteins that is the physiological form of the eukaryotic genome. Chromatin is generally repressive for transcription, especially so during early metazoan development when maternal factors are explicitly in control of new zygotic gene expression. In the important model organism Xenopus laevis, maturing oocytes are transcriptionally active with reduced rates of chromatin assembly, while laid eggs and fertilized embryos have robust rates of chromatin assembly and are transcriptionally repressed. As the DNA-to-cytoplasmic ratio decreases approaching the mid-blastula transition (MBT) and the onset of zygotic transcription activation (ZGA), the chromatin assembly process changes with the concomitant reduction in maternal chromatin components. Chromatin assembly is mediated in part by histone chaperones that store maternal histones and release them into new zygotic chromatin. Here, we review literature on chromatin and transcription in frog embryos and cell-free extracts and highlight key insights demonstrating the roles of maternal and zygotic histone deposition and their relationship with transcriptional regulation. We explore the central historical and recent literature on the use of Xenopus embryos and the key contributions provided by experiments in cell-free oocyte and egg extracts for the interplay between histone chaperones, chromatin assembly, and transcriptional regulation. Ongoing and future studies in Xenopus cell free extracts will likely contribute essential new insights into the interplay between chromatin assembly and transcriptional regulation. PMID:27759155

  6. The Effect of Plasma on Tail Regeneration of Tadpoles Xenopus Laevis

    NASA Astrophysics Data System (ADS)

    June, Joyce; Amadi, Chima; Menon, Jaishri; Martus, Kevin

    2013-03-01

    Healthy wounds require a balanced combination of nutrients and growth factors for healing and tissue regeneration. Nitric oxide, (NO), is also crucial in wound healing processes and linked with production of several cytokines, interaction with other free radicals and influence on microcirculation. Hypothesize is that exposure to plasma will affect wound healing and tail regeneration in tadpoles Xenopus laevis and plasma induced endogenous NO production may have an important role to play at the cellular level. Tail amputation was immediately followed by exposure of the wound to the helium plasma. For histological features, blastema (growing regenerate) was fixed in 4% neutral buffer formalin for paraffin sections. In situ staining for NO was carried out 5 days post amputation. The rate of the regenerating tail was proportional to the plasma exposure time at the expense of metamorphic rate. Histological features show that the tadpoles exposed to the plasma had a higher level of cellular proliferation and microvasculature in blastema. In situ staining for NO indicated its increased endogenous production compared to the control. These findings suggest that accelerated wound healing and tail regeneration following exposure to the plasma may be due to its direct effect on cell proliferation and increased NO production which may be involved in microvascularization. This study was supported, in part, by the NSF Grant 1040108

  7. The developmental expression of the gene for TFIIIA in Xenopus laevis.

    PubMed

    Taylor, W; Jackson, I J; Siegel, N; Kumar, A; Brown, D D

    1986-08-11

    The sequence of the trans-acting positive transcription factor TFIIIA has been deduced by sequencing cDNA and genomic DNA clones. Using DNA from a homozygous diploid animal we show that there is one gene for TFIIIA per haploid genome of X. laevis. Protein sequencing of proteolytic fragments of TFIIIA orients the protein in its interaction with the internal control region (ICR) of the 5S RNA gene. The protein lies along the DNA with its carboxyl terminus at the 5' end of the ICR and its amino terminus at the 3' end. The developmental pattern of TFIIIA and its mRNA during oogenesis and embryogenesis are consistent with the idea that the abundance of TFIIIA plays an essential role in the developmental change in 5S RNA gene expression. The change to almost exclusive somatic 5S RNA gene expression by gastrulation occurs using either the TFIIIA that was synthesized by oocyte and/or TFIIIA synthesized from maternal mRNA.

  8. Valproate-induced neurodevelopmental deficits in Xenopus laevis tadpoles.

    PubMed

    James, Eric J; Gu, Jenny; Ramirez-Vizcarrondo, Carolina M; Hasan, Mashfiq; Truszkowski, Torrey L S; Tan, Yuqi; Oupravanh, Phouangmaly M; Khakhalin, Arseny S; Aizenman, Carlos D

    2015-02-18

    Autism spectrum disorder (ASD) is increasingly thought to result from low-level deficits in synaptic development and neural circuit formation that cascade into more complex cognitive symptoms. However, the link between synaptic dysfunction and behavior is not well understood. By comparing the effects of abnormal circuit formation and behavioral outcomes across different species, it should be possible to pinpoint the conserved fundamental processes that result in disease. Here we use a novel model for neurodevelopmental disorders in which we expose Xenopus laevis tadpoles to valproic acid (VPA) during a critical time point in brain development at which neurogenesis and neural circuit formation required for sensory processing are occurring. VPA is a commonly prescribed antiepileptic drug with known teratogenic effects. In utero exposure to VPA in humans or rodents results in a higher incidence of ASD or ASD-like behavior later in life. We find that tadpoles exposed to VPA have abnormal sensorimotor and schooling behavior that is accompanied by hyperconnected neural networks in the optic tectum, increased excitatory and inhibitory synaptic drive, elevated levels of spontaneous synaptic activity, and decreased neuronal intrinsic excitability. Consistent with these findings, VPA-treated tadpoles also have increased seizure susceptibility and decreased acoustic startle habituation. These findings indicate that the effects of VPA are remarkably conserved across vertebrate species and that changes in neural circuitry resulting from abnormal developmental pruning can cascade into higher-level behavioral deficits.

  9. Formation of extrachromosomal circles from telomeric DNA in Xenopus laevis.

    PubMed

    Cohen, Sarit; Méchali, Marcel

    2002-12-01

    Instability and plasticity of telomeric DNA, which includes extrachromosomal DNA, are usually correlated with the absence of telomerase and with abnormal growth of mammalian cells. Here, we show the formation of extrachromosomal circular DNA of telomeric repeats (tel-eccDNA) during the development of Xenopus laevis. Tel-eccDNA is double-stranded relaxed circles composed of the vertebrate consensus telomeric repeats [TTAGGG](n). Its size varies from <2 to >20 kb and it comprises up to 10% of the total cellular telomere content of the early embryo (pre-MBT stage). The amount of tel-eccDNA is reduced in later developmental stages and in adult tissues. Using a cell-free system derived from Xenopus egg extracts, we show that tel-eccDNA can be formed de novo from the telomere chromosomal tracts of sperm nuclei and naked DNA in a replication-independent manner. These results reveal an unusual plasticity of telomeric DNA during normal development of Xenopus. PMID:12446568

  10. Teratogenic effects of five anticancer drugs on Xenopus laevis embryos.

    PubMed

    Isidori, Marina; Piscitelli, Concetta; Russo, Chiara; Smutná, Marie; Bláha, Luděk

    2016-11-01

    In recent years, the environmental presence of pharmaceuticals - including anticancer drugs - is an emerging issue. Because of the lack of appropriate critical studies about anticancer drug effects in frogs, the aim of the present study was to investigate lethal and teratogenic effects of five anticancer drugs widely used in large quantities, i.e. 5-flourouracil, capecitabine, cisplatin, etoposide, and imatinib, in the embryos of the South African clawed frog, Xenopus laevis, using FETAX - Frog Embryo Teratogenesis Assay in Xenopus. None of the studied anticancer drugs induced statistically significant mortality within the concentrations tested (0.01-50mg/L, depending on the studied compound), and no growth inhibition of embryos after a 96-h exposure was observed. Except for cisplatin, the other pharmaceuticals induced an increase of developmental malformations such as abdominal edema, axial flexure, head, eyes, gut and heart malformations with statistically significant effects observed at the highest concentrations tested (50mg/L for 5-flourouracil; 30mg/L for etoposide and 20mg/L for capecitabine and imatinib). The results indicate that anticancer drugs can affect embryogenesis mechanisms. PMID:27423131

  11. Valproate-Induced Neurodevelopmental Deficits in Xenopus laevis Tadpoles

    PubMed Central

    James, Eric J.; Gu, Jenny; Ramirez-Vizcarrondo, Carolina M.; Hasan, Mashfiq; Truszkowski, Torrey L.S.; Tan, Yuqi; Oupravanh, Phouangmaly M.

    2015-01-01

    Autism spectrum disorder (ASD) is increasingly thought to result from low-level deficits in synaptic development and neural circuit formation that cascade into more complex cognitive symptoms. However, the link between synaptic dysfunction and behavior is not well understood. By comparing the effects of abnormal circuit formation and behavioral outcomes across different species, it should be possible to pinpoint the conserved fundamental processes that result in disease. Here we use a novel model for neurodevelopmental disorders in which we expose Xenopus laevis tadpoles to valproic acid (VPA) during a critical time point in brain development at which neurogenesis and neural circuit formation required for sensory processing are occurring. VPA is a commonly prescribed antiepileptic drug with known teratogenic effects. In utero exposure to VPA in humans or rodents results in a higher incidence of ASD or ASD-like behavior later in life. We find that tadpoles exposed to VPA have abnormal sensorimotor and schooling behavior that is accompanied by hyperconnected neural networks in the optic tectum, increased excitatory and inhibitory synaptic drive, elevated levels of spontaneous synaptic activity, and decreased neuronal intrinsic excitability. Consistent with these findings, VPA-treated tadpoles also have increased seizure susceptibility and decreased acoustic startle habituation. These findings indicate that the effects of VPA are remarkably conserved across vertebrate species and that changes in neural circuitry resulting from abnormal developmental pruning can cascade into higher-level behavioral deficits. PMID:25698756

  12. Compact structure of ribosomal chromatin in Xenopus laevis.

    PubMed Central

    Spadafora, C; Crippa, M

    1984-01-01

    Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X. laevis. It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin. Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion. A greater accessibility of the coding region in comparison to the non-coding spacer was found. In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight. Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion. The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation. Images PMID:6709502

  13. Circadian rhythm of interrenal activity in Xenopus laevis.

    PubMed

    Thurmond, W; Kloas, W; Hanke, W

    1986-02-01

    Young specimens of Xenopus laevis were kept under constant environmental conditions (artifical light from 600 to 1800 hr, feeding between 800 and 900 hr) and the concentrations of aldosterone and corticosterone in the serum were measured every 3 hr. Furthermore, the kidneys containing the interrenals were removed and their corticosteroid release under stimulation by the pars distalis and mammalian ACTH was determined. Under these conditions, the corticosteroid levels in the serum were maximal from 900 to 1200 hr (corticosterone, 7.7 +/- 0.47 ng/ml; aldosterone, 2.8 +/- 0.27 ng/ml) and minimal during the night (corticosterone, 5.2 +/- 0.43 ng/ml; aldosterone, 1.7 +/- 0.18 ng/ml). The basal secretion rate of the interrenals in vitro showed the opposite course (corticosterone, 24 +/- 3 to 72 +/- 8 pg/min/tissue; aldosterone, 46 +/- 5 to 68 +/- 9 pg/min/tissue). Stimulation by the pars distalis and mammalian ACTH clearly increased the secretion rate. After both types of stimulation the original rhythm was lost for aldosterone but still present for corticosterone. The ratio of the amounts of corticosterone/aldosterone was greater than 1.0 in the serum but less than 1.0 in the incubation fluid. It decreased significantly after stimulation in vitro by pars distalis or ACTH.

  14. Kidins220/ARMS is dynamically expressed during Xenopus laevis development.

    PubMed

    Marracci, Silvia; Giannini, Marianna; Vitiello, Marianna; Andreazzoli, Massimiliano; Dente, Luciana

    2013-01-01

    Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a conserved scaffold protein that acts as a downstream substrate for protein kinase D and mediates multiple receptor signalling pathways. Despite the dissecting of the function of this protein in mammals, using both in vitro and in vivo studies, a detailed characterization of its gene expression during early phases of embryogenesis has not been described yet. Here, we have used Xenopus laevis as a vertebrate model system to analyze the gene expression and the protein localization of Kidins220/ARMS. We found its expression was dynamically regulated during development. Kidins220/ARMS mRNA was expressed from neurula to larval stage in different embryonic regions including the nervous system, eye, branchial arches, heart and somites. Similar to the transcript, the protein was present in multiple embryonic domains including the central nervous system, cranial nerves, motor nerves, intersomitic junctions, retinal ganglion cells, lens, otic vesicle, heart and branchial arches. In particular, in some regions such as the retina and somites, the protein displayed a differential localization pattern in stage 42 embryos when compared to the earlier examined stages. Taken together our results suggest that this multidomain protein is involved in distinct spatio-temporal differentiative events.

  15. Evaluation of heterotopic bone formation induced by squalane and bone morphogenetic protein composite.

    PubMed

    Kawakami, T; Kawai, T; Takei, N; Kise, T; Eda, S; Urist, M R

    1997-04-01

    Bone morphogenetic protein is an important molecule whose bioactivity depends on the carrier. Squalane is used in the formulation of various kinds of cosmetics because it is easily emulsified and has the property of spreading well. Thus, squalane might be effective as a bone morphogenetic protein delivery system. As a test for this possibility, gelatin capsules containing squalane and bone morphogenetic protein (bovine derived partially purified) composite were implanted under the hind-quarter perimuscular membrane of ddY mice. Control capsules containing only bone morphogenetic protein were used for controls. The implants were radiographically and histologically examined at 1 to 4 weeks after the operation. According to the radiographic analysis, squalane and bone morphogenetic protein composite and bone morphogenetic protein only control specimens formed widespread heterotopic bone tissues. The amount of heterotopic bone formation in the composite experimental specimens was approximately 40% greater than that in the controls. Histologic examination of experimental and control specimens revealed varying amounts of perichondral ossification by 2 weeks. By 3 and 4 weeks, the bone deposits were colonized by hematopoietic bone marrow. Squalane was effective for the slow local release of bone morphogenetic protein. Furthermore, the squalane and bone morphogenetic protein composite was a reliable osteoinductive biomaterial.

  16. [Xenopus laevis peroxiredoxins: Gene expression during development and characterization of the enzymes].

    PubMed

    Sharapov, M G; Novoselov, V I; Ravin, V K

    2016-01-01

    Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1-6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10-63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1-6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1-6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development. PMID:27239855

  17. Bone morphogenetic protein in pediatric spine fusion surgery

    PubMed Central

    Kerr, Christine; Kerr, Danielle

    2016-01-01

    Background There is a paucity of literature describing the use of bone graft substitutes to achieve fusion in the pediatric spine. Outcomes and complications involving the off-label use of bone morphogenetic protein 2 (BMP-2) in the pediatric spine are not clearly defined. The purpose of this study is to review the existing literature with respect to reported outcomes and complications involving the use of low-dose BMP-2 in pediatric patients. Methods A Medline and PubMed literature search was conducted using the words bone morphogenetic protein, BMP, rh-BMP-2, bone graft substitutes, and pediatric spine. Results To date, there are few published reports on this topic. Complications and appropriate BMP-2 dosage application in the pediatric spine remain unknown. Conclusions This report describes the potential for BMP-2 to achieve successful arthrodesis of the spine in pediatric patients. Usage should be judicious as complications and long-term outcomes of pediatric BMP-2 usage remain undefined in the existing literature.

  18. A gene expression map of the larval Xenopus laevis head reveals developmental changes underlying the evolution of new skeletal elements.

    PubMed

    Square, Tyler; Jandzik, David; Cattell, Maria; Coe, Alex; Doherty, Jacob; Medeiros, Daniel Meulemans

    2015-01-15

    The morphology of the vertebrate head skeleton is highly plastic, with the number, size, shape, and position of its components varying dramatically between groups. While this evolutionary flexibility has been key to vertebrate success, its developmental and genetic bases are poorly understood. The larval head skeleton of the frog Xenopus laevis possesses a unique combination of ancestral tetrapod features and anuran-specific novelties. We built a detailed gene expression map of the head mesenchyme in X. laevis during early larval development, focusing on transcription factor families with known functions in vertebrate head skeleton development. This map was then compared to homologous gene expression in zebrafish, mouse, and shark embryos to identify conserved and evolutionarily flexible aspects of vertebrate head skeleton development. While we observed broad conservation of gene expression between X. laevis and other gnathostomes, we also identified several divergent features that correlate to lineage-specific novelties. We noted a conspicuous change in dlx1/2 and emx2 expression in the second pharyngeal arch, presaging the differentiation of the reduced dorsal hyoid arch skeletal element typical of modern anamniote tetrapods. In the first pharyngeal arch we observed a shift in the expression of the joint inhibitor barx1, and new expression of the joint marker gdf5, shortly before skeletal differentiation. This suggests that the anuran-specific infrarostral cartilage evolved by partitioning of Meckel's cartilage with a new paired joint. Taken together, these comparisons support a model in which early patterning mechanisms divide the vertebrate head mesenchyme into a highly conserved set of skeletal precursor populations. While subtle changes in this early patterning system can affect skeletal element size, they do not appear to underlie the evolution of new joints or cartilages. In contrast, later expression of the genes that regulate skeletal element

  19. Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells.

    PubMed

    Rogers, Melissa B; Shah, Tapan A; Shaikh, Nadia N

    2015-10-01

    The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell behavior. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence the allocation of cells to muscle, fat, cartilage, and bone, the mechanisms that regulate the Bmp2 gene are crucial. Key early mesodermal events that require precise Bmp2 regulation include heart specification and morphogenesis. Originally named for its osteoinductive properties, healing fractures requires BMP2. The human Bmp2 gene also has been linked to osteoporosis and osteoarthritis. In addition, all forms of pathological calcification in the vasculature and in cardiac valves involve the pro-osteogenic BMP2. The diverse tissues, mechanisms, and diseases influenced by BMP2 are too numerous to list here (see OMIM: 112261). However, in all BMP2-influenced pathologies, changes in the behavior and differentiation of pluripotent mesenchymal cells are a recurring theme. Consequently, much effort has been devoted to identifying the molecules and conditions that influence BMP2 synthesis and the complex mechanisms that control Bmp2 gene expression. This review begins with an

  20. Mass transport in morphogenetic processes: A second gradient theory for volumetric growth and material remodeling

    NASA Astrophysics Data System (ADS)

    Ciarletta, P.; Ambrosi, D.; Maugin, G. A.

    2012-03-01

    In this work, we derive a novel thermo-mechanical theory for growth and remodeling of biological materials in morphogenetic processes. This second gradient hyperelastic theory is the first attempt to describe both volumetric growth and mass transport phenomena in a single-phase continuum model, where both stress- and shape-dependent growth regulations can be investigated. The diffusion of biochemical species (e.g. morphogens, growth factors, migration signals) inside the material is driven by configurational forces, enforced in the balance equations and in the set of constitutive relations. Mass transport is found to depend both on first- and on second-order material connections, possibly withstanding a chemotactic behavior with respect to diffusing molecules. We find that the driving forces of mass diffusion can be written in terms of covariant material derivatives reflecting, in a purely geometrical manner, the presence of a (first-order) torsion and a (second-order) curvature. Thermodynamical arguments show that the Eshelby stress and hyperstress tensors drive the rearrangement of the first- and second-order material inhomogeneities, respectively. In particular, an evolution law is proposed for the first-order transplant, extending a well-known result for inelastic materials. Moreover, we define the first stress-driven evolution law of the second-order transplant in function of the completely material Eshelby hyperstress. The theory is applied to two biomechanical examples, showing how an Eshelbian coupling can coordinate volumetric growth, mass transport and internal stress state, both in physiological and pathological conditions. Finally, possible applications of the proposed model are discussed for studying the unknown regulation mechanisms in morphogenetic processes, as well as for optimizing scaffold architecture in regenerative medicine and tissue engineering.

  1. Effects of tributyltin on metamorphosis and gonadal differentiation of Xenopus laevis at environmentally relevant concentrations.

    PubMed

    Shi, Huahong; Zhu, Pan; Guo, Suzhen

    2014-05-01

    Tributyltin (TBT), a well known endocrine disruptor, has high teratogenicity to embryos of amphibian (Xenopus tropicalis). An amphibian metamorphosis assay (AMA) and a complete AMA (CAMA) were conducted for TBT. In AMA, the body weight, the snout-to-vent length and the hind limb length of X. laevis tadpoles were decreased in tributyltin chloride (TBTCl; 12.5-200 ng/L) treatment groups after 7 days exposure. TBT greatly retarded the development of tadpoles, decreased the number of follicle and induced thyroid follicle cell hyperplasia after 19 days exposure. In CAMA, 10 and 100 ng/L TBTCl led to various malformations of gonad, including intersex, segmental aplasia and multiple ovary cavities of X. laevis following exposure from stages 46 to stage 66. The sex ratio was male-biased in TBT treatment groups. These results suggest that TBT delayed the metamorphosis, inhibited the growth of tadpoles and disrupted the gonadal differentiation of X. laevis at environmentally relevant concentrations.

  2. Effects of tributyltin on metamorphosis and gonadal differentiation of Xenopus laevis at environmentally relevant concentrations.

    PubMed

    Shi, Huahong; Zhu, Pan; Guo, Suzhen

    2014-05-01

    Tributyltin (TBT), a well known endocrine disruptor, has high teratogenicity to embryos of amphibian (Xenopus tropicalis). An amphibian metamorphosis assay (AMA) and a complete AMA (CAMA) were conducted for TBT. In AMA, the body weight, the snout-to-vent length and the hind limb length of X. laevis tadpoles were decreased in tributyltin chloride (TBTCl; 12.5-200 ng/L) treatment groups after 7 days exposure. TBT greatly retarded the development of tadpoles, decreased the number of follicle and induced thyroid follicle cell hyperplasia after 19 days exposure. In CAMA, 10 and 100 ng/L TBTCl led to various malformations of gonad, including intersex, segmental aplasia and multiple ovary cavities of X. laevis following exposure from stages 46 to stage 66. The sex ratio was male-biased in TBT treatment groups. These results suggest that TBT delayed the metamorphosis, inhibited the growth of tadpoles and disrupted the gonadal differentiation of X. laevis at environmentally relevant concentrations. PMID:22903176

  3. Effect of titanium dioxide nanomaterials and ultraviolet light coexposure on African clawed frogs (Xenopus laevis).

    PubMed

    Zhang, Junling; Wages, Mike; Cox, Stephen B; Maul, Jonathan D; Li, Yujia; Barnes, Melanie; Hope-Weeks, Louisa; Cobb, George P

    2012-01-01

    Titanium dioxide nanomaterials (nano-TiO(2) ) exhibit stronger photochemical oxidation/reduction capacity compared with their bulk counterparts, but the effectiveness of nano-TiO(2) interaction with ultraviolet (UV) light strongly depends on particle size. In this study, the dependence of nano-TiO(2) toxicity on particle size and interaction with UV light were investigated. Toxicity tests with Xenopus laevis included eight concentrations of nano-TiO(2) in the presence of either white light or UVA (315-400 nm). We quantified viability and growth of Xenopus laevis. Results showed that, regardless of UV light exposure, increasing TiO(2) concentration decreased X. laevis survival (p < 0.05). Coexposure to 5-nm TiO(2) and UVA caused near-significant decreases in X. laevis survival (p = 0.08). Coexposure to 10-nm TiO(2) and UVA significantly decreased X. laevis survival (p = 0.005). However, coexposure to 32-nm TiO(2) and UVA had no statistical effect on X. laevis survival (p = 0.8). For all three particle sizes, whether alone or with UV light, the nano-TiO(2) concentrations significantly affected growth of tadpoles as determined by total body length, snout-vent length, and developmental stage. High-concentration TiO(2) solutions suppressed tadpole body length and delayed developmental stages. Further research to explore reasons for the growth and mortality in tadpoles is still underway in our laboratory. Given the widespread application of nano-TiO(2) , our results may be useful in the management of nano-TiO(2) released from industrial, municipal, and nonpoint sources. PMID:22012895

  4. Quantifying calcium fluxes underlying calcium puffs in Xenopus laevis oocytes

    PubMed Central

    Bruno, Luciana; Solovey, Guillermo; Ventura, Alejandra C.; Dargan, Sheila; Dawson, Silvina Ponce

    2010-01-01

    Summary We determine the calcium fluxes through inositol 1,4,5-trisphosphate receptor/channels underlying calcium puffs of Xenopus laevis oocytes using a simplified version of the algorithm of Ventura et al., 2005 [1]. An analysis of 130 puffs obtained with Fluo-4 indicates that Ca2+ release comes from a region of width ~ 450 nm, that the release duration is peaked around 18ms and that the underlying Ca2+ currents range between 0.12 and 0.95pA. All these parameters are independent of IP3 concentration. We explore what distributions of channels that open during a puff, Np, and what relations between current and number of open channels, I(Np), are compatible with our findings and with the distribution of puff-to-trigger amplitude ratio reported in Rose et al, 2006 [2]. To this end, we use simple “mean field” models in which all channels open and close simultaneously. We find that the variability among clusters plays an important role in shaping the observed puff amplitude distribution and that a model for which I(Np) ~Np for small Np and I(Np)~Np1/α (α>1) for large Np, provides the best agreement. Simulations of more detailed models in which channels open and close stochastically show that this nonlinear behavior can be attributed to the limited time resolution of the observations and to the averaging procedure that is implicit in the mean-field models. These conclusions are also compatible with observations of ~400 puffs obtained using the dye Oregon green. PMID:20097419

  5. Vitellogenin and lipovitellin: zinc proteins of Xenopus laevis oocytes.

    PubMed

    Montorzi, M; Falchuk, K H; Vallee, B L

    1995-08-29

    Xenopus laevis vitellogenin is a plasma protein that contains a total of 5 mol of metal/440 kDa dimer, 2 mol of zinc, and 3 mol of calcium (Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413]. There are no other group IIB or transition metals in the molecule. The zinc atoms are removed instantaneously by 1,10-phenanthroline (OP) (pK 4.8). Once internalized by receptor-mediated endocytosis, vitellogenin is cleaved into multiple polypeptides, i.e., the two lipovitellin subunits (1 and 2) plus phosvitin; these are then stored as microcrystals within yolk platelets. We here show by metal analysis of the individual proteins generated by vitellogenin processing that zinc and calcium occur in different domains of the vitellogenin polypeptide chain. All of the vitellogenin zinc is present in lipovitellin, in amounts equal to 1 mol of zinc/141 kDa. Calcium, in contrast, is detected exclusively in phosvitin which, in addition, contains 3 mol of magnesium/35 kDa, apparently acquired following vitellogenin entry into the oocyte. The zinc in lipovitellin is removed by OP in a concentration-dependent manner with a pK of 4.8, identical to that obtained for vitellogenin, and by exposure to acidic conditions (below pH 5). Following removal of zinc, the two lipovitellin subunits remain associated, suggesting that zinc is not involved in their interaction. On exposure to 1% SDS, lipovitellin does dissociate into 106 and 33 kDa subunits. The presence of stoichiometric quantities of zinc in both vitellogenin and lipovitellin calls for the study of the hitherto unrecognized biochemistry and functions of these proteins in zinc metabolism and development of the frog oocyte and embryo.

  6. Breathing and calling: neuronal networks in the Xenopus laevis hindbrain.

    PubMed

    Zornik, Erik; Kelley, Darcy B

    2007-03-20

    Xenopus laevis is an aquatic anuran with a complex vocal repertoire. Unlike terrestrial frogs, vocalizations are independent of respiration, and a single muscle group--the laryngeal dilators--produces underwater calls. We sought to identify the premotor neural network that underlies vocal behaviors. Vocal patterns generated by premotor networks control laryngeal motor neurons in cranial nucleus (n.) IX-X. Glottal motor neurons, active during respiration, are also present in n.IX-X. We used horseradish peroxidase (HRP), Lucifer yellow, and fluorescently conjugated dextrans to characterize the organization of n.IX-X and to trace premotor neuron projections. Premotor nuclei include the inferior reticular formation (Ri) adjacent to n.IX-X and the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM), the primary descending input to n.IX-X. Intramuscular HRP injections revealed a spatially segregated pattern, with glottal motor neurons in anterior n.IX-X and laryngeal motor neurons in the caudal portion of the nucleus. Dextran injections identified commissural n.IX-X neurons that project to the contralateral motor nucleus and DTAM-projecting n.IX-X neurons. Both neuronal types are clustered in anteromedial n.IX-X, closely associated with glottal motor neurons. Ri neurons project to ipsilateral and contralateral DTAM. Projections from DTAM target n.IX-X bilaterally, and all four identified subtypes receive DTAM input. In contrast, Ri neurons receive little input from DTAM. We hypothesize that connectivity between neurons in n.IX-X, Ri and DTAM may provide mechanisms to generate laryngeal and glottal activity patterns and that DTAM may coordinate vocal and respiratory motor pools, perhaps acting to switch between these two mutually exclusive behaviors.

  7. Characterization of nuclear protein kinases of Xenopus laevis oocytes

    SciTech Connect

    Leiva, L.; Gonzalez, C.; Allende, C.; Allende, J.

    1986-05-01

    Xenopus laevis oocytes contain large nuclei (germinal vesicles) that can be isolated in very pure form and which permit the study of enzymatic activities present in these organelles. Incubation of pure oocyte nuclear homogenates with /sup 32/P in a buffered solution containing 5 mM MgCl/sub 2/ results in the phosphorylation of a large number of proteins by endogenous protein kinases. This phosphorylation is not affected by the addition of cyclic nucleotides or calcium ion and calmodulin. On the other hand the nuclear kinases are considerably stimulated by spermine and spermidine and strongly inhibited by heparin (10 ..mu..g/ml). Addition of exogenous protein substrates shows that the major oocyte kinases are very active with casein and phosvitin as substrates but do not phosphorylate histones or protamines. DEAE-Sephadex chromatography of the nuclear extract fractionates the casein phosphorylating activity in two main peaks. The first peak is not retained on the column equilibrated with 0.1 M NH/sub 2/SO/sub 4/ and uses exclusively ATP as phosphate donor and is insensitive to polyamines or heparin. The second peak which corresponds to 70% of the casein phosphorylation elutes at 0.27 M NH/sub 2/SO/sub 4/ and uses both ATP and GTP as phosphate donors and is greatly stimulated by polyamines and completely inhibited by 10 ..mu..g/ml heparin. On this evidence the authors conclude that the major protein kinase peak corresponds to casein kinase type II which has been found in mammalian nuclei.

  8. Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1*

    PubMed Central

    Wohl, Alexander P.; Troilo, Helen; Collins, Richard F.; Baldock, Clair

    2016-01-01

    Since the discovery of bone morphogenetic proteins (BMPs) as pluripotent cytokines extractable from bone matrix, it has been speculated how targeting of BMPs to the extracellular matrix (ECM) modulates their bioavailability. Understanding these processes is crucial for elucidating pathomechanisms of connective tissue disorders characterized by ECM deficiency and growth factor dysregulation. Here, we provide evidence for a new BMP targeting and sequestration mechanism that is controlled by the ECM molecule fibrillin-1. We present the nanoscale structure of the BMP-7 prodomain-growth factor complex using electron microscopy, small angle x-ray scattering, and circular dichroism spectroscopy, showing that it assumes an open V-like structure when it is bioactive. However, upon binding to fibrillin-1, the BMP-7 complex is rendered into a closed ring shape, which also confers latency to the growth factor, as demonstrated by bioactivity measurements. BMP-7 prodomain variants were used to map the critical epitopes for prodomain-growth factor and prodomain-prodomain binding. Together, these data show that upon prodomain binding to fibrillin-1, the BMP-7 complex undergoes a conformational change, which denies access of BMP receptors to the growth factor. PMID:27059954

  9. Biochemical study of prolactin binding sites in Xenopus laevis brain and choroid plexus

    SciTech Connect

    Muccioli, G.; Guardabassi, A.; Pattono, P. )

    1990-03-01

    The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrence of prolactin specific binding sites in Xenopus laevis choroid plexus cells.

  10. Parasites of the African clawed frog, Xenopus laevis, in southern California, U.S.A

    USGS Publications Warehouse

    Kuperman, Boris I.; Matey, Victoria E.; Fisher, Richard N.; Ervin, Edward L.; Warburton, Manna L.; Bakhireva, Ludmila; Lehman, Cynthia A.

    2004-01-01

    A total of 230 feral African clawed frogs, Xenopus laevis, from 3 localities in southern California were examined for parasites. The following species were found: 3 species of Protozoa, Nyctotherussp., Balantidium xenopodis, Protoopalina xenopodus; 2 species of Monogenea, Protopolystoma xenopodis, Gyrdicotylus gallieni; 1 species of Digenea, Clinostomum sp. (as metacercariae); 1 species of Cestoda, Cephalochlamys namaquensis; 2 species of Nematoda, Contracaecum sp. (as larvae), Eustrongylides sp. (as larvae); and 1 species of Acanthocephala, Acanthocephalus sp. (as cystacanth). Of these, the protozoans P. xenopodus and B. xenopodis, both monogeneans, and the cestode have an African origin. Contracaecum sp., Eustrongylides sp., and Acanthocephalus sp. have not been previously reported from X. laevis.

  11. Biomimetic design processes in architecture: morphogenetic and evolutionary computational design.

    PubMed

    Menges, Achim

    2012-03-01

    Design computation has profound impact on architectural design methods. This paper explains how computational design enables the development of biomimetic design processes specific to architecture, and how they need to be significantly different from established biomimetic processes in engineering disciplines. The paper first explains the fundamental difference between computer-aided and computational design in architecture, as the understanding of this distinction is of critical importance for the research presented. Thereafter, the conceptual relation and possible transfer of principles from natural morphogenesis to design computation are introduced and the related developments of generative, feature-based, constraint-based, process-based and feedback-based computational design methods are presented. This morphogenetic design research is then related to exploratory evolutionary computation, followed by the presentation of two case studies focusing on the exemplary development of spatial envelope morphologies and urban block morphologies.

  12. [Peculiarities of morphogenetical development of erwiniophage ZF40 virulent mutants ].

    PubMed

    Korol', N A; Romaniuk, L V; Ostapchuk, A N; Ivanitsa, T V; Kushkina, A I; Tovkach, F I

    2011-01-01

    The distortion of morphopoiesis or tail attachment to the capsid is a characteristic feature of morphogenetical development not only of a reproductive infection but also of the lysogenic induction of the defective bacteriophage Erwinia carotovora subsp. carotovora (Ecc). A model system for studying morphogenetical development and assembling of the virion was created on the basis of the phage ZF40 and its two virulent mutants ZF40-421 and ZF40(5/5), as well as the indicator culture Ecc M2-4/50 R1 being nontraditional host for these phages. It has helped to establish that the diameter of the phage capsid is not a conservative value. The presence of capsids of two types with the average diameters 60.3 and 65.0 nm is characteristic of the virmutant ZF40c(5/5)/50RI, while in the course of morphogenesis the phage ZF40-421/50RI forms only one type of heads of 65 nm in size. These heads are probably not firmly connected to the tails since the degree of the secondary destruction of the virions of the phage Zf40-421/50RI is considerably higher, than that of the virions of the phage ZF40c(5/5)/50RI. The number of capsids being 60.3 nm in diameter prevails considerably in the latter. The both virulent mutants as a whole are essentially more stable than their isogenic partners obtained on Ecc RC5297 which helps to make a conclusion about considerable influence of specific bacterial proteins of the host-cell on morphogenesis and morphopoiesis. PMID:21598661

  13. Lethal and sublethal effects of three insecticides on two developmental stages of Xenopus laevis and comparison with other amphibians.

    PubMed

    Yu, Shuangying; Wages, Mike R; Cai, Qingsong; Maul, Jonathan D; Cobb, George P

    2013-09-01

    It has been suggested that Xenopus laevis is less sensitive than other amphibians to some chemicals, and therefore, that the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) may have limited use in risk assessments for other amphibians. However, comparisons are based mostly on results of FETAX, which emphasizes embryos. Larval X. laevis may be more sensitive to chemicals than embryos and may serve as a better life stage in risk assessments. The present study was conducted to determine the lethal and sublethal effects of 3 insecticides (malathion, endosulfan, and α-cypermethrin) on X. laevis embryos and larvae and to compare toxicity of X. laevis with that of other amphibians. All 3 insecticides have different modes of action, and they caused mortality, malformations, and growth inhibition in both developmental stages. Compared with embryos, larvae were more sensitive to endosulfan and α-cypermethrin but not to malathion. Xenopus laevis larvae had low sensitivity to endosulfan, median sensitivity to malathion, and high sensitivity to α-cypermethrin/cypermethrin relative to other larval amphibians. Our results suggest that X. laevis larvae may generate more protective toxicity estimates in risk assessments than embryos. Xenopus laevis may have limited use in evaluating risk of organochlorine insecticides to other amphibians but may provide useful toxicity thresholds for pyrethroid and perhaps organophosphorus insecticides.

  14. Unequal contribution of native South African phylogeographic lineages to the invasion of the African clawed frog, Xenopus laevis, in Europe

    PubMed Central

    Courant, Julien; Herrel, Anthony; Rebelo, Rui; Rödder, Dennis; Measey, G. John; Backeljau, Thierry

    2016-01-01

    Due to both deliberate and accidental introductions, invasive African Clawed Frog (Xenopus laevis) populations have become established worldwide. In this study, we investigate the geographic origins of invasive X. laevis populations in France and Portugal using the phylogeographic structure of X. laevis in its native South African range. In total, 80 individuals from the whole area known to be invaded in France and Portugal were analysed for two mitochondrial and three nuclear genes, allowing a comparison with 185 specimens from the native range. Our results show that native phylogeographic lineages have contributed differently to invasive European X. laevis populations. In Portugal, genetic and historical data suggest a single colonization event involving a small number of individuals from the south-western Cape region in South Africa. In contrast, French invasive X. laevis encompass two distinct native phylogeographic lineages, i.e., one from the south-western Cape region and one from the northern regions of South Africa. The French X. laevis population is the first example of a X. laevis invasion involving multiple lineages. Moreover, the lack of population structure based on nuclear DNA suggests a potential role for admixture within the invasive French population. PMID:26855879

  15. Neural transduction in Xenopus laevis lateral line system.

    PubMed

    Strelioff, D; Honrubia, V

    1978-03-01

    1. The process of neural excitation in hair cell systems was studied in an in vitro preparation of the Xenopus laevis (African clawed toad) lateral line organ. A specially designed stimulus chamber was used to apply accurately controlled pressure, water movement, or electrical stimuli, and to record the neural responses of the two afferent fibers innervating each organ or stitch. The objective of the study was to determine the characteristics of the neural responses to these stimuli, and thus gain insight into the transduction process. 2. A sustained deflection of the hair cell cilia due to a constant flow of water past the capula resulted in a maintained change in the mean firing rate (MFR) of the afferent fibers. The data also demonstrated that the neural response was proportional to the velocity of the water flow and indicated that both deflection and movement of the cilia were the effective physiological stimuli for this hair cell system. 3. The preparations responded to sinusoidal water movements (past the capula) over the entire frequency range of the stimulus chamber, 0.1-130 Hz, and were most sensitive between 10 and 40 Hz. The variation of the MFR and the percent modulation indicated that the average dynamic range of each organ was 23.5 dB. 4. The thresholds, if any, for sustained pressure changes and for sinusoidal pressure variations in the absence of water movements were very high. Due to the limitations of the stimulus chamber it was not possible to generate pressure stimuli of sufficient magnitude to elicit a neural response without also generating suprathreshold water-movement stimuli. Sustained pressures had no detectable effect on the neural response to water-movement stimuli. 5. The preparations were very sensitive to electrical potentials applied across the toad skin on which the hair cells were located. Potentials which made the ciliated surfaces of the hair cells positive with respect to their bases increased the MFR of the fibers, whereas

  16. The maturation-inducing hormone 17a-20b-dihydroxy-4pregnen-3-one regulates gene expression of inhibin A and bambi (bone morphogenetic protein and activin membrane bound inhibitor) in the rainbow trout ovary

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transforming growth factor-beta (TGFb) superfamily members are important paracrine and autocrine regulators of ovarian development and steroidogenesis in mammals and birds, but their reproductive roles in fish are not well understood. The activin system, Tgfb, and bone morphogenetic protein 15 (Bmp...

  17. METAMORPHIC INHIBITION OF XENOPUS LAEVIS BY SODIUM PERCHLORATE: EFFECTS ON DEVELOPMENT AND THYROID HISTOLOGY

    EPA Science Inventory

    The perchlorate anion inhibits thyroid hormone (TH) synthesis via inhibition of the sodium-iodide symporter. It is, therefore, a good model chemical to aid in the development of a bioassay to screen chemicals for effects on thyroid function. Xenopus laevis larvae were exposed to ...

  18. Effects of ZnO nanomaterials on Xenopus laevis growth and development.

    PubMed

    Nations, Shawna; Long, Monique; Wages, Mike; Canas, Jaclyn; Maul, Jonathan D; Theodorakis, Chris; Cobb, George P

    2011-02-01

    The objectives of this study were to quantify uptake and developmental effects of zinc oxide nanomaterials (nano-ZnO) on Xenopus laevis throughout the metomormosis process. To accomplish this, X. laevis were exposed to aqueous suspensions of 40-100 nm nano-ZnO beginning in-ovo and proceeding through metamorphosis. Nanomaterials were dispersed via sonication methods into reconstituted moderately hard water test solutions. A flow-through system was utilized to decrease the likelihood of depletion in ZnO concentration. Exposure to 2 mg/L nano-ZnO significantly increased mortality incidence to 40% and negatively affected metamorphosis of X. laevis. Tadpoles exposed to 2 mg/L nano-ZnO developed slower as indicated by tadpoles with an average stage of 56 at the conclusion of the study which was significantly lower than the control tadpole stages. No tadpoles exposed to 2 mg/L of nano-ZnO completed metamorphosis by the conclusion of the study. Tadpoles exposed to 0.125 mg/L nano-ZnO experienced faster development along with larger body measurements indicating that low dose exposure to nano-ZnO can stimulate growth and metamorphosis of X. laevis. PMID:20801509

  19. Identification, characterization, and expression of dentin matrix protein 1 gene in Xenopus laevis.

    PubMed

    Yonekura, Tomoko; Homma, Hiromi; Sakurai, Atsuo; Moriguchi, Mitsuko; Miake, Yasuo; Toyosawa, Satoru; Shintani, Seikou

    2013-12-01

    Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

  20. On the natural diet of Daphnia laevis in the eutrophic Pampulha reservoir (Belo Horizonte, Minas Gerais).

    PubMed

    Eskinazi-Sant'Anna, E M; Maia-Barbosa, P M; Barbosa, F A R

    2002-08-01

    The aim of this study was to assess the major food items ingested by adult specimens of Daphnia laevis within the eutrophic Pampulha reservoir in Belo Horizonte, Minas Gerais, Brazil. The gut content was analyzed after addition of sodium hypochlorite and also through the examination of dissected guts under scanning electron microscopy. The results showed that Chlorophyceae was the main food item ingested, representing c. 80.5% of the total ingested food. Moreover, Eutetramorus fottii, Coelastrum pseudomicroporum and Oocystis lacustris, the dominant phytoplankton species within the reservoir, were the most frequent cells found in the gut contents. Euglenophyta also represented an important food item accounting for 15% of the ingested material, including mainly Trachelomonas volvocina and Euglena oxyuris, although less abundant in the reservoir (< 10% of total phytoplankton). Blue-green algae occurred at much lower percentages in the guts than in the phytoplankton. A small amount of undigested Microcystis aeruginosa colonies were also found in the gut content of D. laevis. Scanning electron microscopy results showed that, besides phytoplankton cells, a great amount of abiogenic material was also ingested. The amount of this inorganic material increased considerably in the tract (from 15% to 75% of the gut content), when a peak of D. laevis was observed in the reservoir. Our assumption is that the ingestion of this inorganic material can be a strategy used by D. laevis to obtain additional food supply.

  1. Identification and expression of an atypical isoform of metallothionein in the African clawed frog Xenopus laevis.

    PubMed

    Scudiero, Rosaria; Tussellino, Margherita; Carotenuto, Rosa

    2015-05-01

    Exploiting the annotation of the western clawed frog Silurana tropicalis genome, we identified a new metallothionein (MT) gene, exhibiting all the features to be considered an active gene, but with an atypical coding region, showing only 17 cysteine residues instead of the canonical 20 cysteines of vertebrate metallothioneins and two anomalous cysteine triplets. However, the presence of a gene in the genome does not ensure its effective expression. By using conventional and Real-Time PCR analyses, we demonstrated that this atypical MT is constitutively expressed throughout the life cycle of the African clawed frog Xenopus laevis; moreover, this gene is highly expressed in the adult liver, the major site of MT expression and synthesis in vertebrates. To our knowledge, the X. laevis MT described in this paper is the first sequence of a vertebrate MT showing only 17 cysteine residues, arranged in two Cys-Cys-Cys motifs. Phylogenetic analyses also demonstrated that the atypical X. laevis MT merges in the anuran clade, but is the most derived sequence among tetrapods MTs. Finally, Tajima's Relative Rate Test suggested a different evolutionary rate between the canonical X. laevis MT and this novel isoform.

  2. CONCENTRATION DEPENDENT ACCUMULATION OF [3H]-DELTAMETHRIN IN XENOPUS LAEVIS OOCYTES.

    EPA Science Inventory

    Pyrethroid insecticides such as deltamethrin have been demonstrated to target and disrupt voltage-sensitive sodium channels (VSSCs). VSSCs were expressed in Xenopus laevis oocytes and used to study the effects of deltamethrin on VSSCs. This study evaluated the amount of deltameth...

  3. Identification and Analysis of Regulatory Elements in Porcine Bone Morphogenetic Protein 15 Gene Promoter.

    PubMed

    Wan, Qianhui; Wang, Yaxian; Wang, Huayan

    2015-10-27

    Bone morphogenetic protein 15 (BMP15) is secreted by the mammalian oocytes and is indispensable for ovarian follicular development, ovulation, and fertility. To determine the regulation mechanism of BMP15 gene, the regulatory sequence of porcine BMP15 was investigated in this study. The cloned BMP15 promoter retains the cell-type specificity, and is activated in cells derived from ovarian tissue. The luciferase assays in combination with a series of deletion of BMP15 promoter sequence show that the -427 to -376 bp region of BMP15 promoter is the primary regulatory element, in which there are a number of transcription factor binding sites, including LIM homeobox 8 (LHX8), newborn ovary homeobox gene (NOBOX), and paired-like homeodomain transcription factor 1 (PITX1). Determination of tissue-specific expression reveals that LHX8, but not PITX1 and NOBOX, is exclusively expressed in pig ovary tissue and is translocated into the cell nuclei. Overexpression of LHX8 in Chinese hamster ovary (CHO) cells could significantly promote BMP15 promoter activation. This study confirms a key regulatory element that is located in the proximal region of BMP15 promoter and is regulated by the LHX8 factor.

  4. Identification and Analysis of Regulatory Elements in Porcine Bone Morphogenetic Protein 15 Gene Promoter

    PubMed Central

    Wan, Qianhui; Wang, Yaxian; Wang, Huayan

    2015-01-01

    Bone morphogenetic protein 15 (BMP15) is secreted by the mammalian oocytes and is indispensable for ovarian follicular development, ovulation, and fertility. To determine the regulation mechanism of BMP15 gene, the regulatory sequence of porcine BMP15 was investigated in this study. The cloned BMP15 promoter retains the cell-type specificity, and is activated in cells derived from ovarian tissue. The luciferase assays in combination with a series of deletion of BMP15 promoter sequence show that the −427 to −376 bp region of BMP15 promoter is the primary regulatory element, in which there are a number of transcription factor binding sites, including LIM homeobox 8 (LHX8), newborn ovary homeobox gene (NOBOX), and paired-like homeodomain transcription factor 1 (PITX1). Determination of tissue-specific expression reveals that LHX8, but not PITX1 and NOBOX, is exclusively expressed in pig ovary tissue and is translocated into the cell nuclei. Overexpression of LHX8 in Chinese hamster ovary (CHO) cells could significantly promote BMP15 promoter activation. This study confirms a key regulatory element that is located in the proximal region of BMP15 promoter and is regulated by the LHX8 factor. PMID:26516845

  5. Characterization of the hypothalamus of Xenopus laevis during development. II. The basal regions.

    PubMed

    Domínguez, Laura; González, Agustín; Moreno, Nerea

    2014-04-01

    The expression patterns of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the basal hypothalamus of Xenopus laevis throughout development by means of combined immunohistochemical and in situ hybridization techniques. The connectivity of the main subdivisions was investigated by in vitro tracing techniques with dextran amines. The basal hypothalamic region is topologically rostral to the basal diencephalon and is composed of the tuberal (rostral) and mammillary (caudal) subdivisions, according to the prosomeric model. It is dorsally bounded by the optic chiasm and the alar hypothalamus, and caudally by the diencephalic prosomere p3. The tuberal hypothalamus is defined by the expression of Nkx2.1, xShh, and Isl1, and rostral and caudal portions can be distinguished by the distinct expression of Otp rostrally and Nkx2.2 caudally. In the mammillary region the xShh/Nkx2.1 combination defined the rostral mammillary area, expressing Nkx2.1, and the caudal retromammillary area, expressing xShh. The expression of xLhx1, xDll4, and Otp in the mammillary area and Isl1 in the tuberal region highlights the boundary between the two basal hypothalamic territories. Both regions are strongly connected with subpallial regions, especially those conveying olfactory/vomeronasal information, and also possess abundant intrahypothalamic connections. They show reciprocal connections with the diencephalon (mainly the thalamus), project to the midbrain tectum, and are bidirectionally related to the rhombencephalon. These results illustrate that the basal hypothalamus of anurans shares many features of specification, regionalization, and hodology with amniotes, reinforcing the idea of a basic bauplan in the organization of this prosencephalic region in all tetrapods.

  6. Does atrazine influence larval development and sexual differentiation in Xenopus laevis?

    PubMed

    Kloas, Werner; Lutz, Ilka; Springer, Timothy; Krueger, Henry; Wolf, Jeff; Holden, Larry; Hosmer, Alan

    2009-02-01

    Debate and controversy exists concerning the potential for the herbicide atrazine to cause gonadal malformations in developing Xenopus laevis. Following review of the existing literature the U.S. Environmental Protection Agency required a rigorous investigation conducted under standardized procedures. X. laevis tadpoles were exposed to atrazine at concentrations of 0.01, 0.1, 1, 25, or 100 microg/l from day 8 postfertilization (dpf) until completion of metamorphosis or dpf 83, whichever came first. Nearly identical experiments were performed in two independent laboratories: experiment 1 at Wildlife International, Ltd. and experiment 2 at the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB). Both experiments employed optimized animal husbandry procedures and environmental conditions in validated flow-through exposure systems. The two experiments demonstrated consistent survival, growth, and development of X. laevis tadpoles, and all measured parameters were within the expected ranges and were comparable in negative control and atrazine-treated groups. Atrazine, at concentrations up to 100 microg/l, had no effect in either experiment on the percentage of males or the incidence of mixed sex as determined by histological evaluation. In contrast, exposure of larval X. laevis to 0.2 microg 17beta-estradiol/l as the positive control resulted in gonadal feminization. Instead of an even distribution of male and female phenotypes, percentages of males:females:mixed sex were 19:75:6 and 22:60:18 in experiments 1 and 2, respectively. These studies demonstrate that long-term exposure of larval X. laevis to atrazine at concentrations ranging from 0.01 to 100 microg/l does not affect growth, larval development, or sexual differentiation.

  7. [Morphogenetic foundations for increased evolutionary complexity in the organization of thecate hydroids shoots (Cnidaria, Hydroidomedusa, Leptomedusae)].

    PubMed

    Kosevich, I A

    2012-01-01

    The morphogenetic approach is applied to analyze the diversity of spatial organization of shoots in thecate hydroids (Cnidaria, Hydroidomedusa, Leptomedusae). The main tendencies and constraints of increased evolutionary complexity in thecate hydroids colonies are uncovered.

  8. Bone graft substitutes and bone morphogenetic proteins for osteoporotic fractures: what is the evidence?

    PubMed

    Van Lieshout, Esther M M; Alt, Volker

    2016-01-01

    Despite improvements in implants and surgical techniques, osteoporotic fractures remain challenging to treat. Among other major risk factors, decreased expression of morphogenetic proteins has been identified for impaired fracture healing in osteoporosis. Bone grafts or bone graft substitutes are often used for stabilizing the implant and for providing a scaffold for ingrowth of new bone. Both synthetic and naturally occurring biomaterials are available. Products generally contain hydroxyapatite, tricalcium phosphate, dicalcium phosphate, calcium phosphate cement, calcium sulfate (plaster of Paris), or combinations of the above. Products have been used for the treatment of osteoporotic fractures of the proximal humerus, distal radius, vertebra, hip, and tibia plateau. Although there is generally consensus that screw augmentation increased the biomechanical properties and implant stability, the results of using these products for void filling are not unequivocal. In osteoporotic patients, Bone Morphogenetic Proteins (BMPs) have the potential impact to improve fracture healing by augmenting the impaired molecular and cellular mechanisms. However, the clinical evidence on the use of BMPs in patients with osteoporotic fractures is poor as there are no published clinical trials, case series or case studies. Even pre-clinical literature on in vitro and in vivo data is weak as most articles focus on the beneficial role for BMPs for restoration of the underlying pathophysiological factors of osteoporosis but do not look at the specific effects on osteoporotic fracture healing. Limited data on animal experiments suggest stimulation of fracture healing in ovariectomized rats by the use of BMPs. In conclusion, there is only limited data on the clinical relevance and optimal indications for the use of bone graft substitute materials and BMPs on the treatment of osteoporotic fractures despite the clinical benefits of these materials in other clinical indications. Given the

  9. Sustained release of bone morphogenetic protein 2 via coacervate improves the osteogenic potential of muscle-derived stem cells.

    PubMed

    Li, Hongshuai; Johnson, Noah Ray; Usas, Arvydas; Lu, Aiping; Poddar, Minakshi; Wang, Yadong; Huard, Johnny

    2013-09-01

    Muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle by a modified preplate technique exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. MDSCs retrovirally transduced to express bone morphogenetic proteins (BMPs) can differentiate into osteocytes and chondrocytes and enhance bone and articular cartilage repair in vivo, a feature that is not observed with nontransduced MDSCs. These results emphasize that MDSCs require prolonged exposure to BMPs to undergo osteogenic and chondrogenic differentiation. A sustained BMP protein delivery approach provides a viable and potentially more clinically translatable alternative to genetic manipulation of the cells. A unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD), was used to bind, protect, and sustain the release of bone morphogenetic protein-2 (BMP2) in a temporally and spatially controlled manner. Prolonged exposure to BMP2 released by the PEAD:heparin delivery system promoted the differentiation of MDSCs to an osteogenic lineage in vitro and induced the formation of viable bone at an ectopic site in vivo. This new strategy represents an alternative approach for bone repair mediated by MDSCs while bypassing the need for gene therapy.

  10. Bone morphogenetic protein-2 activates NADPH oxidase to increase endoplasmic reticulum stress and human coronary artery smooth muscle cell calcification.

    PubMed

    Liberman, Marcel; Johnson, Rebecca C; Handy, Diane E; Loscalzo, Joseph; Leopold, Jane A

    2011-09-30

    Bone morphogenetic protein-2 (BMP-2) increases oxidant stress and endoplasmic reticulum (ER) stress to stimulate differentiation of osteoblasts; however, the role of these signaling pathways in the transition of smooth muscle cells to a calcifying osteoblast-like phenotype remains incompletely characterized. We, therefore, treated human coronary artery smooth muscle cells (HCSMC) with BMP-2 (100ng/mL) and found an increase in NADPH oxidase activity and oxidant stress that occurred via activation of the bone morphogenetic protein receptor 2 and Smad 1 signaling. BMP-2-mediated oxidant stress also increased endoplasmic reticulum (ER) stress demonstrated by increased expression of GRP78, phospho-IRE1α, and the transcription factor XBP1. Analysis of a 1kb segment of the Runx2 promoter revealed an XBP1 binding site; electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that XBP1 bound to the Runx2 promoter at this site in BMP-2-treated HCSMC. Inhibition of oxidant stress or ER stress decreased Runx2 expression, intracellular calcium deposition, and mineralization of BMP-2-treated HCSMC. Thus, in HCSMC, BMP-2 increases oxidant stress and ER stress to increase Runx2 expression and promote vascular smooth muscle cell calcification.

  11. Bone Morphogenetic Protein Signaling Regulates Development and Activation of CD4(+) T Cells.

    PubMed

    Kuczma, Michal; Kraj, Piotr

    2015-01-01

    Bone morphogenetic proteins (BMPs) are growth factors belonging to the TGF-β (transforming growth factor β) superfamily. BMPs were found to regulate multiple cell processes such as proliferation, survival, differentiation, and apoptosis. They were originally described to play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites but were found to play a significant role in embryogenesis and development of multiple tissues and organs. Activities of BMPs are regulated by a number of secreted proteins, which modulate their availability to bind cellular receptors. The functions of individual BMPs are highly redundant due to binding the same receptors and inducing overlapping signal transduction pathways. Recently, BMPs were found to regulate cells of the innate and adaptive immune system. BMPs are involved in thymic development of T cells at the early, double negative, as well as later, double positive, stages of thymopoesis. They specifically modulate thymic development of regulatory T cells (T(reg)). In the periphery, BMPs affect T cell activation, promoting generation of T(reg) cells. We found that mice deficient for one of the receptors activated by BMPs demonstrated slower growth of transplantable melanoma tumors.

  12. Bone morphogenetic protein 2 stimulates endochondral ossification by regulating periosteal cell fate during bone repair

    PubMed Central

    Yu, Yan Yiu; Lieu, Shirley; Lu, Chuanyong; Colnot, Céline

    2010-01-01

    Bone repair depends on the coordinated action of numerous growth factors and cytokines to stimulate new skeletal tissue formation. Among all the growth factors involved in bone repair, Bone Morphogenetic Proteins (BMPs) are the only molecules now used therapeutically to enhance healing. Although BMPs are known as strong bone inducers, their role in initiating skeletal repair is not entirely elucidated. The aim of this study was to define the role of BMP2 during the early stages of bone regeneration and more specifically in regulating the fate of skeletal progenitors. During healing of non-stabilized fractures via endochondral ossification, exogenous BMP2 increased the deposition and resorption of cartilage and bone, which was correlated with a stimulation of osteoclastogenesis but not angiogenesis in the early phase of repair. During healing of stabilized fractures, which normally occurs via intramembranous ossification, exogenous BMP2 induced cartilage formation suggesting a role in regulating cell fate decisions. Specifically, the periosteum was found to be a target of exogenous BMP2 as shown by activation of the BMP pathway in this tissue. Using cell lineage analyses, we further show that BMP2 can direct cell differentiation towards the chondrogenic lineage within the periosteum but not the endosteum, indicating that skeletal progenitors within periosteum and endosteum respond differently to BMP signals. In conclusion, BMP2 plays an important role in the early stages of repair by recruiting local sources of skeletal progenitors within periosteum and endosteum and by determining their differentiation towards the chondrogenic and osteogenic lineages. PMID:20348041

  13. Imaging bone morphogenetic protein 7 induced cell cycle arrest in experimental gliomas.

    PubMed

    Klose, Anke; Waerzeggers, Yannic; Monfared, Parisa; Vukicevic, Slobodan; Kaijzel, Eric L; Winkeler, Alexandra; Wickenhauser, Claudia; Löwik, Clemens W G M; Jacobs, Andreas H

    2011-03-01

    Bone morphogenetic protein 7 (BMP-7) belongs to the superfamily of transforming growth factor β-like cytokines, which can act either as tumor suppressors or as tumor promoters depending on cell type and differentiation. Our investigations focused on analyzing the effects of BMP-7 during glioma cell proliferation in vitro and in vivo. BMP-7 treatment decreased the proliferation of Gli36ΔEGFR-LITG glioma cells up to 50%through a cell cycle arrest in the G(1) phase but not by induction of apoptosis. This effect was mediated by the modulation of the expression and phosphorylation of cyclin-dependent kinase 2, cyclin-dependent kinase inhibitor p21, and downstream retinoblastoma protein. Furthermore, in vivo optical imaging of luciferase activity of Gli36ΔEGFR-LITG cells implanted intracranially into nude mice in the presence or absence of BMP-7 treatment corroborated the antiproliferative effects of this cytokine. This report clearly underlines the tumor-suppressive role of BMP-7 in glioma-derived cells. Taken together, our results indicate that manipulating the BMP/transforming growth factor β signaling cascade may serve as a new strategy for imaging-guided molecular-targeted therapy of malignant gliomas.

  14. Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis.

    PubMed

    Murphy, Noelle; Gaynor, Katherine U; Rowan, Simon C; Walsh, Sinead M; Fabre, Aurelie; Boylan, John; Keane, Michael P; McLoughlin, Paul

    2016-03-01

    Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor β1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor β stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury.

  15. Expression of mutant bone morphogenetic protein receptor II worsens pulmonary hypertension secondary to pulmonary fibrosis.

    PubMed

    Bryant, Andrew J; Robinson, Linda J; Moore, Christy S; Blackwell, Thomas R; Gladson, Santhi; Penner, Niki L; Burman, Ankita; McClellan, Lucas J; Polosukhin, Vasiliy V; Tanjore, Harikrishna; McConaha, Melinda E; Gleaves, Linda A; Talati, Megha A; Hemnes, Anna R; Fessel, Joshua P; Lawson, William E; Blackwell, Timothy S; West, James D

    2015-12-01

    Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis.

  16. Expression of mutant bone morphogenetic protein receptor II worsens pulmonary hypertension secondary to pulmonary fibrosis

    PubMed Central

    Robinson, Linda J.; Moore, Christy S.; Blackwell, Thomas R.; Gladson, Santhi; Penner, Niki L.; Burman, Ankita; McClellan, Lucas J.; Polosukhin, Vasiliy V.; Tanjore, Harikrishna; McConaha, Melinda E.; Gleaves, Linda A.; Talati, Megha A.; Hemnes, Anna R.; Fessel, Joshua P.; Lawson, William E.; Blackwell, Timothy S.; West, James D.

    2015-01-01

    Abstract Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis. PMID:26697175

  17. Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency

    PubMed Central

    2013-01-01

    Background The complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD. Methods We sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure. Results Sequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools. Conclusions A mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD. PMID:24289245

  18. Inkjet-Based Biopatterning of Bone Morphogenetic Protein-2 to Spatially Control Calvarial Bone Formation

    PubMed Central

    Miller, Eric D.; DeCesare, Gary E.; Usas, Arvydas; Lensie, Emily L.; Bykowski, Michael R.; Huard, Johnny; Weiss, Lee E.; Losee, Joseph E.; Campbell, Phil G.

    2010-01-01

    The purpose of this study was to demonstrate spatial control of osteoblast differentiation in vitro and bone formation in vivo using inkjet bioprinting technology and to create three-dimensional persistent bio-ink patterns of bone morphogenetic protein-2 (BMP-2) and its modifiers immobilized within microporous scaffolds. Semicircular patterns of BMP-2 were printed within circular DermaMatrix™ human allograft scaffold constructs. The contralateral halves of the constructs were unprinted or printed with BMP-2 modifiers, including the BMP-2 inhibitor, noggin. Printed bio-ink pattern retention was validated using fluorescent or 125I-labeled bio-inks. Mouse C2C12 progenitor cells cultured on patterned constructs differentiated in a dose-dependent fashion toward an osteoblastic fate in register to BMP-2 patterns. The fidelity of spatial restriction of osteoblastic differentiation at the boundary between neighboring BMP-2 and noggin patterns improved in comparison with patterns without noggin. Acellular DermaMatrix constructs similarly patterned with BMP-2 and noggin were then implanted into a mouse calvarial defect model. Patterns of bone formation in vivo were comparable with patterned responses of osteoblastic differentiation in vitro. These results demonstrate that three-dimensional biopatterning of a growth factor and growth factor modifier within a construct can direct cell differentiation in vitro and tissue formation in vivo in register to printed patterns. PMID:20028232

  19. Regulation of cyclin E stability in Xenopus laevis embryos

    NASA Astrophysics Data System (ADS)

    Brandt-(Webb), Yekaterina

    Cyclin-Cdk complexes positively regulate cell cycle progression. Cyclins are regulatory subunits that bind to and activate cyclin-dependent kinases or Cdks. Cyclin E associates with Cdk2 to mediate G1/S phase transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how this protein is downregulated during development in order to determine if these mechanisms are disrupted during tumorigenesis, leading to its overexpression. Using Xenopus laevis embryos as a model, we have shown previously that during the first 12 embryonic cell cycles Cyclin E levels remain constant yet Cdk2 activity oscillates twice per cell cycle. Cyclin E is abruptly destabilized by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT). Based on work our work and work by others, we have hypothesized that differential phosphorylation and a change in localization result in Cyclin E degradation by the 26S proteasome at the MBT. To test this, we generated a series of point mutations in conserved threonine/serine residues implicated in degradation of human Cyclin E. Using Western blot analysis, we show that similarly to human Cyclin E, mutation of these residues to unphosphorylatable alanine stabilizes Cyclin E past the MBT when they are expressed in vivo. Cyclin E localization was studied by immunofluorescence analysis of endogenous and exogenous protein in pre-MBT, MBT, and post-MBT embryos. In addition, we developed a novel method of conjugating recombinant His6-tagged Cyclin E to fluorescent (CdSe)ZnS nanoparticles (quantum dots) capped with dihydrolipoic acid. Confocal microscopy was used to visualize His6Cyclin E-quantum dot complexes inside embryo cells in real time. We found that re-localization at the MBT from the cytoplasm to the nucleus

  20. THYROID AXIS INHIBITION IN XENOPUS LAEVIS: DEVELOPMENT OF AN AMPHIBIAN-BASED SCREENING ASSAY FOR THYROID DISRUPTION

    EPA Science Inventory

    In response to the initial EDSTAC recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. These experiments highlighted key limitations associated with reliance on tail resorption as a meas...

  1. CONCENTRATION DEPENDENT ACCUMULATION OF [3H]-DELTAMETHRIN IN SODIUM CHANNEL N AV1.2 EXPRESSING XENOPUS LAEVIS OOCYTES.

    EPA Science Inventory

    Disruption of neuronal voltage-sensitive sodium channels (VSSCs) by pyrethroid insecticides such as deltamethrin (DLT) has been widely studied using Xenopus laevis oocytes transfected with VSSC. However, the extent of pyrethroid accumulation in VSSC-expressing oocytes is unknown....

  2. Programming the emergence in morphogenetically architected complex systems.

    PubMed

    Varenne, Franck; Chaigneau, Pierre; Petitot, Jean; Doursat, René

    2015-09-01

    Large sets of elements interacting locally and producing specific architectures reliably form a category that transcends the usual dividing line between biological and engineered systems. We propose to call them morphogenetically architected complex systems (MACS). While taking the emergence of properties seriously, the notion of MACS enables at the same time the design (or "meta-design") of operational means that allow controlling and even, paradoxically, programming this emergence. To demonstrate our claim, we first show that among all the self-organized systems studied in the field of Artificial Life, the specificity of MACS essentially lies in the close relation between their emergent properties and functional properties. Second, we argue that to be a MACS a system does not need to display more than weak emergent properties. Third, since the notion of weak emergence is based on the possibility of simulation, whether computational or mechanistic via machines, we see MACS as good candidates to help design artificial self-architected systems (such as robotic swarms) but also harness and redesign living ones (such as synthetic bacterial films). PMID:26024971

  3. Bone morphogenetic protein signalling in heritable versus idiopathic pulmonary hypertension

    PubMed Central

    Dewachter, Laurence; Adnot, Serge; Guignabert, Christophe; Tu, Ly; Marcos, Elisabeth; Fadel, Elie; Humbert, Marc; Dartevelle, Philippe; Simonneau, Gérald; Naeije, Robert; Eddahibi, Saadia

    2009-01-01

    Mutations in gene encoding for bone morphogenetic protein type 2 receptor (BMPR-2) have been reported in pulmonary arterial hypertension (PAH), but their functional relevance remains incompletely understood. BMP receptors expression was evaluated in human lungs and in cultured pulmonary artery smooth muscle cells (PASMCs) isolated from 19 idiopathic PAH patients and 9 heritable PAH patients with demonstrated BMPR-2 mutations. BMP4-treated PASMCs were assessed for Smad and p38MAPK signaling associated to mitosis and apoptosis. Lung tissue and PASMCs from heritable PAH patients presented with decreased BMPR-2 expression and variable increases in BMPR-1A and BMPR-1B expressions, while a less important decreased BMPR-2 expression was observed in PASMCs from idiopathic PAH patients. Heritable PAH PASMCs showed no increased phosphorylation of Smad1/5/8 in the presence of BMP4, which actually activated the p38MAPK pathway. Individual responses varied from one mutation to another. PASMCs from PAH patients presented with an in vitro proliferative pattern, which could be inhibited by BMP4 in idiopathic PAH, not in heritable PAH. PASMCs from idiopathic PAH and more so from heritable presented an inhibition of BMP4-induced apoptosis. Most heterogenous BMPR-2 mutations are associated with defective Smad signaling compensed for by an activation of p38MAPK signaling, accounting for PASMC proliferation and deficient apoptosis. PMID:19324947

  4. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  5. Pleiotrophin regulates bone morphogenetic protein (BMP)-induced ectopic osteogenesis.

    PubMed

    Sato, Yasuko; Takita, Hiroko; Ohata, Noboru; Tamura, Masato; Kuboki, Yoshinori

    2002-06-01

    We previously isolated pleiotrophin (PTN) from bovine bone as a protein and showed that it stimulated osteoblastic growth and differentiation. Further details of its function, however, have not been fully clarified. The aim of this paper was to elucidate the effects of PTN on bone morphogenetic protein (BMP)-induced ectopic osteogenesis. Recombinant human BMP (rhBMP)-2 (1.2 microg) was combined with a fibrous glass membrane, which had been established as an effective carrier. Various amounts of the purified bovine PTN (5, 10, 50, and 100 microg) or rhPTN (5 and 10 microg) were added to the rhBMP-2/carrier composites and implanted into rats subcutaneously as reported. It was found that the amount of bone induced in the system increased with the addition of 10 microg of either purified PTN or rhPTN. However, the amount of bone decreased with the addition of 50 or 100 microg of purified PTN dose-dependently, as judged by both alkaline phosphatase activity and calcium content in the retrieved implants. It was concluded that purified PTN or rhPTN, at ratios of concentration of 10-100 microg of PTN to 1.2 microg of rhBMP-2 in the carrier, regulated the ectopic bone-inducing activity of rhBMP-2.

  6. Recurrence methods for the identification of morphogenetic patterns.

    PubMed

    Facchini, Angelo; Mocenni, Chiara

    2013-01-01

    This paper addresses the problem of identifying the parameters involved in the formation of spatial patterns in nonlinear two dimensional systems. To this aim, we perform numerical experiments on a prototypical model generating morphogenetic Turing patterns, by changing both the spatial frequency and shape of the patterns. The features of the patterns and their relationship with the model parameters are characterized by means of the Generalized Recurrence Quantification measures. We show that the recurrence measures Determinism and Recurrence Entropy, as well as the distribution of the line lengths, allow for a full characterization of the patterns in terms of power law decay with respect to the parameters involved in the determination of their spatial frequency and shape. A comparison with the standard two dimensional Fourier transform is performed and the results show a better performance of the recurrence indicators in identifying a reliable connection with the spatial frequency of the patterns. Finally, in order to evaluate the robustness of the estimation of the power low decay, extensive simulations have been performed by adding different levels of noise to the patterns. PMID:24066062

  7. Morphological evidence for a morphogenetic field in gastropod mollusc eggs.

    PubMed

    Tyler, S E; Butler, R D; Kimber, S J

    1998-01-01

    Eggs of the marine gastropod Crepidula fornicata examined by confocal imaging of FITC-lectin binding to the surface, and cryoscopic-SEM both reveal a surface architecture of linear structures organized around the animal-vegetal axis, which is spatially related to the anterior-posterior (a-p) axis of the subsequent embryo. A series of structures is also orientated with reference to specific micromere quartets formed during spiral cleavage. Thus, the surface architecture may provide a visible marker for a morphogenetic field which generates the a-p axis and organizes the cleavage pattern. Moreover, this architecture is co-extensive with that found on the vegetal, polar lobe-bearing region of eggs, as described by others, and which varies between gastropod taxa with varied types of body form. Confocal imaging reveals a distinct localization of F-actin to the architecture of the lobe region. However, the integrity of this F-actin is not responsible for the maintenance of the surface architecture. The significance of these findings to our understanding of the generation of diversity within the Gastropoda and general ontogenic mechanisms is discussed.

  8. Role of morphogenetic proteins in skeletal tissue engineering and regeneration.

    PubMed

    Reddi, A H

    1998-03-01

    Morphogenesis is the developmental cascade of pattern formation and body plan establishment, culminating in the adult form. It has formed the basis for the emerging discipline of tissue engineering, which uses principles of molecular developmental biology and morphogenesis gleaned through studies on inductive signals, responding stem cells, and the extracellular matrix to design and construct spare parts that restore function to the human body. Among the many organs in the body, bone has considerable powers for regeneration and is a prototype model for tissue engineering. Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. This model mimics sequential limb morphogenesis and has permitted the isolation of bone morphogens, such as bone morphogenetic proteins (BMPs), from demineralized adult bone matrix. BMPs initiate, promote, and maintain chondrogenesis and osteogenesis, but are also involved in the morphogenesis of organs other than bone. The symbiosis of the mechanisms underlying bone induction and differentiation is critical for tissue engineering and is governed by both biomechanics (physical forces) and context (microenvironment/extracellular matrix), which can be duplicated by biomimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhesion glycoproteins, including fibronectins and laminin. Rules of tissue architecture elucidated in bone morphogenesis may provide insights into tissue engineering and be universally applicable for all organs/tissues, including bones and joints. PMID:9528003

  9. Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

    PubMed Central

    Miyamoto, Kei; Simpson, David; Gurdon, John B.

    2015-01-01

    Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development. PMID:25742326

  10. Sexually dimorphic expression of Dmrt1 and γH2AX in germ stem cells during gonadal development in Xenopus laevis.

    PubMed

    Fujitani, Kazuko; Otomo, Asako; Wada, Mikako; Takamatsu, Nobuhiko; Ito, Michihiko

    2016-04-01

    In many animals, primordial germ cells (PGCs) migrate into developing gonads. There, they proliferate and differentiate into female and male germ stem cells (GSCs), oogonia and spermatogonia, respectively. Few studies have focused on the molecular mechanisms underlying the development of GSC sex determination. Here, we investigated the expression of the transcription factor Dmrt1 and a phosphorylated form of the histone variant H2AX (γH2AX) during gonadal development in Xenopus laevis. During early sexual differentiation, Dmrt1 was expressed in the GSCs of the ZW (female) and ZZ (male) gonads as well as somatic cells of the ZZ gonads. Notably, the PGCs and primary GSCs contained large, unstructured nuclei, whereas condensed, rounder nuclei appeared only in primary oogonia during tadpole development. After metamorphosis, Dmrt1 showed its expression in secondary spermatogonia, but not in secondary oogonia. Like Dmrt1, γH2AX was expressed in the nuclei of primary GSCs in early developing gonads. However, after metamorphosis, γH2AX expression continued in primary and secondary spermatogonia, but was barely detected in the condensed nuclei of primary oogonia. Taken together, these observations indicate that spermatogonia tend to retain PGC characteristics, compared to oogonia, which undergo substantial changes during gonadal differentiation in X. laevis. Our findings suggest that Dmrt1 and γH2AX may contribute to the maintenance of stem cell identity by controlling gene expression and epigenetic changes, respectively. PMID:27239441

  11. Bone morphogenetic protein 7 polarizes THP-1 cells into M2 macrophages.

    PubMed

    Rocher, Crystal; Singla, Reetu; Singal, Pawan K; Parthasarathy, Sampath; Singla, Dinender K

    2012-07-01

    It was hypothesized that monocyte treatment with bone morphogenetic protein 7 (BMP7) would significantly enhance monocyte polarization into M2 macrophages as well as increasing the levels of anti-inflammatory cytokines. In a cell culture system using monocytes (human acute monocytic leukemia cell line THP-1), we studied the effects of BMP7 on monocytes polarizing into M2 macrophages. The data demonstrate that THP-1 cells contain a BMP type II receptor (BMPR2), and that its activation is significantly (p < 0.05) increased following treatment with BMP7. Furthermore, there was an increase of M2 macrophages, BMPR2, and anti-inflammatory cytokines interleukin (IL)-10 and IL-1ra compared with the respective controls. Moreover, treatment with BMP7 caused a significant (p < 0.05) decrease in the levels of pro-inflammatory cytokines IL-6, tumour necrosis factor (TNF-α), and monocyte chemotactic protein-1 (MCP-1), compared with the controls. In conclusion, we suggest for the first time that BMP7 has a unique potential to polarize monocytes into M2 macrophages, required for tissue repair, which will have significant applications for the treatment of atherosclerosis. PMID:22720873

  12. Recombinant Human Bone Morphogenetic Protein-2 in Development and Progression of Oral Squamous Cell Carcinoma.

    PubMed

    Zaid, Khaled Waleed; Chantiri, Mansour; Bassit, Ghassan

    2016-01-01

    Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-β superfamily, regulate many cellular activities including cell migration, differentiation, adhesion, proliferation and apoptosis. Use of recombinant human bone morphogenic protein?2 (rhBMP?2) in oral and maxillofacial surgery has seen a tremendous increase. Due to its role in many cellular pathways, the influence of this protein on carcinogenesis in different organs has been intensively studied over the past decade. BMPs also have been detected to have a role in the development and progression of many tumors, particularly disease-specific bone metastasis. In oral squamous cell carcinoma - the tumor type accounting for more than 90% of head and neck malignancies- aberrations of both BMP expression and associated signaling pathways have a certain relation with the development and progression of the disease by regulating a range of biological functions in the altered cells. In the current review, we discuss the influence of BMPs -especially rhBMP-2- in the development and progression of oral squamous cell carcinoma. PMID:27039814

  13. [MORPHOLOGICAL CHARACTERISTICS OF OSSEOINTEGRATION AFTERAPPLICATION OF TITANIUM IMPLANTS WITH BIOACTIVE COATING AND RECOMBINANT BONE MORPHOGENETIC PROTEIN].

    PubMed

    Gaifullin, N M; Karyagina, A S; Gromov, A V; Terpilovskiy, A A; Malanin, D A; Demeshchenko, M V; Novochadov, V V

    2016-01-01

    Experiments were carried out on 22 albino male Wistar rats to study the morphological peculiarities of osseointegration of titanium grafts with bioactive surface stimulated additionally with bone plastic material "Gamalant-paste-FORTE Plus" containing recombinant human bone morphogenetic protein-2 (rhBMP-2). In 9 rats the implants were placed into femoral bones after local treatment of bone canal with rhBMP-2-containing material. Another 9 animals were implanted but received no treatment, 4 rats formed the group of intact control. Zone of osseointegration was studied 4, 8 and 12 weeks after graft placement using histological and morphometric methods as well as immune histochemistry to demonstrate osteonectin, CD68, MMP-9, and TIMP-1. The study showed that preliminary treatment of bone canal with rhBMP-2-containing material preceding implant placement was accompanied by an additional osteoinductive effect. More intense and outrunning bone formation in the area of osseointegration was observed, together with remodeling and compaction of the contiguous cancellous bone, thus providing the necessary balance between MMP-9 and TIMP-1 with a high level of each factor expression. PMID:27487669

  14. Bone Morphogenetic Protein 2 Signaling Negatively Modulates Lymphatic Development in Vertebrate Embryos

    PubMed Central

    Dunworth, William P.; Cardona-Costa, Jose; Bozkulak, Esra Cagavi; Kim, Jun-Dae; Meadows, Stryder; Fischer, Johanna C.; Wang, Yeqi; Cleaver, Ondine; Qyang, Yibing; Ober, Elke A.; Jin, Suk-Won

    2014-01-01

    Rationale The emergence of lymphatic endothelial cells (LECs) seems to be highly regulated during development. Although several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown. Objective Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. Methods and Results BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2 signaling in zebrafish embryos and mouse embryonic stem cell–derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox protein 1 during development. Conclusions Our data identify BMP2 as a key negative regulator for the emergence of the lymphatic lineage during vertebrate development. PMID:24122719

  15. Treatment with bone morphogenetic protein 2 limits infarct size after myocardial infarction in mice.

    PubMed

    Ebelt, Henning; Hillebrand, Ina; Arlt, Stephan; Zhang, Ying; Kostin, Sawa; Neuhaus, Herbert; Müller-Werdan, Ursula; Schwarz, Elisabeth; Werdan, Karl; Braun, Thomas

    2013-04-01

    Various strategies have been devised to reduce the clinical consequences of myocardial infarction, including acute medical care, revascularization, stem cell transplantations, and more recently, prevention of cardiomyocyte cell death. Activation of embryonic signaling pathways is a particularly interesting option to complement these strategies and to improve the functional performance and survival rate of cardiomyocytes. Here, we have concentrated on bone morphogenetic protein 2 (BMP-2), which induces ectopic formation of beating cardiomyocytes during development in the mesoderm and protects neonatal cardiomyocytes from ischemia-reperfusion injury. In a mouse model of acute myocardial infarction, an i.v. injection of BMP-2 reduced infarct size in mice when given after left anterior descending artery ligation. Mice treated with BMP-2 are characterized by a reduced rate of apoptotic cardiomyocytes both in the border zone of the infarcts and in the remote myocardium. In vitro, BMP-2 increases the frequency of spontaneously beating neonatal cardiomyocytes and the contractile performance under electrical pacing at 2 Hz, preserves cellular adenosine triphosphate stores, and decreases the rate of apoptosis despite the increased workload. In addition, BMP-2 specifically induced phosphorylation of Smad1/5/8 proteins and protected adult cardiomyocytes from long-lasting hypoxia-induced cellular damage and oxidative stress without activation of the cardiodepressant transforming growth factor-β pathway. Our data suggest that BMP-2 treatment may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by improving the contractility of cardiomyocytes and preventing cardiomyocyte cell death.

  16. Cross Talk between Insulin and Bone Morphogenetic Protein Signaling Systems in Brown Adipogenesis ▿ †

    PubMed Central

    Zhang, Hongbin; Schulz, Tim J.; Espinoza, Daniel O.; Huang, Tian Lian; Emanuelli, Brice; Kristiansen, Karsten; Tseng, Yu-Hua

    2010-01-01

    Both insulin and bone morphogenetic protein (BMP) signaling systems are important for adipocyte differentiation. Analysis of gene expression in BMP7-treated fibroblasts revealed a coordinated change in insulin signaling components by BMP7. To further investigate the cross talk between insulin and BMP signaling systems in brown adipogenesis, we examined the effect of BMP7 in insulin receptor substrate 1 (IRS-1)-deficient brown preadipocytes, which exhibit a severe defect in differentiation. Treatment of these cells with BMP7 for 3 days prior to adipogenic induction restored differentiation and expression of brown adipogenic markers. The high level of adipogenic inhibitor preadipocyte factor 1 (Pref-1) in IRS-1-null cells was markedly reduced by 3 days of BMP7 treatment, and analysis of the 1.3-kb pref-1 promoter revealed 9 putative Smad binding elements (SBEs), suggesting that BMP7 could directly suppress Pref-1 expression, thereby allowing the initiation of the adipogenic program. Using a series of sequential deletion mutants of the pref-1 promoter linked to the luciferase gene and chromatin immunoprecipitation, we demonstrate that the promoter-proximal SBE (−192/−184) was critical in mediating BMP7's suppressive effect on pref-1 transcription. Together, these data suggest cross talk between the insulin and BMP signaling systems by which BMP7 can rescue brown adipogenesis in cells with insulin resistance. PMID:20584981

  17. Bone morphogenetic protein (BMP) signaling regulates mitotic checkpoint protein levels in human breast cancer cells.

    PubMed

    Yan, Hualong; Zhu, Songcheng; Song, Chenlin; Liu, Naifa; Kang, Jiuhong

    2012-04-01

    Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection. PMID:22234345

  18. Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

    PubMed Central

    Cyr-Depauw, Chanèle; Northey, Jason J.; Tabariès, Sébastien; Annis, Matthew G.; Dong, Zhifeng; Cory, Sean; Hallett, Michael; Rennhack, Jonathan P.; Andrechek, Eran R.

    2016-01-01

    ShcA is an important mediator of ErbB2- and transforming growth factor β (TGF-β)-induced breast cancer cell migration, invasion, and metastasis. We show that in the context of reduced ShcA levels, the bone morphogenetic protein (BMP) antagonist chordin-like 1 (Chrdl1) is upregulated in numerous breast cancer cells following TGF-β stimulation. BMPs have emerged as important modulators of breast cancer aggressiveness, and we have investigated the ability of Chrdl1 to block BMP-induced increases in breast cancer cell migration and invasion. Breast cancer-derived conditioned medium containing elevated concentrations of endogenous Chrdl1, as well as medium containing recombinant Chrdl1, suppresses BMP4-induced signaling in multiple breast cancer cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling. PMID:26976638

  19. Use of bone morphogenetic proteins in mesenchymal stem cell stimulation of cartilage and bone repair

    PubMed Central

    Scarfì, Sonia

    2016-01-01

    The extracellular matrix-associated bone morphogenetic proteins (BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively participate to kidney development, digit and limb formation, angiogenesis, tissue fibrosis and tumor development. Since their discovery, they have attracted attention for their fascinating perspectives in the regenerative medicine and tissue engineering fields. BMPs have been employed in many preclinical and clinical studies exploring their chondrogenic or osteoinductive potential in several animal model defects and in human diseases. During years of research in particular two BMPs, BMP2 and BMP7 have gained the podium for their use in the treatment of various cartilage and bone defects. In particular they have been recently approved for employment in non-union fractures as adjunct therapies. On the other hand, thanks to their potentialities in biomedical applications, there is a growing interest in studying the biology of mesenchymal stem cell (MSC), the rules underneath their differentiation abilities, and to test their true abilities in tissue engineering. In fact, the specific differentiation of MSCs into targeted cell-type lineages for transplantation is a primary goal of the regenerative medicine. This review provides an overview on the current knowledge of BMP roles and signaling in MSC biology and differentiation capacities. In particular the article focuses on the potential clinical use of BMPs and MSCs concomitantly, in cartilage and bone tissue repair. PMID:26839636

  20. [MORPHOLOGICAL CHARACTERISTICS OF OSSEOINTEGRATION AFTERAPPLICATION OF TITANIUM IMPLANTS WITH BIOACTIVE COATING AND RECOMBINANT BONE MORPHOGENETIC PROTEIN].

    PubMed

    Gaifullin, N M; Karyagina, A S; Gromov, A V; Terpilovskiy, A A; Malanin, D A; Demeshchenko, M V; Novochadov, V V

    2016-01-01

    Experiments were carried out on 22 albino male Wistar rats to study the morphological peculiarities of osseointegration of titanium grafts with bioactive surface stimulated additionally with bone plastic material "Gamalant-paste-FORTE Plus" containing recombinant human bone morphogenetic protein-2 (rhBMP-2). In 9 rats the implants were placed into femoral bones after local treatment of bone canal with rhBMP-2-containing material. Another 9 animals were implanted but received no treatment, 4 rats formed the group of intact control. Zone of osseointegration was studied 4, 8 and 12 weeks after graft placement using histological and morphometric methods as well as immune histochemistry to demonstrate osteonectin, CD68, MMP-9, and TIMP-1. The study showed that preliminary treatment of bone canal with rhBMP-2-containing material preceding implant placement was accompanied by an additional osteoinductive effect. More intense and outrunning bone formation in the area of osseointegration was observed, together with remodeling and compaction of the contiguous cancellous bone, thus providing the necessary balance between MMP-9 and TIMP-1 with a high level of each factor expression.

  1. Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

    PubMed

    Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

    2015-03-01

    Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway.

  2. The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors

    SciTech Connect

    Kalen, Anders; Wahlstroem, Ola; Linder, Cecilia Halling; Magnusson, Per

    2008-10-17

    Platelet derivates and platelet rich plasma have been used to stimulate bone formation and wound healing because of the rich content of potent growth factors. However, not all reports have been conclusive since some have not been able to demonstrate a positive effect. We investigated the interindividual variation of bone morphogenetic proteins (BMPs) in platelets from healthy donors, and the pH-dependent effect on the release of BMPs in preparations of lysed platelets in buffer (LPB). Platelet concentrates from 31 healthy donors were prepared in pH 4.3 and pH 7.4 buffers and investigated with respect to BMP-2, -4, -6, and -7. BMP-2 and BMP-4 were significantly more common in acidic LPBs in comparison with neutral preparations. We also observed a considerable variation among platelet donors with respect to the release of BMPs at pH 4.3 and 7.4. In conclusion, a considerable variation was found among platelet donors, which may be of importance considering the ambiguous results previously reported on osteoblast proliferation and differentiation.

  3. The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

    PubMed Central

    Chen, Justin L.; Qian, Hongwei; Liu, Yingying; Bernardo, Bianca C.; Beyer, Claudia; Watt, Kevin I.; Thomson, Rachel E.; Connor, Timothy; Turner, Bradley J.; McMullen, Julie R.; Larsson, Lars; McGee, Sean L.; Harrison, Craig A.

    2013-01-01

    Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders. PMID:24145169

  4. Effect of selective heparin desulfation on preservation of bone morphogenetic protein-2 bioactivity after thermal stress.

    PubMed

    Seto, Song P; Miller, Tobias; Temenoff, Johnna S

    2015-02-18

    Bone morphogenetic protein-2 (BMP-2) plays an important role in bone and cartilage formation and is of interest in regenerative medicine. Heparin can interact electrostatically with BMP-2 and thus has been explored for controlled release and potential stabilization of this growth factor in vivo. However, in its natively sulfated state, heparin has potent anticoagulant properties that may limit its use. Desulfation reduces anticoagulant properties, but may impact heparin's ability to interact and protect BMP-2 from denaturation. The goal of this study was to characterize three selectively desulfated heparin species (N-desulfated (Hep(-N)), 6-O,N-desulfated (Hep(-N,-6O)), and completely desulfated heparin (Hep(-))) and determine if the sulfation level of heparin affected the level of BMP-2 bioactivity after heat treatment at 65 °C. BMP-2 bioactivity was evaluated using the established C2C12 cell assay. The resulting alkaline phosphatase activity data demonstrated that native heparin maintained a significant amount of BMP-2 bioactivity and the effect appeared to be heparin concentration dependent. Although all three had the same molecular charge as determined by zeta potential measurements, desulfated heparin derivatives Hep(-N) and Hep(-N,-6O) were not as effective as native heparin in maintaining BMP-2 bioactivity (only ~35% of original activity remained in both cases). These findings can be used to better select desulfated heparin species that exhibit low anticoagulant activity while extending the half-life of BMP-2 in solution and in delivery systems.

  5. Hydrogel Delivery of Mesenchymal Stem Cell–Expressing Bone Morphogenetic Protein-2 Enhances Bone Defect Repair

    PubMed Central

    Hsiao, Hui-Yi; Yang, Shu-Rui; Brey, Eric M.; Chu, I-Ming

    2016-01-01

    Background: The application of bone tissue engineering for repairing bone defects has gradually shown some satisfactory progress. One of the concerns raising scientific attention is the poor supply of growth factors. A number of growth factor delivery approaches have been developed for promoting bone formation. However, there is no systematic comparison of those approaches on efficiency of neobone formation. In this study, the approaches using periosteum, direct supply of growth factors, or gene transfection of growth factors were evaluated to determine the osteogenic capacity on the repair of bone defect. Methods: In total, 42 male 21-week-old Sprague-Dawley rats weighing 250 to 400 g were used as the bone defect model to evaluate the bone repair efficiency. Various tissue engineered constructs of poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogel with periosteum, with external supply of bone morphogenetic protein-2 (BMP2), or with BMP2-transfected bone marrow–derived mesenchymal stem cells (BMMSCs) were filled in a 7-mm bone defect region. Animals were euthanized at 3 months, and the hydrogel constructs were harvested. The evaluation with histological staining and radiography analysis were performed for the volume of new bone formation. Results: The PEG-PLLA scaffold with BMMSCs promotes bone regeneration with the addition of periosteum. The group with BMP2-transfected BMMSCs demonstrated the largest volume of new bone among all the testing groups. Conclusions: Altogether, the results of this study provide the evidence that the combination of PEG-PLLA hydrogels with BMMSCs and sustained delivery of BMP2 resulted in the maximal bone regeneration.

  6. Hydrogel Delivery of Mesenchymal Stem Cell–Expressing Bone Morphogenetic Protein-2 Enhances Bone Defect Repair

    PubMed Central

    Hsiao, Hui-Yi; Yang, Shu-Rui; Brey, Eric M.; Chu, I-Ming

    2016-01-01

    Background: The application of bone tissue engineering for repairing bone defects has gradually shown some satisfactory progress. One of the concerns raising scientific attention is the poor supply of growth factors. A number of growth factor delivery approaches have been developed for promoting bone formation. However, there is no systematic comparison of those approaches on efficiency of neobone formation. In this study, the approaches using periosteum, direct supply of growth factors, or gene transfection of growth factors were evaluated to determine the osteogenic capacity on the repair of bone defect. Methods: In total, 42 male 21-week-old Sprague-Dawley rats weighing 250 to 400 g were used as the bone defect model to evaluate the bone repair efficiency. Various tissue engineered constructs of poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogel with periosteum, with external supply of bone morphogenetic protein-2 (BMP2), or with BMP2-transfected bone marrow–derived mesenchymal stem cells (BMMSCs) were filled in a 7-mm bone defect region. Animals were euthanized at 3 months, and the hydrogel constructs were harvested. The evaluation with histological staining and radiography analysis were performed for the volume of new bone formation. Results: The PEG-PLLA scaffold with BMMSCs promotes bone regeneration with the addition of periosteum. The group with BMP2-transfected BMMSCs demonstrated the largest volume of new bone among all the testing groups. Conclusions: Altogether, the results of this study provide the evidence that the combination of PEG-PLLA hydrogels with BMMSCs and sustained delivery of BMP2 resulted in the maximal bone regeneration. PMID:27622106

  7. Effects of Elevated In Ovo Selenium Exposure on Late Stage Development of Xenopus laevis Tadpoles.

    PubMed

    Massé, Anita J; Muscatello, Jorgelina R; Janz, David M

    2016-10-01

    The effects of elevated in ovo selenium (Se) exposure on late stage larval Xenopus laevis development were investigated. Adult females were fed diets augmented with selenomethionine for 68 days and bred with untreated males to obtain embryos with measured concentrations of 1.6 (control), 10.8, 28.1 and 81.7 μg Se/g dry mass. Embryos were reared under uncontaminated conditions until 50 % of individuals within an aquarium had completed metamorphosis. The highest in ovo Se exposure group exhibited greater froglet body mass and snout to vent length while having a higher proportion of tadpoles at earlier stages of development. No differences were detected among treatment groups for mortality or metamorphic timing during the rearing period. This research suggests that in ovo Se exposure has minimal effect on the survival and development of late stage larval X. laevis in a controlled laboratory environment with adequate food availability. PMID:27412338

  8. The optic vesicle promotes cornea to lens transdifferentiation in larval Xenopus laevis

    PubMed Central

    Cannata, Stefano M; Bernardini, Sergio; Filoni, Sergio; Gargioli, Cesare

    2008-01-01

    The outer cornea and pericorneal epidermis (lentogenic area) of larval Xenopus laevis are the only epidermal regions competent to regenerate a lens under the influence of the retinal inducer. However, the head epidermis of the lentogenic area can acquire the lens-regenerating competence following transplantation of an eye beneath it. In this paper we demonstrate that both the outer cornea and the head epidermis covering a transplanted eye are capable of responding not only to the retinal inducer of the larval eye but also to the inductive action of the embryonic optic vesicle by synthesizing crystallins. As the optic vesicle is a very weak lens inductor, which promotes crystallin synthesis only on the lens biased ectoderm of the embryo, these results indicate that the lens-forming competence in the outer cornea and epidermis of larval X. laevis corresponds to the persistence and acquisition of a condition similar to that of the embryonic biased ectoderm. PMID:18430089

  9. Expression of the rabbit intestinal N2 Na+/nucleoside transporter in Xenopus laevis oocytes.

    PubMed Central

    Jarvis, S M; Griffith, D A

    1991-01-01

    Polyadenylated [poly(A)+] mRNA isolated from rabbit small-intestinal mucosa was injected into Xenopus laevis oocytes, and expression of the N2 Na+/nucleoside co-transporter was assayed by measuring Na(+)-dependent thymidine uptake. Expression of Na(+)-dependent thymidine uptake steadily increased after mRNA injection and was on average increased 11-fold by day 6 over background. Na(+)-dependent thymidine uptake was saturable (apparent Km approximately 30 microM at 22 degrees C) and inhibited by uridine and cytidine, but not by guanosine and inosine. These properties of the expressed thymidine transport strongly suggest that the epithelial N2 Na+/nucleoside co-transporter can be expressed in X. laevis oocytes. PMID:1898349

  10. Diagnosis of Aeromonas hydrophila, Mycobacterium species, and Batrachochytrium dendrobatidis in an African Clawed Frog (Xenopus laevis)

    PubMed Central

    Hill, William A; Newman, Shelley J; Craig, Linden; Carter, Christopher; Czarra, Jane; Brown, J Paige

    2010-01-01

    Here we describe diagnosis of concurrent infection with Aeromonas hydrophila, Mycobacterium spp., and Batrachochytrium dendrobatidis in a wild female Xenopus laevis captured in Chile and transported to the United States. After approximately 130 d in the laboratory, the frog was presented for dysecdysis and obtundation. After euthanasia, tissues were submitted for histopathologic evaluation and PCR analysis for B. dendrobatidis and Ranavirus. Clinically significant gross lesions included cutaneous ulcerations on the lip, right forelimb, and ventral chest. Microscopic findings included regionally extensive splenic necrosis, diffuse pneumonia, and fibrinous coelomitis all containing intralesional bacteria. PCR analysis yielded positive results for B. dendrobatidis only. Bacterial culture of the ulcerated skin and liver yielded A. hydrophila. Infection with Contracaecum spp. was diagnosed as an incidental finding. To our knowledge, this case is the first report of simultaneous infection with Aeromonas hydrophila, Mycobacterium spp., and Batrachochytrium dendrobatidis in a laboratory-maintained X. laevis captured from the wild. PMID:20353698

  11. Isolation of Chlamydia psittaci from naturally infected African clawed frogs (Xenopus laevis).

    PubMed Central

    Wilcke, B W; Newcomer, C E; Anver, M R; Simmons, J L; Nace, G W

    1983-01-01

    An inclusion-forming agent was isolated from the livers of commercially raised African clawed frogs (Xenopus laevis) involved in an epizootic of high morbidity and mortality. Original isolation was made in McCoy cells. This agent was identified as Chlamydia psittaci based on the formation of typical intracytoplasmic inclusions which developed within 48 h, were not stained by iodine, and were resistant to sulfadiazine. The isolate from one particular frog (designated as strain 178) was further studied and found to be lethal for 7-day-old embryonated chicken eggs after intra-yolk sac inoculation. This strain was demonstrated not to be pathogenic for mice when inoculated intraperitoneally. The cell culture isolate of C. psittaci was transmitted to uninfected X. laevis, causing disease and death. Images PMID:6347897

  12. Effects of Elevated In Ovo Selenium Exposure on Late Stage Development of Xenopus laevis Tadpoles.

    PubMed

    Massé, Anita J; Muscatello, Jorgelina R; Janz, David M

    2016-10-01

    The effects of elevated in ovo selenium (Se) exposure on late stage larval Xenopus laevis development were investigated. Adult females were fed diets augmented with selenomethionine for 68 days and bred with untreated males to obtain embryos with measured concentrations of 1.6 (control), 10.8, 28.1 and 81.7 μg Se/g dry mass. Embryos were reared under uncontaminated conditions until 50 % of individuals within an aquarium had completed metamorphosis. The highest in ovo Se exposure group exhibited greater froglet body mass and snout to vent length while having a higher proportion of tadpoles at earlier stages of development. No differences were detected among treatment groups for mortality or metamorphic timing during the rearing period. This research suggests that in ovo Se exposure has minimal effect on the survival and development of late stage larval X. laevis in a controlled laboratory environment with adequate food availability.

  13. Urotensin II upregulates migration and cytokine gene expression in leukocytes of the African clawed frog, Xenopus laevis.

    PubMed

    Tomiyama, Shiori; Nakamachi, Tomoya; Uchiyama, Minoru; Matsuda, Kouhei; Konno, Norifumi

    2015-05-15

    Urotensin II (UII) exhibits diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response via the UII receptor (UTR) in mammals. However, in amphibians the function of the UII-UTR system remains unknown. In the present study, we investigated the potential immune function of UII using leukocytes isolated from the African clawed frog, Xenopus laevis. Stimulation of male frogs with lipopolysaccharide increased mRNA expression of UII and UTR in leukocytes, suggesting that inflammatory stimuli induce activation of the UII-UTR system. Migration assays showed that both UII and UII-related peptide enhanced migration of leukocytes in a dose-dependent manner, and that UII effect was inhibited by the UTR antagonist urantide. Inhibition of Rho kinase with Y-27632 abolished UII-induced migration, suggesting that it depends on the activation of RhoA/Rho kinase. Treatment of isolated leukocytes with UII increased the expression of several cytokine genes including tumor necrosis factor-α, interleukin-1β, and macrophage migration inhibitory factor, and the effects were abolished by urantide. These results suggest that in amphibian leukocytes the UII-UTR system is involved in the activation of leukocyte migration and cytokine gene expression in response to inflammatory stimuli.

  14. Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

    SciTech Connect

    Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.

    1988-05-03

    DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, (..gamma..-/sup 32/P)ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.

  15. Impacts of Climate Change on the Global Invasion Potential of the African Clawed Frog Xenopus laevis

    PubMed Central

    Ihlow, Flora; Courant, Julien; Secondi, Jean; Herrel, Anthony; Rebelo, Rui; Measey, G. John; Lillo, Francesco; De Villiers, F. André; Vogt, Solveig; De Busschere, Charlotte; Backeljau, Thierry; Rödder, Dennis

    2016-01-01

    By altering or eliminating delicate ecological relationships, non-indigenous species are considered a major threat to biodiversity, as well as a driver of environmental change. Global climate change affects ecosystems and ecological communities, leading to changes in the phenology, geographic ranges, or population abundance of several species. Thus, predicting the impacts of global climate change on the current and future distribution of invasive species is an important subject in macroecological studies. The African clawed frog (Xenopus laevis), native to South Africa, possesses a strong invasion potential and populations have become established in numerous countries across four continents. The global invasion potential of X. laevis was assessed using correlative species distribution models (SDMs). SDMs were computed based on a comprehensive set of occurrence records covering South Africa, North America, South America and Europe and a set of nine environmental predictors. Models were built using both a maximum entropy model and an ensemble approach integrating eight algorithms. The future occurrence probabilities for X. laevis were subsequently computed using bioclimatic variables for 2070 following four different IPCC scenarios. Despite minor differences between the statistical approaches, both SDMs predict the future potential distribution of X. laevis, on a global scale, to decrease across all climate change scenarios. On a continental scale, both SDMs predict decreasing potential distributions in the species’ native range in South Africa, as well as in the invaded areas in North and South America, and in Australia where the species has not been introduced. In contrast, both SDMs predict the potential range size to expand in Europe. Our results suggest that all probability classes will be equally affected by climate change. New regional conditions may promote new invasions or the spread of established invasive populations, especially in France and Great

  16. Impacts of Climate Change on the Global Invasion Potential of the African Clawed Frog Xenopus laevis.

    PubMed

    Ihlow, Flora; Courant, Julien; Secondi, Jean; Herrel, Anthony; Rebelo, Rui; Measey, G John; Lillo, Francesco; De Villiers, F André; Vogt, Solveig; De Busschere, Charlotte; Backeljau, Thierry; Rödder, Dennis

    2016-01-01

    By altering or eliminating delicate ecological relationships, non-indigenous species are considered a major threat to biodiversity, as well as a driver of environmental change. Global climate change affects ecosystems and ecological communities, leading to changes in the phenology, geographic ranges, or population abundance of several species. Thus, predicting the impacts of global climate change on the current and future distribution of invasive species is an important subject in macroecological studies. The African clawed frog (Xenopus laevis), native to South Africa, possesses a strong invasion potential and populations have become established in numerous countries across four continents. The global invasion potential of X. laevis was assessed using correlative species distribution models (SDMs). SDMs were computed based on a comprehensive set of occurrence records covering South Africa, North America, South America and Europe and a set of nine environmental predictors. Models were built using both a maximum entropy model and an ensemble approach integrating eight algorithms. The future occurrence probabilities for X. laevis were subsequently computed using bioclimatic variables for 2070 following four different IPCC scenarios. Despite minor differences between the statistical approaches, both SDMs predict the future potential distribution of X. laevis, on a global scale, to decrease across all climate change scenarios. On a continental scale, both SDMs predict decreasing potential distributions in the species' native range in South Africa, as well as in the invaded areas in North and South America, and in Australia where the species has not been introduced. In contrast, both SDMs predict the potential range size to expand in Europe. Our results suggest that all probability classes will be equally affected by climate change. New regional conditions may promote new invasions or the spread of established invasive populations, especially in France and Great Britain

  17. Epistylididae ectoparasites in a colony of African clawed frogs (Xenopus laevis).

    PubMed

    Pritchett, Kathleen R; Sanders, George E

    2007-03-01

    This report describes the discovery and treatment of a multiagent infection in a captive colony of adult, female Xenopus laevis. Animals were determined to be infected with Saprolegnia sp, a relatively common fungal parasite in laboratory-housed frogs, and a less common ectoparasite, Epistylis sp, that had been described only once before in frogs. We discuss the diagnosis, pathology, and treatment of Epistylis and the importance of water-quality monitoring and husbandry in the care of these research animals.

  18. Impacts of Climate Change on the Global Invasion Potential of the African Clawed Frog Xenopus laevis.

    PubMed

    Ihlow, Flora; Courant, Julien; Secondi, Jean; Herrel, Anthony; Rebelo, Rui; Measey, G John; Lillo, Francesco; De Villiers, F André; Vogt, Solveig; De Busschere, Charlotte; Backeljau, Thierry; Rödder, Dennis

    2016-01-01

    By altering or eliminating delicate ecological relationships, non-indigenous species are considered a major threat to biodiversity, as well as a driver of environmental change. Global climate change affects ecosystems and ecological communities, leading to changes in the phenology, geographic ranges, or population abundance of several species. Thus, predicting the impacts of global climate change on the current and future distribution of invasive species is an important subject in macroecological studies. The African clawed frog (Xenopus laevis), native to South Africa, possesses a strong invasion potential and populations have become established in numerous countries across four continents. The global invasion potential of X. laevis was assessed using correlative species distribution models (SDMs). SDMs were computed based on a comprehensive set of occurrence records covering South Africa, North America, South America and Europe and a set of nine environmental predictors. Models were built using both a maximum entropy model and an ensemble approach integrating eight algorithms. The future occurrence probabilities for X. laevis were subsequently computed using bioclimatic variables for 2070 following four different IPCC scenarios. Despite minor differences between the statistical approaches, both SDMs predict the future potential distribution of X. laevis, on a global scale, to decrease across all climate change scenarios. On a continental scale, both SDMs predict decreasing potential distributions in the species' native range in South Africa, as well as in the invaded areas in North and South America, and in Australia where the species has not been introduced. In contrast, both SDMs predict the potential range size to expand in Europe. Our results suggest that all probability classes will be equally affected by climate change. New regional conditions may promote new invasions or the spread of established invasive populations, especially in France and Great Britain.

  19. Reproduction, larval growth, and reproductive development in African clawed frogs (Xenopus laevis) exposed to atrazine.

    PubMed

    Du Preez, Louis H; Kunene, Nisile; Everson, Gideon J; Carr, James A; Giesy, John P; Gross, Timothy S; Hosmer, Alan J; Kendall, Ronald J; Smith, Ernest E; Solomon, Keith R; Van Der Kraak, Glen J

    2008-03-01

    Reproductive success and development of F2 offspring from F1 adult African clawed frogs (Xenopus laevis) exposed to atrazine throughout larval development and as sexually mature adults was examined. Larval X. laevis were exposed to one of four nominal concentrations of atrazine (0, 1, 10, 25 microg atrazine/l) beginning 96 hr after fertilization and continuing through two years post-metamorphosis. Clutch size and survival of offspring were used as measurement endpoints to gauge reproductive success of the F1 frogs. Larval survivorship and time to metamorphosis were used to gauge developmental success of the F2 offspring from atrazine-exposed frogs. Testes in F1 and F2 frogs were examined for incidence of anomalies, such as testicular ovarian follicles, and sex ratios in F2 offspring were investigated to determine if exposure to atrazine caused trans-generational effects (effects on F2 individuals due to exposure of F1 individuals). There were no effects of any of the studied concentrations of atrazine on clutch size of F1 frogs. There were also no effects on hatching success or time to metamorphosis. Sex ratios did not differ between F2 offspring among treatments. There was no evidence to suggest a transgenerational effect of atrazine on spawning success or reproductive development of X. laevis. This is consistent with the presence of robust populations of X. laevis in areas where they are exposed to atrazine that has been used for several decades for weed control in production of corn. Our observations also are consistent with the results of most other studies of frogs where no effects were found to be associated with exposure to atrazine. Our data do not support the hypothesis that atrazine significantly affects reproductive fitness and development of frogs.

  20. A morphogenetic quantitative approach to species-typical behaviour.

    PubMed

    Irsigler, F J

    1991-01-01

    The numerical ratio of the allocortical, late-myelinized infra-temporal sector (Flechsig) to the neocortical convexity of the temporal lobe in the human brain is proposed as a quantitative invariant of the human species, called "Neocorticalisation Index NI". Its morphogenetic nature derives from (1) the dual, that is the reptilian-palaeomammalian origin of the primate cortex (Kappers 1909, Vogt 1910. Kuhlenbeck 1924, Filimonoff 1947, Northcutt 1967, Stephan 1975). The two layers differentiate at independent rates forming a transitional belt, called "periallocortex" (limbic) and "mesocortex" (insular). They approach the most the general cyto- and myeloarchitectonic scheme of Brodmann (1909) and C. and O. Vogt (1919), and in the human brain are the latest to reach maturity (Flechsig 1920, Kahle 1969, Sanides 1975); (2) measurements in fossilized human brain endocasts (Taung, Rhodesian man) reported earlier (Irsigler 1984a and b); (3) clinico-pathological evidence gained from cases of the Pick-type of presenile dementia confirming the anthropogenetic character of the frontotemporal "basal forebrain" (Spatz and his school since 1937, Lüers 1947, Jakob 1979). The latter is considered to be in the mammals the major counterpart of the hormonally and reciprocally controlled reptilian forebrain (Schepers 1948, Sanides 1970, MacLean 1978). The results of NI measurements in 128 human brains are tabulated and interpreted in the light of species-typical behaviour. It turns out that the left hemisphere is the one possessing the greater share of allocorticity showing precocious development in the non-European (Negro) infant. There are gender-related differences of the NI parameters. The Indo-Europeans (Caucasians) undergo, in their ontogeny, a kind of "secondary" allocorticalisation in both hemispheres which, hypothetically, may be correlated with the "substantializing" (reifying) property inherent in their languages.

  1. Developmental Design of Synthetic Bacterial Architectures by Morphogenetic Engineering.

    PubMed

    Pascalie, Jonathan; Potier, Martin; Kowaliw, Taras; Giavitto, Jean-Louis; Michel, Olivier; Spicher, Antoine; Doursat, René

    2016-08-19

    (divergence of the homology). Such morphogenetic phenotypes open the way to more complex shapes made of a recursive array of core bodies and limbs and, most importantly, to an evolutionary developmental exploration of unplanned functional forms.

  2. Developmental Design of Synthetic Bacterial Architectures by Morphogenetic Engineering.

    PubMed

    Pascalie, Jonathan; Potier, Martin; Kowaliw, Taras; Giavitto, Jean-Louis; Michel, Olivier; Spicher, Antoine; Doursat, René

    2016-08-19

    (divergence of the homology). Such morphogenetic phenotypes open the way to more complex shapes made of a recursive array of core bodies and limbs and, most importantly, to an evolutionary developmental exploration of unplanned functional forms. PMID:27244532

  3. Swimming kinematics and respiratory behaviour of Xenopus laevis larvae raised in altered gravity

    NASA Technical Reports Server (NTRS)

    Fejtek, M.; Souza, K.; Neff, A.; Wassersug, R.

    1998-01-01

    We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

  4. Overland movement in African clawed frogs (Xenopus laevis): a systematic review.

    PubMed

    Measey, John

    2016-01-01

    African clawed frogs (Xenopus laevis) are often referred to as 'purely aquatic' but there are many publications which suggest extensive overland movements. Previous reviews which considered the topic have not answered the following questions: (1) is there evidence for overland dispersal in native and invasive ranges; (2) what is the range of distances moved overland; (3) when does overland movement occur; and (4) is there evidence of breeding migratory behaviour? A systematic review was chosen to synthesise and critically analyse all literature on the overland movement in Xenopus laevis. Database searches resulted in 57 documents which revealed a paucity of empirical studies, with 28 containing no data, and 19 having anecdotal content. Overwhelming evidence shows that both native and invasive populations of X. laevis move overland, with well documented examples for several other members of the genus (X. borealis, X. gilli, X. muelleri, X. fraseriand X. tropicalis). Reports of distances moved overland were from 40 m to 2 km, with no apparent difference between native and invasive ranges. Overland movements are not confined to wet seasons or conditions, but the literature suggests that moving overland does not occur in the middle of the day. Migrations to temporary water-bodies for breeding have been suggested, but without any corroborating data. PMID:27688972

  5. Effects of depleted uranium on survival, growth, and metamorphosis in the african clawed frog (Xenopus laevis)

    USGS Publications Warehouse

    Mitchell, S.E.; Caldwell, C.A.; Gonzales, G.; Gould, W.R.; Arimoto, R.

    2005-01-01

    Embryos (stage 8-47, Nieuwkoop and Faber) of the African clawed frog (Xenopus laevis) were subjected to water-borne depleted uranium (DU) concentrations that ranged from 4.8 to 77.7 mg/Lusing an acute 96-h frog embryo teratogenesis assay-Xenopus (FETAX). In a chronic 64-d assay, X. laevis (from embryo through metamorphosis; stages 8-66) were subjected to concentrations of DU that ranged from 6.2 to 54.3 mg/L Our results indicate DU is a non teratogenic metal. No effects on mortality, malformations, or growth were observed in the 96-h FETAX with concentrations of DU that ranged from 4.8 to 77.7 mg/L From stage 8 to stage 47, X. laevis tadpoles do not actively feed and the gills are not well developed. Thus, uptake of DU was reduced despite exposure to elevated concentrations. The 64-d assay resulted in no concentration response for either mortality or malformations; however, a delay in metamorphosis was observed in tadpoles subjected to elevated DU concentrations (from 13.1 to 54.3 mg/L) compared to tadpoles in both the well-water control and reference. The delay in metamorphosis was likely due to increasing body burden of DU that ranged from 0.98 to 2.82 mg/kg. Copyright?? Taylor & Francis Inc.

  6. Spatiotemporal expression profile of no29/nucleophosmin3 in the intestine of Xenopus laevis during metamorphosis.

    PubMed

    Motoi, Natsuki; Hasebe, Takashi; Suzuki, Ken-Ichi T; Ishizuya-Oka, Atsuko

    2011-06-01

    A Xenopus laevis homolog of nucleophosmin/nucleoplasmin3 (NPM3), no29, has been previously identified as a thyroid hormone (TH)-response gene during TH-induced metamorphosis. X. laevis has another NPM3 homolog (npm3) in the pseudo-tetraploid genome, whereas X. tropicalis possesses one ortholog in the diploid genome. To assess the possible roles of these NPM3 homologs in amphibian metamorphosis, we have analyzed their expression profiles in X. laevis tadpoles. Levels of no29 and npm3 mRNA are rapidly up-regulated by exogenous TH in various organs of the premetamorphic tadpoles. Notably, in the small intestine, no29 and npm3 mRNA levels are transiently up-regulated during metamorphic climax, when progenitor/stem cells of the adult epithelium appear and actively proliferate. In situ hybridization analysis has revealed that the no29 transcript is specifically localized in adult epithelial progenitor/stem cells of the intestine during natural and TH-induced metamorphosis. Double-staining for in situ hybridization and immunohistochemistry has shown co-expression of no29 mRNA and no38 protein (an ortholog of NPM1), which is known to interact with NPM3 and to regulate cell proliferation in mammals. Thus, no29/npm3 might serve as a stem cell marker in the intestine during metamorphosis.

  7. Effects of Aroclor 1254 on oxidative stress in developing Xenopus laevis tadpoles.

    PubMed

    Gillardin, Virginie; Silvestre, Frédéric; Divoy, Céline; Thomé, Jean-Pierre; Kestemont, Patrick

    2009-02-01

    Over the last decades, amphibians decline has been reported worldwide. Exposure to polychlorinated biphenyls (PCBs) is one of the possible causes in addition to climate changes, UV-radiation or habitat destruction. In the present study, we tested the hypothesis that PCBs could induce oxidative stress in young tadpoles. Developing Xenopus laevis were exposed from 2- to 5-d postfertilization (pf) to 0.1 or 1 mg/l of Aroclor 1254. Lipid peroxidation and antioxidant systems (SOD, CAT, GST, GPx, GR activities and t-GSH level) were investigated in whole organisms. Exposure to both concentrations did not impact on the survival and development whereas the average body weight decreased. Exposure to 1 mg/l of Aroclor 1254 induced a significant (p<0.05) increase of GST activity when compared to controls 0 and DMSO. The other antioxidant enzymes and LPO evaluation remained unchanged. Our results demonstrate that exposure of X. laevis tadpoles to environmental concentrations of Aroclor 1254 interfere with normal growth. They also highlight that very young X. laevis tadpoles express antioxidant systems. PMID:18407353

  8. Thyroid disruption effects of environmental level perfluorooctane sulfonates (PFOS) in Xenopus laevis.

    PubMed

    Cheng, Yan; Cui, Yuan; Chen, Hui-ming; Xie, Wen-ping

    2011-11-01

    Perfluorooctane sulfonate (PFOS), one of the emerging persistent organic pollutants (POPs), has caused growing international concern especially related to the potential disruption in the development and function of thyroid system. Xenopus laevis is an amphibian species widely used as a suitable amphibian model for thyroid disruption research. To study the thyroid disruption effects related to PFOS exposure at environmental low levels, X. laevis tadpoles were exposed to 0.1, 1, 10 and 100 μg/l PFOS in water respectively from stage 46/47 to stage 62. The results showed that the time to metamorphosis (presented by forelimb emergence, FLE) did not significantly change with PFOS exposure, but exhibited an increasing trend (except for 10 μg/l exposure). Partial colloid depletion was observed for PFOS exposure, but no significant histological abnormality was observed in treatment groups. In addition, PFOS exposure resulted in up-regulation of thyroid hormone-regulated genes-thyroid receptor beta A (TRβA), basic transcription element-binding protein (BTEB) and type II deiodinase (DI2) mRNA expression, presented as an inverted U-shaped dose response pattern. However, the mRNA expression of type III deiodinase (DI3) remained unaffected compared with the control. These results demonstrated that PFOS might disrupt the thyroid system in X. laevis tadpoles regarding FLE changes and regulation alternation of thyroid hormone-regulated genes. Our study has raised new concerns for possible thyroid disruption of PFOS in amphibians at environmental relevant levels. PMID:21809121

  9. β-hexosaminidase from Xenopus laevis eggs and oocytes: from gene to immunochemical characterization.

    PubMed

    Morales, Enrique S; Krapf, Darío; Botta, Pablo E; Cabada, Marcelo O; Arranz, Silvia E

    2012-12-01

    Glycosidases are present both in sperm and eggs in vertebrates and have been associated with different fertilization steps as gamete binding, egg coat penetration, and polyspermy prevention. In this manuscript, we have analyzed the activity of different glycosidases of Xenopus laevis eggs. The main activity corresponded to N-acetyl-β-D-glucosaminidase (Hex), which was reported to participate both in gamete binding and polyspermy prevention among phylogenetically distant animals. We have raised homologous antibodies against a recombinant N-terminal fragment of a X. laevis Hex, and characterized egg's Hex both by Western blot and immunohistochemical assays. Noteworthy, Hex was mainly localized to the cortex of animal hemisphere of full-grown oocytes and oviposited eggs, and remained unaltered after fertilization. Hex is constituted by different pair arrangements of two subunits (α and β), giving rise to three possible Hex isoforms: A (αβ), B (ββ), and S (αα). However, no information was available regarding molecular identity of Hex in amphibians. We present for the first time the primary sequences of two isoforms of X. laevis Hex. Interestingly, our results suggest that α- and β-like subunits that constitute Hex isoforms could be synthesized from a same gene in Xenopus, by alternative exon use. This finding denotes an evolutionary divergence with mammals, where α and β Hex subunits are synthesized from different genes on different chromosomes. PMID:22753314

  10. Xenopus laevis oocytes infected with multi-drug–resistant bacteria: implications for electrical recordings

    PubMed Central

    O'Connell, Denice; Mruk, Karen; Rocheleau, Jessica M.

    2011-01-01

    The Xenopus laevis oocyte has been the workhorse for the investigation of ion transport proteins. These large cells have spawned a multitude of novel techniques that are unfathomable in mammalian cells, yet the fickleness of the oocyte has driven many researchers to use other membrane protein expression systems. Here, we show that some colonies of Xenopus laevis are infected with three multi-drug–resistant bacteria: Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Oocytes extracted from infected frogs quickly (3–4 d) develop multiple black foci on the animal pole, similar to microinjection scars, which render the extracted eggs useless for electrical recordings. Although multi-drug resistant, the bacteria were susceptible to amikacin and ciprofloxacin in growth assays. Supplementing the oocyte storage media with these two antibiotics prevented the appearance of the black foci and afforded oocytes suitable for whole-cell recordings. Given that P. fluorescens associated with X. laevis has become rapidly drug resistant, it is imperative that researchers store the extracted oocytes in the antibiotic cocktail and not treat the animals harboring the multi-drug–resistant bacteria. PMID:21788613

  11. Overland movement in African clawed frogs (Xenopus laevis): a systematic review

    PubMed Central

    2016-01-01

    African clawed frogs (Xenopus laevis) are often referred to as ‘purely aquatic’ but there are many publications which suggest extensive overland movements. Previous reviews which considered the topic have not answered the following questions: (1) is there evidence for overland dispersal in native and invasive ranges; (2) what is the range of distances moved overland; (3) when does overland movement occur; and (4) is there evidence of breeding migratory behaviour? A systematic review was chosen to synthesise and critically analyse all literature on the overland movement in Xenopus laevis. Database searches resulted in 57 documents which revealed a paucity of empirical studies, with 28 containing no data, and 19 having anecdotal content. Overwhelming evidence shows that both native and invasive populations of X. laevis move overland, with well documented examples for several other members of the genus (X. borealis, X. gilli, X. muelleri, X. fraseriand X. tropicalis). Reports of distances moved overland were from 40 m to 2 km, with no apparent difference between native and invasive ranges. Overland movements are not confined to wet seasons or conditions, but the literature suggests that moving overland does not occur in the middle of the day. Migrations to temporary water-bodies for breeding have been suggested, but without any corroborating data. PMID:27688972

  12. Quantification of X. laevis vitellogenin by liquid chromatography tandem mass spectrometry.

    PubMed

    Luna, Leah G; Coady, Katherine

    2016-02-01

    Over the last several decades, there has been an increase in public awareness and regulatory activity in regard to the presence of emerging contaminants in the environment that may have the potential to interact with the endocrine system of exposed wildlife. Alterations in vitellogenin (VTG), a high density yolk precursor protein, can indicate endocrine activity in oviparous species, including many fish and amphibians. While various methodologies and experiments have been performed to characterize baseline VTG concentrations among commonly studied fish species, fewer methodologies for accurately quantifying amphibian VTG are available. Since there is relatively little information available on background VTG levels in male and female frogs, the present investigation set out to quantify baseline levels of VTG in juvenile as well as adult male and female African clawed frogs (Xenopus laevis) using a newly developed liquid chromatography tandem mass spectrometry method. This new methodology for quantifying VTG in X. laevis frog blood plasma can be applied in mechanistic and toxicity studies with X. laevis to better characterize potential endocrine modes of action.

  13. Overland movement in African clawed frogs (Xenopus laevis): a systematic review

    PubMed Central

    2016-01-01

    African clawed frogs (Xenopus laevis) are often referred to as ‘purely aquatic’ but there are many publications which suggest extensive overland movements. Previous reviews which considered the topic have not answered the following questions: (1) is there evidence for overland dispersal in native and invasive ranges; (2) what is the range of distances moved overland; (3) when does overland movement occur; and (4) is there evidence of breeding migratory behaviour? A systematic review was chosen to synthesise and critically analyse all literature on the overland movement in Xenopus laevis. Database searches resulted in 57 documents which revealed a paucity of empirical studies, with 28 containing no data, and 19 having anecdotal content. Overwhelming evidence shows that both native and invasive populations of X. laevis move overland, with well documented examples for several other members of the genus (X. borealis, X. gilli, X. muelleri, X. fraseriand X. tropicalis). Reports of distances moved overland were from 40 m to 2 km, with no apparent difference between native and invasive ranges. Overland movements are not confined to wet seasons or conditions, but the literature suggests that moving overland does not occur in the middle of the day. Migrations to temporary water-bodies for breeding have been suggested, but without any corroborating data.

  14. Thrombopoietin induces production of nucleated thrombocytes from liver cells in Xenopus laevis

    PubMed Central

    Tanizaki, Yuta; Ichisugi, Megumi; Obuchi-Shimoji, Miyako; Ishida-Iwata, Takako; Tahara-Mogi, Ayaka; Meguro-Ishikawa, Mizue; Kato, Takashi

    2015-01-01

    The development of mammalian megakaryocytes (MKs) and platelets, which are thought to be absent in non-mammals, is primarily regulated by the thrombopoietin (TPO)/Mpl system. Although non-mammals possess nucleated thrombocytes instead of platelets, the features of nucleated thrombocyte progenitors remain to be clarified. Here, we provide the general features of TPO using Xenopus laevis TPO (xlTPO). Hepatic and splenic cells were cultured in liquid suspension with recombinant xlTPO. These cells differentiated into large, round, polyploid CD41-expressing cells and were classified as X. laevis MKs, comparable to mammalian MKs. The subsequent culture of MKs after removal of xlTPO produced mature, spindle-shaped thrombocytes that were activated by thrombin, thereby altering their morphology. XlTPO induced MKs in cultured hepatic cells for at least three weeks; however, this was not observed in splenic cells; this result demonstrates the origin of early haematopoietic progenitors in the liver rather than the spleen. Additionally, xlTPO enhanced viability of peripheral thrombocytes, indicating the xlTPO-Mpl pathway stimulates anti-apoptotic in peripheral thrombocytes. The development of thrombocytes from MKs via the TPO-Mpl system in X. laevis plays a crucial role in their development from MKs, comparable to mammalian thrombopoiesis. Thus, our results offer insight into the cellular evolution of platelets/MKs in vertebrates. (200/200). PMID:26687619

  15. Swimming kinematics and respiratory behaviour of xenopus laevis larvae raised in altered gravity

    PubMed

    Fejtek; Souza; Neff; Wassersug

    1998-05-21

    We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

  16. Swimming kinematics and respiratory behaviour of Xenopus laevis larvae raised in altered gravity.

    PubMed

    Fejtek, M; Souza, K; Neff, A; Wassersug, R

    1998-06-01

    We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

  17. Effects of Chinese domestic polychlorinated biphenyls (PCBs) on gonadal differentiation in Xenopus laevis.

    PubMed Central

    Qin, Zhan-Fen; Zhou, Jing-Ming; Chu, Shao-Gang; Xu, Xiao-Bai

    2003-01-01

    To determine whether polychlorinated biphenyls (PCBs) influence gonadal differentiation in Xenopus laevis, tadpoles were exposed to two Chinese domestic PCBs (PCB3 and PCB5 from Nieuwkoop and Faber stage 46/47 to complete metamorphosis. Gonads were characterized using a dissecting microscope. The control X. laevis had normal ovaries or testes in gross morphology, whereas obviously abnormal testes including ovotestes were found in PCB3- and PCB5-exposed groups. Ovotestes were characterized by morphologic ovaries in the cranial and caudal parts and morphologic testes in the middle part. PCBs did not alter the percentage of females but reduced the percentage of males with morphologically normal testes. The histologic structure of gonads was examined by a series of sections. Morphologically normal and abnormal testes from a few frogs exposed to PCBs were interspersed with oocytes in histologic sections. These testes exhibited looser structure with fewer seminiferous tubes, spermatogonia, and spermatozoa than in controls. The findings suggest that PCB3 and PCB5 have significant feminization effects on gonadal differentiation in X. laevis and that this species is sensitive to endocrine disruption and may be used as a good model to study endocrine disruption. PMID:12676614

  18. Cell Type-specific Translational Profiling in the Xenopus laevis Retina

    PubMed Central

    Watson, F.L.; Mills, E. A.; Wang, X.; Guo, C.; Chen, D.F.; Marsh-Armstrong, N.

    2013-01-01

    Background Translating Ribosome Affinity Purification (TRAP), a method recently developed to generate cell type-specific translational profiles, relies on creating transgenic lines of animals in which a tagged ribosomal protein is placed under regulatory control of a cell type-specific promoter. An antibody is then used to affinity purify the tagged ribosomes so that cell type-specific mRNAs can be isolated from whole tissue lysates. Results Here, cell type-specific transgenic lines were generated to enable TRAP studies for retinal ganglion cells and rod photoreceptors in the Xenopus laevis retina. Using real time quantitative PCR for assessing expression levels of cell type-specific mRNAs, the TRAP method was shown to selectively isolate mRNAs expressed in the targeted cell and was efficient at purifying mRNAs expressed at both high and low levels. Statistical measures used to distinguish cell type-specific RNAs from low level background and non-specific RNAs showed TRAP to be highly effective in Xenopus. Conclusions TRAP can be used to purify mRNAs expressed in rod photoreceptors and retinal ganglion cells in Xenopus laevis. The generated transgenic lines will enable numerous studies into the development, disease and injury of the Xenopus laevis retina. PMID:23074098

  19. Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease

    PubMed Central

    Li, Fei Feng; Deng, Xia; Zhou, Jing; Yan, Peng; Zhao, Er Ying; Liu, Shu Lin

    2016-01-01

    Congenital heart disease (CHD) is a complex illness with high rates of morbidity and mortality. In embryonic development, the heart is the first formed organ, which is strictly controlled by gene regulatory networks, including transcription factors, signaling pathways, epigenetic factors and microRNAs. Bone morphogenetic protein (BMP)-2 and -4 are essential in cardiogenesis as they can induce the expression of transcription factors, NKX2-5 and GATA binding protein 4, which are important in the development of the heart. The inhibition of BMP-2 and 4- inhibits the late expression of NKX2-5 and affects cardiac differentiation. The aim of the present study was to investigate whether BMP-2 and -4 variations may be associated with CHD in Chinese Han populations. The rs1049007, rs235768 and rs17563 single nucleotide polymorphisms (SNPs), which are genetic variations located within the translated region of the BMP-2 and -4, were evaluated in 230 patients with CHD from the Chinese Han population and 160 non CHD control individuals. Statistical analyses were performed using the χ2 test, implemented using SPSS software (version 13.0). The Hardy Weinberg equilibrium test was performed on the population using online Online Encyclopedia for Genetic Epidemiology studies software, and multiple-sequence alignments of the BMP proteins were performed using Vector NTI software. No statistically significant associations were identified between these genetic variations and the risk of CHD (rs1049007, P value=0.560; rs235768, P value=0.972; rs17563, P value=0.787). In addition, no correlation was found between the patients with CHD and the non-CHD control individuals. Therefore, the rs1049007, rs235768 and rs17563 genetic variations of BMP-2 were not associated with CHD in the Chinese Han population. PMID:27357418

  20. Beyond osteogenesis: an in vitro comparison of the potentials of six bone morphogenetic proteins.

    PubMed

    Rivera, Jessica C; Strohbach, Cassandra A; Wenke, Joseph C; Rathbone, Christopher R

    2013-01-01

    Bone morphogenetic proteins (BMPs) other than the clinically available BMP-2 and BMP-7 may be useful for improving fracture healing through both increasing osteogenesis and creating a favorable healing environment by altering cytokine release by endogenous cells. Given the spectrum of potential applications for BMPs, the objective of this study was to evaluate various BMPs under a variety of conditions to provide further insight into their therapeutic capabilities. The alkaline phosphatase (ALP) activity of both C2C12 and human adipose-derived stem cells (hASCs) was measured after exposure of increasing doses of recombinant human BMP-2, -4, -5, -6, -7, or -9 for 3 and 7 days. BMPs-2, -4, -5, -6, -7, and -9 were compared in terms of their ability to affect the release of stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (b-FGF) from human bone marrow stromal cells (hBMSCs). Gene expression of ALP, osteocalcin, SDF-1, VEGF, and b-FGF following shRNA-mediated knockdown of BMP-2 and BMP-6 in hBMSCs or human osteoblasts under osteogenic differentiation conditions was also evaluated. Collectively, BMPs-6 and -9 produced the greatest osteogenic differentiation of C2C12 and hASCs as determined by ALP. The hBMSC secretion of SDF-1 was most affected by BMP-5, VEGF by BMP-4, and b-FGF by BMP-2. The knockdown of BMP-2 in BMSCs had no effect on any of the genes measured whereas BMP-6 knockdown in hBMSCs caused a significant increase in VEGF gene expression. BMP-2 and BMP-6 knockdown in human osteoblasts caused significant increases in VEGF gene expression and trends toward decreases in osteocalcin expression. These findings support efforts to study other BMPs as potential bone graft supplements, and to consider combined BMP delivery for promotion of multiple aspects of fracture healing. PMID:24101902

  1. Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease.

    PubMed

    Li, Fei-Feng; Deng, Xia; Zhou, Jing; Yan, Peng; Zhao, Er-Ying; Liu, Shu-Lin

    2016-08-01

    Congenital heart disease (CHD) is a complex illness with high rates of morbidity and mortality. In embryonic development, the heart is the first formed organ, which is strictly controlled by gene regulatory networks, including transcription factors, signaling pathways, epigenetic factors and microRNAs. Bone morphogenetic protein (BMP)-2 and -4 are essential in cardiogenesis as they can induce the expression of transcription factors, NKX2‑5 and GATA binding protein 4, which are important in the development of the heart. The inhibition of BMP‑2 and ‑4 inhibits the late expression of NKX2-5 and affects cardiac differentiation. The aim of the present study was to investigate whether BMP-2 and ‑4 variations may be associated with CHD in Chinese Han populations. The rs1049007, rs235768 and rs17563 single nucleotide polymorphisms (SNPs), which are genetic variations located within the translated region of the BMP-2 and -4, were evaluated in 230 patients with CHD from the Chinese Han population and 160 non-CHD control individuals. Statistical analyses were performed using the χ2 test, implemented using SPSS software (version 13.0). The Hardy-Weinberg equilibrium test was performed on the population using online Online Encyclopedia for Genetic Epidemiology studies software, and multiple-sequence alignments of the BMP proteins were performed using Vector NTI software. No statistically significant associations were identified between these genetic variations and the risk of CHD (rs1049007, P‑value=0.560; rs235768, P‑value=0.972; rs17563, P‑value=0.787). In addition, no correlation was found between the patients with CHD and the non‑CHD control individuals. Therefore, the rs1049007, rs235768 and rs17563 genetic variations of BMP-2 were not associated with CHD in the Chinese Han population. PMID:27357418

  2. Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

    PubMed

    Tsuchiya, Yugo; Pham, Uyen; Hu, Wanzhou; Ohnuma, Shin-Ichi; Gout, Ivan

    2014-01-01

    Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis. PMID:24831956

  3. Chytrid fungus infections in laboratory and introduced Xenopus laevis populations: assessing the risks for U.K. native amphibians

    PubMed Central

    Tinsley, Richard C.; Coxhead, Peter G.; Stott, Lucy C.; Tinsley, Matthew C.; Piccinni, Maya Z.; Guille, Matthew J.

    2015-01-01

    The chytrid fungus Batrachochytrium dendrobatidis (Bd) is notorious amongst current conservation biology challenges, responsible for mass mortality and extinction of amphibian species. World trade in amphibians is implicated in global dissemination. Exports of South African Xenopus laevis have led to establishment of this invasive species on four continents. Bd naturally infects this host in Africa and now occurs in several introduced populations. However, no previous studies have investigated transfer of infection into co-occurring native amphibian faunas. A survey of 27 U.K. institutions maintaining X. laevis for research showed that most laboratories have low-level infection, a risk for native species if animals are released into the wild. RT-PCR assays showed Bd in two introduced U.K. populations of X. laevis, in Wales and Lincolnshire. Laboratory and field studies demonstrated that infection levels increase with stress, especially low temperature. In the U.K., native amphibians may be exposed to intense transmission in spring when they enter ponds to spawn alongside X. laevis that have cold-elevated Bd infections. Exposure to cross-infection has probably been recurrent since the introduction of X. laevis, >20 years in Lincolnshire and 50 years in Wales. These sites provide an important test for assessing the impact of X. laevis on Bd spread. However, RT-PCR assays on 174 native amphibians (Bufo, Rana, Lissotriton and Triturus spp.), sympatric with the Bd-infected introduced populations, showed no foci of self-sustaining Bd transmission associated with X. laevis. The abundance of these native amphibians suggested no significant negative population-level effect after the decades of co-occurrence. PMID:25843959

  4. Thermal acclimation of locomotor performance in tadpoles and adults of the aquatic frog Xenopus laevis.

    PubMed

    Wilson, R S; James, R S; Johnston, I A

    2000-03-01

    Among amphibians, the ability to compensate for the effects of temperature on the locomotor system by thermal acclimation has only been reported in larvae of a single species of anuran. All other analyses have examined predominantly terrestrial adult life stages of amphibians and found no evidence of thermal acclimatory capacity. We examined the ability of both tadpoles and adults of the fully aquatic amphibian Xenopus laevis to acclimate their locomotor system to different temperatures. Tadpoles were acclimated to either 12 degrees C or 30 degrees C for 4 weeks and their burst swimming performance was assessed at four temperatures between 5 degrees C and 30 degrees C. Adult X. laevis were acclimated to either 10 degrees C or 25 degrees C for 6 weeks and their burst swimming performance and isolated muscle performance was determined at six temperatures between 5 degrees C and 30 degrees C. Maximum swimming performance of cold-acclimated X. laevis tadpoles was greater at cool temperatures and lower at the highest temperature in comparison with the warm-acclimated animals. At the test temperature of 12 degrees C, maximum swimming velocity of tadpoles acclimated to 12 degrees C was 38% higher than the 30 degrees C-acclimation group, while at 30 degrees C, maximum swimming velocity of the 30 degrees C-acclimation group was 41% faster than the 12 degrees C-acclimation group. Maximum swimming performance of adult X. laevis acclimated to 10 degrees C was also higher at the lower temperatures than the 25 degrees C acclimated animals, but there was no difference between the treatment groups at higher temperatures. When tested at 10 degrees C, maximum swimming velocity of the 10 degrees C-acclimation group was 67% faster than the 25 degrees C group. Isolated gastrocnemius muscle fibres from adult X. laevis acclimated to 10 degrees C produced higher relative tetanic tensions and decreased relaxation times at 10 degrees C in comparison with animals acclimated to 25 degrees C

  5. A covalently dimerized recombinant human bone morphogenetic protein-15 variant identifies bone morphogenetic protein receptor type 1B as a key cell surface receptor on ovarian granulosa cells.

    PubMed

    Pulkki, Minna M; Mottershead, David G; Pasternack, Arja H; Muggalla, Pranuthi; Ludlow, Helen; van Dinther, Maarten; Myllymaa, Samu; Koli, Katri; ten Dijke, Peter; Laitinen, Mika; Ritvos, Olli

    2012-03-01

    Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. Biochemical characterization of ligand-receptor interactions was performed with affinity-labeling experiments using [(125)I]iodinated BMP15 variants. Both ligand variants were shown to form homodimers and to stimulate Smad1/5/8 signaling and inhibin B production in human granulosa cells in a similar manner. [(125)I]Iodination of both ligands was achieved, but only the covalent dimer variant retained receptor binding capacity. The [(125)I]BMP15(S356C) variant bound preferentially to endogenous BMP receptor 1B (BMPR1B) and BMPR2 receptors on COV434 cells. Binding experiments in COS cells with overexpression of these receptors confirmed that the [(125)I]BMP15(S356C) variant binds to BMPR1B and BMPR2 forming the BMP15 signaling complex. The results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss

  6. Phylogeny of subclass Scuticociliatia (Protozoa, Ciliophora) using combined data inferred from genetic, morphological, and morphogenetic evidence

    NASA Astrophysics Data System (ADS)

    Yi, Zhenzhen; Wang, Yangang; Lin, Xiaofeng; Al-Rasheid, Khaled A. S.; Song, Weibo

    2010-07-01

    Gene sequence-based genealogies of scuticociliates are different from those produced by morphological analyses. For this reason, 11 representative scuticociliates and two ambiguously related genera were chosen to test the ability of combined phylogenetic analyses using both gene sequences and morphological/morphogenetic characteristics. Analyses of both the SSrRNA gene sequences and the combined datasets revealed a consistent branching pattern. While the terminal branches and the order level relationships were generally well resolved, the family level relationships remain unresolved. However, two other trees based on ITS1-5.8S-ITS2 region sequences and morphological/morphogenetic characters showed limited information, due to a lack of informative sites in these two datasets. Our data suggest, however, that the combined analysis of morphological/morphogenetic characters and gene sequences did produce some changes to the phylogenetic estimates of this group.

  7. Prominent Amphibian (Xenopus laevis) Tadpole Type III Interferon Response to the Frog Virus 3 Ranavirus

    PubMed Central

    Grayfer, Leon; De Jesús Andino, Francisco

    2015-01-01

    ABSTRACT Ranaviruses (Iridoviridae) are posing an increasing threat to amphibian populations, with anuran tadpoles being particularly susceptible to these viral infections. Moreover, amphibians are the most basal phylogenetic class of vertebrates known to possess both type I and type III interferon (IFN)-mediated immunity. Moreover, little is known regarding the respective roles of the IFN mediators in amphibian antiviral defenses. Accordingly, we transcriptionally and functionally compared the amphibian Xenopus laevis type I (IFN) and III (IFN-λ) IFNs in the context of infections by the ranavirus frog virus 3 (FV3). X. laevis IFN and IFN-λ displayed distinct tissue expression profiles. In contrast to our previous findings that X. laevis tadpoles exhibit delayed and modest type I IFN responses to FV3 infections compared to the responses of adults, here we report that tadpoles mount timely and robust type III IFN gene responses. Recombinant forms of these cytokines (recombinant X. laevis IFN [rXlIFN] and rXlIFN-λ) elicited antiviral gene expression in the kidney-derived A6 cell line as well as in tadpole leukocytes and tissues. However, rXlIFN-λ was less effective than rXlIFN in preventing FV3 replication in A6 cells and tadpoles and inferior at promoting tadpole survival. Intriguingly, FV3 impaired A6 cell and tadpole kidney type III IFN receptor gene expression. Furthermore, in A6 cultures rXlIFN-λ conferred equal or greater protection than rXlIFN against recombinant viruses deficient for the putative immune evasion genes, the viral caspase activation and recruitment domain (vCARD) or a truncated vIF-2α gene. Thus, in contrast to previous assumptions, tadpoles possess intact antiviral defenses reliant on type III IFNs, which are overcome by FV3 pathogens. IMPORTANCE Anuran tadpoles, including those of Xenopus laevis, are particularly susceptible to infection by ranavirus such as FV3. We investigated the respective roles of X. laevis type I and type III

  8. Atrazine and malathion shorten the maturation process of Xenopus laevis oocytes and have an adverse effect on early embryo development.

    PubMed

    Ji, Qichao; Lee, Jessica; Lin, Yu-Huey; Jing, Guihua; Tsai, L Jillianne; Chen, Andrew; Hetrick, Lindsay; Jocoy, Dylan; Liu, Junjun

    2016-04-01

    The use of pesticides has a negative impact on the environment. Amphibians have long been regarded as indicator species to pollutants due to their permeable skin and sensitivity to the environment. Studies have shown that population declines of some amphibians are directly linked with exposure to agricultural contaminants. In the past, much of the studies have focused on the toxic effect of contaminants on larvae (tadpoles), juvenile and adult frogs. However, due to the nature of their life cycle, amphibian eggs and early embryos are especially susceptible to the contaminants, and any alteration during the early reproductive stages may have a profound effect on the health and population of amphibians. In this study, we analyzed the effect of atrazine and malathion, two commonly used pesticides, on Xenopus laevis oocyte maturation and early embryogenesis. We found that both atrazine and malathion shortened the frog oocyte maturation process and resulted in reduced Emi2 levels at cytostatic factor-mediated metaphase arrest, and a high level of Emi2 is critically important for oocyte maturation. Furthermore, frog embryos fertilized under the influence of atrazine and/or malathion displayed a higher rate of abnormal division that eventually led to embryo death during early embryogenesis. PMID:26686575

  9. The effects of temperature, desiccation, and body mass on the locomotion of the terrestrial isopod, Porcellio laevis.

    PubMed

    Dailey, Tara M; Claussen, Dennis L; Ladd, Gregory B; Buckner, Shizuka T

    2009-06-01

    Locomotion in terrestrial isopods is strongly influenced by body size and by abiotic factors. We determined the speeds of isopods of differing masses within a linear racetrack at temperatures ranging from 15 to 35 degrees C. We also predicted maximum speeds based on the Froude number concept as originally applied to vertebrates. In addition we used a circular thermal gradient to examine the temperature preferences of isopods, and we measured the effects of desiccation on locomotion. Measured speeds of the isopods progressively increased with temperature with an overall Q(10) of 1.64 and scaling exponents ranging from 0.38 to 0.63. The predicted maximum speeds were remarkably close to the measured speeds at the highest test temperature although the scaling exponents were closer to 0.15. The isopods did not exhibit a strong thermal preference within the gradient; however, they did generally avoid temperatures above 25 degrees C. Moderate desiccation had no apparent effect on locomotor performance, but there was a progressive decrease in speed once animals had lost more than 10% of their initial body mass. Though largely restricted to moist habitats, P. laevis can easily withstand short exposures to desiccating conditions, and they are capable of effective locomotion over a wide range of temperatures. Since they are nonconglobating, active escape appears to be their primary defense when threatened under exposed conditions. Although their maximum speeds may be limited both by temperature and by their inability to change gait, these speeds are clearly adequate for survival.

  10. Atrazine and malathion shorten the maturation process of Xenopus laevis oocytes and have an adverse effect on early embryo development.

    PubMed

    Ji, Qichao; Lee, Jessica; Lin, Yu-Huey; Jing, Guihua; Tsai, L Jillianne; Chen, Andrew; Hetrick, Lindsay; Jocoy, Dylan; Liu, Junjun

    2016-04-01

    The use of pesticides has a negative impact on the environment. Amphibians have long been regarded as indicator species to pollutants due to their permeable skin and sensitivity to the environment. Studies have shown that population declines of some amphibians are directly linked with exposure to agricultural contaminants. In the past, much of the studies have focused on the toxic effect of contaminants on larvae (tadpoles), juvenile and adult frogs. However, due to the nature of their life cycle, amphibian eggs and early embryos are especially susceptible to the contaminants, and any alteration during the early reproductive stages may have a profound effect on the health and population of amphibians. In this study, we analyzed the effect of atrazine and malathion, two commonly used pesticides, on Xenopus laevis oocyte maturation and early embryogenesis. We found that both atrazine and malathion shortened the frog oocyte maturation process and resulted in reduced Emi2 levels at cytostatic factor-mediated metaphase arrest, and a high level of Emi2 is critically important for oocyte maturation. Furthermore, frog embryos fertilized under the influence of atrazine and/or malathion displayed a higher rate of abnormal division that eventually led to embryo death during early embryogenesis.

  11. Species differences in the expression and activity of bone morphogenetic protein 15.

    PubMed

    Al-Musawi, Sara L; Walton, Kelly L; Heath, Derek; Simpson, Courtney M; Harrison, Craig A

    2013-02-01

    Oocyte-derived bone morphogenetic protein 15 (BMP15) regulates ovulation rate and female fertility in a species-specific manner, being important in humans and sheep and largely superfluous in mice. To understand these species differences, we have compared the expression and activity of human, murine, and ovine BMP15. In HEK293F cells, human BMP15 is highly expressed (120 ng/ml), ovine BMP15 is poorly expressed (15 ng/ml), and murine BMP15 is undetectable. Because BMP15 synthesis is dependent upon interactions between the N-terminal prodomain and the C-terminal mature domain, we used site-directed mutagenesis to identify four prodomain residues (Glu(46), Glu(47), Leu(49), and Glu(50)) that mediate the high expression of human BMP15. Substituting these residues into the prodomains of murine and ovine BMP15 led to significant increases in growth factor expression; however, maximal expression was achieved only when the entire human prodomain was linked to the mature domains of the other species. Using these chimeric constructs, we produced and purified murine and ovine BMP15 and showed that in a COV434 granulosa cell bioassay, these molecules displayed little activity relative to human BMP15 (EC(50) 0.2nM). Sequence analysis suggested that the disparity in activity could be due to species differences at the type I receptor binding interface. Indeed, murine BMP15 activity was restored when specific residues through this region (Pro(329)/Tyr(330)) were replaced with the corresponding residues (Arg(329)/Asp(330)) from human BMP15. The identified differences in the expression and activity of BMP15 likely underlie the relative importance of this growth factor between species.

  12. Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

    PubMed

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L; Ritter, Lesley J; White, Melissa A; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

  13. Bone morphogenetic protein modulator BMPER is highly expressed in malignant tumors and controls invasive cell behavior.

    PubMed

    Heinke, J; Kerber, M; Rahner, S; Mnich, L; Lassmann, S; Helbing, T; Werner, M; Patterson, C; Bode, C; Moser, M

    2012-06-14

    Bone morphogenetic proteins (BMPs) are growth factors that exert important functions in cell proliferation, migration and differentiation. Till date, multiple human tumors have been reported to display a dysregulation of several members of the BMP pathway that is associated with enhanced malignant tumor growth and metastasis. BMPER (BMP endothelial cell precursor-derived regulator) is a direct BMP modulator that is necessary for BMPs to exert their full-range signaling activity. Moreover, BMPER is expressed by endothelial cells and their progenitors, and has pro-angiogenic features in these cells. Here, we describe the expression of BMPER in human specimens of lung, colon and cervix carcinomas and cell lines derived from such carcinomas. In contrast to healthy tissues, BMPER is highly expressed upon malignant deterioration. Functionally, loss of BMPER in the lung tumor cell line A549 impairs proliferation, migration, invasion as well as tumor cell-induced endothelial cell sprout formation. In contrast, stimulation of A549 cells with exogenous BMPER had no further effect. We found that the BMPER effect may be transduced by regulation of the BMP target transcription factor inhibitor of DNA binding 1 (Id1) and matrix metalloproteinases (MMPs) 9 and 2. These facilitators of cell migration are downregulated when BMPER is absent. To prove the relevance of our in vitro results in vivo, we generated Lewis lung carcinoma cells with impaired BMPER expression and implanted them into the lungs of C57BL/6 mice. In this model, the absence of BMPER resulted in severely reduced tumor growth and tumor angiogenesis. Taken together, these data unequivocally demonstrate that the BMP modulator BMPER is highly expressed in malignant tumors and tumor growth is dependent on the presence of BMPER.

  14. Bone morphogenetic protein modulator BMPER is highly expressed in malignant tumors and controls invasive cell behavior

    PubMed Central

    Heinke, J; Kerber, M; Rahner, S; Mnich, L; Lassmann, S; Helbing, T; Werner, M; Patterson, C; Bode, C; Moser, M

    2012-01-01

    Bone morphogenetic proteins (BMPs) are growth factors that exert important functions in cell proliferation, migration and differentiation. Till date, multiple human tumors have been reported to display a dysregulation of several members of the BMP pathway that is associated with enhanced malignant tumor growth and metastasis. BMPER (BMP endothelial cell precursor-derived regulator) is a direct BMP modulator that is necessary for BMPs to exert their full-range signaling activity. Moreover, BMPER is expressed by endothelial cells and their progenitors, and has pro-angiogenic features in these cells. Here, we describe the expression of BMPER in human specimens of lung, colon and cervix carcinomas and cell lines derived from such carcinomas. In contrast to healthy tissues, BMPER is highly expressed upon malignant deterioration. Functionally, loss of BMPER in the lung tumor cell line A549 impairs proliferation, migration, invasion as well as tumor cell-induced endothelial cell sprout formation. In contrast, stimulation of A549 cells with exogenous BMPER had no further effect. We found that the BMPER effect may be transduced by regulation of the BMP target transcription factor inhibitor of DNA binding 1 (Id1) and matrix metalloproteinases (MMPs) 9 and 2. These facilitators of cell migration are down-regulated when BMPER is absent. To prove the relevance of our in vitro results in vivo, we generated Lewis lung carcinoma cells with impaired BMPER expression and implanted them into the lungs of C57BL/6 mice. In this model, the absence of BMPER resulted in severely reduced tumor growth and tumor angiogenesis. Taken together, these data unequivocally demonstrate that the BMP modulator BMPER is highly expressed in malignant tumors and tumor growth is dependent on the presence of BMPER. PMID:22020334

  15. Influence of bone morphogenetic protein-2 on spiral ganglion neurite growth in vitro.

    PubMed

    Volkenstein, Stefan; Brors, D; Hansen, S; Minovi, A; Laub, M; Jennissen, H P; Dazert, S; Neumann, A

    2009-09-01

    Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a growth factor of the transforming growth factor-beta superfamily. Members of this protein family are involved in the development of various mammalian tissues, including the inner ear. As their notations indicate, they also have well-known effects on bone formation and regeneration. In this study, we examined the influence of rhBMP-2 on spiral ganglion (SG) neurite growth in vitro and showed the presence of its most preferred receptor BMPR-IB in spiral ganglion cells both in vitro and in vivo. SG explants of postnatal day 4 rats were analysed for neurite length and number after organotypical cell culture for 72 h, fixation and immunolabeling. Different concentrations of rhBMP-2 were used in a serum-free culture media. Neurite growth was compared with control groups that lacked stimulative effects; with neutrophin-3 (NT-3), which is a well-established positive stimulus on neurite length and number; and with combinations of these parameters. The results display that neurite number and total neurite length per explant in particular concentrations of rhBMP-2 increased by a maximum factor of two, while the mean neurite length was not affected. NT-3 demonstrated a much more potent effect, delivering a maximum increase of a factor of five. Furthermore, a combination of both growth factors shows a predominant effect on NT-3. Immunohistological detection of BMPR-IB was successful both in cell culture explants and in paraffin-embedded sections of animals of different ages. The results show that rhBMP-2 is, among other growth factors, a positive stimulus for SG neurite growth in vitro. Most growth factors are unstable and cannot be attached to surfaces without loss of their biological function. In contrast, rhBMP-2 can be attached to metal surfaces without loss of activity. Our findings suggest in vivo studies and a future clinical application of rhBMP-2 in cochlear implant technology to improve the tissue

  16. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

    SciTech Connect

    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  17. A New Nomenclature of Xenopus laevis Chromosomes Based on the Phylogenetic Relationship to Silurana/Xenopus tropicalis.

    PubMed

    Matsuda, Yoichi; Uno, Yoshinobu; Kondo, Mariko; Gilchrist, Michael J; Zorn, Aaron M; Rokhsar, Daniel S; Schmid, Michael; Taira, Masanori

    2015-01-01

    Xenopus laevis (XLA) is an allotetraploid species which appears to have undergone whole-genome duplication after the interspecific hybridization of 2 diploid species closely related to Silurana/Xenopus tropicalis (XTR). Previous cDNA fluorescence in situ hybridization (FISH) experiments have identified 9 sets of homoeologous chromosomes in X. laevis, in which 8 sets correspond to chromosomes 1-8 of X. tropicalis (XTR1-XTR8), and the last set corresponds to a fusion of XTR9 and XTR10. In addition, recent X. laevis genome sequencing and BAC-FISH experiments support this physiological relationship and show no gross chromosome translocation in the X. laevis karyotype. Therefore, for the benefit of both comparative cytogenetics and genome research, we here propose a new chromosome nomenclature for X. laevis based on the phylogenetic relationship and chromosome length, i.e. XLA1L, XLA1S, XLA2L, XLA2S, and so on, in which the numbering of XLA chromosomes corresponds to that in X. tropicalis and the postfixes 'L' and 'S' stand for 'long' and 'short' chromosomes in the homoeologous pairs, which can be distinguished cytologically by their relative size. The last chromosome set is named XLA9L and XLA9S, in which XLA9 corresponds to both XTR9 and XTR10, and hence, to emphasize the phylogenetic relationship to X. tropicalis, XLA9_10L and XLA9_10S are also used as synonyms.

  18. In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.

    PubMed

    Sekiguchi, J M; Kmiec, E B

    1992-02-25

    Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.

  19. Bone morphogenetic protein 15 may promote follicle selection in the hen.

    PubMed

    Stephens, C S; Johnson, P A

    2016-09-01

    In the hen, optimal ovulation rate depends on selection of a single follicle into the pre-ovulatory hierarchy. Follicle selection is associated with increased oocyte growth and changes in gene expression in granulosa cells surrounding the oocyte, in preparation for ovulation. This study investigated the expression, function and regulation of bone morphogenetic protein-15 (BMP15) during follicle development in the hen. BMP15 mRNA expression was analyzed in the ooplasm and granulosa cells of 3mm follicles and was confirmed to be primarily in the ooplasm. BMP15 was detected by immunoblotting in 6 and 8mm follicles near the time of follicle selection. Expression of mRNA for BMP15 receptors (BMPR1B and BMPR2) in granulosa cells increased with follicle size, indicating that BMP15 may play an important role around follicle selection. The function of BMP15 was examined by culturing granulosa cells from 3-5mm and 6-8mm follicles with recombinant human BMP15 (rhBMP15). BMP15 increased expression of follicle stimulating hormone receptor (FSHR) mRNA and decreased anti-Müllerian hormone (AMH) mRNA and occludin (OCLN), factors associated with follicle maturation and growth in the hen. Hormonal regulation of BMP15 was assessed by whole follicle culture with estradiol (E2) which increased BMP15 mRNA expression. The distinct expression pattern of BMP15 and its receptors, coupled with the effects of BMP15 to increase FSHR mRNA and decrease AMH mRNA and OCLN mRNA and protein expression suggest that the oocyte may have a role in follicle selection in the chicken. PMID:27340039

  20. Hyaluronan Regulates Bone Morphogenetic Protein-7-dependent Prevention and Reversal of Myofibroblast Phenotype*

    PubMed Central

    Midgley, Adam C.; Duggal, Lucy; Jenkins, Robert; Hascall, Vincent; Steadman, Robert; Phillips, Aled O.; Meran, Soma

    2015-01-01

    Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na+/H+ exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology. PMID:25716319

  1. Bone Morphogenetic Proteins: Promising Molecules for Bone Healing, Bioengineering, and Regenerative Medicine.

    PubMed

    Carreira, Ana Claudia Oliveira; Zambuzzi, Willian Fernando; Rossi, Mariana Correa; Astorino Filho, Renato; Sogayar, Mari Cleide; Granjeiro, José Mauro

    2015-01-01

    Bone morphogenetic proteins (BMPs), glycoproteins secreted by some cells, are members of the TGF-β superfamily that have been implicated in a wide variety of roles. Currently, about 20 different BMPs have been identified and grouped into subfamilies, according to similarities with respect to their amino acid sequences. It has been shown that BMPs are secreted growth factors involved in mesenchymal stem cell differentiation, also being reported to control the differentiation of cancer stem cells. BMPs initiate signaling from the cell surface by binding to two different receptors (R: Type I and II). The heterodimeric formation of type I R and II R may occur before or after BMP binding, inducing signal transduction pathways through SMADs. BMPs may also signal through SMAD-independent pathways via mitogen-activated protein kinases (ERK, p38MAPKs, JNK). BMPs may act in an autocrine or paracrine manner, being regulated by specific antagonists, namely: noggin and chordin. Genetic engineering allows the production of large amounts of BMPs for clinical use, and clinical trials have shown the benefits of FDA-approved recombinant human BMPs 2 and 7. Several materials from synthetic to natural sources have been tested as BMP carriers, ranging from hydroxyapatite, and organic polymers to collagen. Bioactive membranes doped with BMPs are promising options, acting to accelerate and enhance osteointegration. The development of smart materials, mainly based on biopolymers and bone-like calcium phosphates, appears to provide an attractive alternative for delivering BMPs in an adequately controlled fashion. BMPs have revealed a promising future for the fields of Bioengineering and Regenerative Medicine. In this chapter, we review and discuss the data on BMP structure, mechanisms of action, and possible clinical applications.

  2. Bone morphogenetic protein 15 may promote follicle selection in the hen.

    PubMed

    Stephens, C S; Johnson, P A

    2016-09-01

    In the hen, optimal ovulation rate depends on selection of a single follicle into the pre-ovulatory hierarchy. Follicle selection is associated with increased oocyte growth and changes in gene expression in granulosa cells surrounding the oocyte, in preparation for ovulation. This study investigated the expression, function and regulation of bone morphogenetic protein-15 (BMP15) during follicle development in the hen. BMP15 mRNA expression was analyzed in the ooplasm and granulosa cells of 3mm follicles and was confirmed to be primarily in the ooplasm. BMP15 was detected by immunoblotting in 6 and 8mm follicles near the time of follicle selection. Expression of mRNA for BMP15 receptors (BMPR1B and BMPR2) in granulosa cells increased with follicle size, indicating that BMP15 may play an important role around follicle selection. The function of BMP15 was examined by culturing granulosa cells from 3-5mm and 6-8mm follicles with recombinant human BMP15 (rhBMP15). BMP15 increased expression of follicle stimulating hormone receptor (FSHR) mRNA and decreased anti-Müllerian hormone (AMH) mRNA and occludin (OCLN), factors associated with follicle maturation and growth in the hen. Hormonal regulation of BMP15 was assessed by whole follicle culture with estradiol (E2) which increased BMP15 mRNA expression. The distinct expression pattern of BMP15 and its receptors, coupled with the effects of BMP15 to increase FSHR mRNA and decrease AMH mRNA and OCLN mRNA and protein expression suggest that the oocyte may have a role in follicle selection in the chicken.

  3. Bone morphogenetic protein 15 (BMP15) acts as a BMP and Wnt inhibitor during early embryogenesis.

    PubMed

    Di Pasquale, Elisa; Brivanlou, Ali H

    2009-09-18

    Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor beta (TGFbeta) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of beta-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure.

  4. Mode of heparin attachment to nanocrystalline hydroxyapatite affects its interaction with bone morphogenetic protein-2.

    PubMed

    Goonasekera, Chandhi S; Jack, Kevin S; Bhakta, Gajadhar; Rai, Bina; Luong-Van, Emma; Nurcombe, Victor; Cool, Simon M; Cooper-White, Justin J; Grøndahl, Lisbeth

    2015-01-01

    Heparin has a high affinity for bone morphogenetic protein-2 (BMP-2), which is a key growth factor in bone regeneration. The aim of this study was to investigate how the rate of release of BMP-2 was affected when adsorbed to nanosized hydroxyapatite (HAP) particles functionalized with heparin by different methods. Heparin was attached to the surface of HAP, either via adsorption or covalent coupling, via a 3-aminopropyltriethoxysilane (APTES) layer. The chemical composition of the particles was evaluated using X-ray photoelectron spectroscopy and elemental microanalysis, revealing that the heparin grafting densities achieved were dependent on the curing temperature used in the fabrication of APTES-modified HAP. Comparable amounts of heparin were attached via both covalent coupling and adsorption to the APTES-modified particles, but characterization of the particle surfaces by zeta potential and Brunauer-Emmett-Teller measurements indicated that the conformation of the heparin on the surface was dependent on the method of attachment, which in turn affected the stability of heparin on the surface. The release of BMP-2 from the particles after 7 days in phosphate-buffered saline found that 31% of the loaded BMP-2 was released from the APTES-modified particles with heparin covalently attached, compared to 16% from the APTES-modified particles with the heparin adsorbed. Moreover, when heparin was adsorbed onto pure HAP, it was found that the BMP-2 released after 7 days was 5% (similar to that from unmodified HAP). This illustrates that by altering the mode of attachment of heparin to HAP the release profile and total release of BMP-2 can be manipulated. Importantly, the BMP-2 released from all the heparin particle types was found by the SMAD 1/5/8 phosphorylation assay to be biologically active. PMID:26474791

  5. Bone formation in the presence of platelet-rich plasma vs. bone morphogenetic protein-7.

    PubMed

    Roldán, J Camilo; Jepsen, Søren; Miller, Joanna; Freitag, Sandra; Rueger, David C; Açil, Yahya; Terheyden, Hendrik

    2004-01-01

    Growth factors contained in platelet-rich plasma (PRP) have recently been proposed to enhance maturation of bone grafts and, in combination with anorganic bovine bone, to support repair in the treatment of small bone defects in maxillofacial surgery. Bone morphogenetic proteins (BMP) carried in a matrix may be able to replace the autologous bone graft in the treatment of critical size defects. However, no studies have compared the bone stimulating capacity of PRP and BMP. Likewise there is no data comparing the effects of PRP in either an autologous bone graft or in anorganic bovine bone. We augmented the mandible of Wistar rats (n = 28) on both sides with either anorganic bovine bone (Bio-Oss) or autologous rib bone. On the test side we applied either 20 microl of autologous PRP or 10 microl of rhBMP-7 (4 groups, n = 7). In addition, bone induction was evaluated in an extraskeletal site (n = 14). A polychrome sequential labeling was performed. The animals were sacrificed by intra-vital perfusion on day 50. Undecalcified ground sections were evaluated by microradiography, digitized histomorphometry and under fluorescent light. The qualitative analysis of fluorochrome labels suggested that PRP and rhBMP-7 accelerated bone growth. However, histomorphometric analysis revealed no significant differences in the area of newly mineralized bone under either the influence of PRP or rhBMP-7 on autologous bone graft. Likewise, the addition of PRP to anorganic bovine bone showed no statistical difference to the control group. The strongest bone stimulating effect was seen for the combination of rhBMP-7 with anorganic bovine bone (p = 0.028). In the extraskeletal model, newly formed bone was evident in the presence of rhBMP-7, but not of PRP. In conclusion, according to the histomorphometry, the addition of platelet-rich plasma failed to enhance bone formation on anorganic bovine bone and on autologous bone grafts.

  6. MiR-503 inhibits adipogenesis by targeting bone morphogenetic protein receptor 1a

    PubMed Central

    Man, Xiao-Fei; Tan, Shu-Wen; Tang, Hao-Neng; Guo, Yue; Tang, Chen-Yi; Tang, Jun; Zhou, Ci-La; Zhou, Hou-De

    2016-01-01

    Adipogenesis plays a key role in the regulation of whole-body energy homeostasis and is critically related to obesity. To overcome obesity and its associated disorders, it is necessary to elucidate the molecular mechanisms involved in adipogenesis. An adipogenesis-related miRNA array analysis demonstrated that miR-503 was differentially expressed before and after adipocyte differentiation; however, the exact role of miR-503 in adipocyte differentiation is unclear. Thus, the objective of this study was to further examine miR-503 in adipocyte differentiation. We found significantly decreased expression of miR-503 during adipocyte differentiation process. Using bioinformatic analysis, miR-503 was identified as a potential regulator of Bone Morphogenetic Protein Receptor 1a (BMPR1a). We then validated BMPR1a as the target of miR-503 using a dual luciferase assay, and found decreased miR-503 and increased BMPR1a expression during adipogenesis. Overexpression of miR-503 in preadipocytes repressed expression of BMPR1a and adipogenic-related factors such as CCAAT/enhancer binding protein a (C/EBPα), proliferator-activated receptor-gamma (PPARγ), and adipocyte protein 2 (AP2). In addition, miR-503 overexpression impaired the phosphoinositol-3 kinase (PI3K)/Akt pathway. Inhibition of miR-503 had the opposite effect. Additionally, BMPR1a interference by siRNA attenuated adipocyte differentiation and the accumulation of lipid droplets via downregulating the PI3K/Akt signaling pathway. Our study provides the first evidence of the role miR-503 plays in adipocyte differentiation by regulating BMPR1a via the PI3K/Akt pathway, which may become a novel target for obesity therapy. PMID:27398155

  7. Prefabrication of bone by use of a vascularized periosteal flap and bone morphogenetic protein.

    PubMed

    Vögelin M D, E; Jones, N F; Lieberman, J R; Baker, J M; Tsingotjidou, A S; Brekke, J H

    2002-01-01

    The purpose of this pilot study was to prefabricate a vascularized bone graft by using a vascularized periosteal flap containing osteoprogenitor cells, a structural matrix, and recombinant human bone morphogenetic protein-2 (rhBMP-2). In a rat model, a periosteal flap vascularized by the saphenous artery and vein was dissected off the medial surface of the tibia. This flap consisted of three layers-periosteum, muscle, and fascia-and was tubed on itself to form a watertight chamber that was then transferred on its vascular pedicle to the groin. A total of 78 vascularized periosteal chambers were constructed in 39 animals and divided into 10 groups. In group 1, the periosteal chamber was left empty. Groups 2, 3, and 4 consisted of the periosteal flap and rhBMP-2, but in group 3, the proximal vascular pedicle was ligated, and in group 4, the flap was harvested without the periosteal layer and turned inside out. Groups 5 through 10 consisted of the vascularized periosteal flap containing several different structural matrices (calcium alginate spheres, polylactic acid, or demineralized bone matrix) with or without rhBMP-2. Animals were killed at 2, 4, or 8 weeks in each group. The presence and density of any new bone formation was evaluated both radiologically and histologically. Significant bone formation was seen only in those periosteal flaps containing rhBMP-2 and either the calcium alginate or polylactic acid matrix. New bone formation increased both radiologically and histologically from 2 weeks to 8 weeks only in the periosteal flaps containing the polylactic acid matrix and rhBMP-2. This preliminary study therefore suggests that four factors-blood supply, osteoprogenitor cells in the periosteal layer, a biodegradable matrix, and rhBMP-2-are required for optimal prefabrication of a vascularized bone graft.

  8. Mode of heparin attachment to nanocrystalline hydroxyapatite affects its interaction with bone morphogenetic protein-2.

    PubMed

    Goonasekera, Chandhi S; Jack, Kevin S; Bhakta, Gajadhar; Rai, Bina; Luong-Van, Emma; Nurcombe, Victor; Cool, Simon M; Cooper-White, Justin J; Grøndahl, Lisbeth

    2015-12-16

    Heparin has a high affinity for bone morphogenetic protein-2 (BMP-2), which is a key growth factor in bone regeneration. The aim of this study was to investigate how the rate of release of BMP-2 was affected when adsorbed to nanosized hydroxyapatite (HAP) particles functionalized with heparin by different methods. Heparin was attached to the surface of HAP, either via adsorption or covalent coupling, via a 3-aminopropyltriethoxysilane (APTES) layer. The chemical composition of the particles was evaluated using X-ray photoelectron spectroscopy and elemental microanalysis, revealing that the heparin grafting densities achieved were dependent on the curing temperature used in the fabrication of APTES-modified HAP. Comparable amounts of heparin were attached via both covalent coupling and adsorption to the APTES-modified particles, but characterization of the particle surfaces by zeta potential and Brunauer-Emmett-Teller measurements indicated that the conformation of the heparin on the surface was dependent on the method of attachment, which in turn affected the stability of heparin on the surface. The release of BMP-2 from the particles after 7 days in phosphate-buffered saline found that 31% of the loaded BMP-2 was released from the APTES-modified particles with heparin covalently attached, compared to 16% from the APTES-modified particles with the heparin adsorbed. Moreover, when heparin was adsorbed onto pure HAP, it was found that the BMP-2 released after 7 days was 5% (similar to that from unmodified HAP). This illustrates that by altering the mode of attachment of heparin to HAP the release profile and total release of BMP-2 can be manipulated. Importantly, the BMP-2 released from all the heparin particle types was found by the SMAD 1/5/8 phosphorylation assay to be biologically active.

  9. Calcium Phosphate Scaffolds Combined with Bone Morphogenetic Proteins or Mesenchymal Stem Cells in Bone Tissue Engineering

    PubMed Central

    Sun, Han; Yang, Hui-Lin

    2015-01-01

    Objective: The purpose of this study was to review the current status of calcium phosphate (CaP) scaffolds combined with bone morphogenetic proteins (BMPs) or mesenchymal stem cells (MSCs) in the field of bone tissue engineering (BTE). Date Sources: Data cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014, with highly regarded older publications also included. The terms BTE, CaP, BMPs, and MSC were used for the literature search. Study Selection: Reviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved, reviewed, analyzed, and summarized. Results: An ideal BTE product contains three elements: Scaffold, growth factors, and stem cells. CaP-based scaffolds are popular because of their outstanding biocompatibility, bioactivity, and osteoconductivity. However, they lack stiffness and osteoinductivity. To solve this problem, composite scaffolds of CaP with BMPs have been developed. New bone formation by CaP/BMP composites can reach levels similar to those of autografts. CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness. In addition, a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft. Conclusions: Novel BTE products possess remarkable osteoconduction and osteoinduction capacities, and exhibit balanced degradation with osteogenesis. Further work should yield safe, viable, and efficient materials for the repair of bone lesions. PMID:25881610

  10. Induction of mortality and malformation in Xenopus laevis embryos by water sources associated with field frog deformities.

    PubMed Central

    Burkhart, J G; Helgen, J C; Fort, D J; Gallagher, K; Bowers, D; Propst, T L; Gernes, M; Magner, J; Shelby, M D; Lucier, G

    1998-01-01

    Water samples from several ponds in Minnesota were evaluated for their capacity to induce malformations in embryos of Xenopus laevis. The FETAX assay was used to assess the occurrence of malformations following a 96-hr period of exposure to water samples. These studies were conducted following reports of high incidences of malformation in natural populations of frogs in Minnesota wetlands. The purpose of these studies was to determine if a biologically active agent(s) was present in the waters and could be detected using the FETAX assay. Water samples from ponds with high incidences of frog malformations (affected sites), along with water samples from ponds with unaffected frog populations (reference sites), were studied. Initial experiments clearly showed that water from affected sites induced mortality and malformation in Xenopus embryos, while water from reference sites had little or no effect. Induction of malformation was dose dependent and highly reproducible, both with stored samples and with samples taken at different times throughout the summer. The biological activity of the samples was reduced or eliminated when samples were passed through activated carbon. Limited evidence from these samples indicates that the causal factor(s) is not an infectious organism nor are ion concentrations or metals responsible for the effects observed. Results do indicate that the water matrix has a significant effect on the severity of toxicity. Based on the FETAX results and the occurrence of frog malformations observed in the field, these studies suggest that water in the affected sites contains one or more unknown agents that induce developmental abnormalities in Xenopus. These same factors may contribute to the increased incidence of malformation in native species. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9831545

  11. Radioimmunoassay of bone morphogenetic protein in serum: a tissue-specific parameter of bone metabolism

    SciTech Connect

    Urist, M.R.; Hudak, R.T.

    1984-05-01

    Bone morphogenetic protein (BMP), a paracrine agent inducing cartilage and bone cell differentiation, circulates in the blood and is detectable by BMP radioimmunoassay. Serum BMP levels are higher in growing children and patients with Paget's disease than in normal adults. These observations are interpreted as evidence of a BMP function in the physiology of bone in health and disease.

  12. Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine.

    PubMed

    Sai, Linlin; Dong, Zhihua; Li, Ling; Guo, Qiming; Jia, Qiang; Xie, Lin; Bo, Cunxiang; Liu, Yanzhong; Qu, Binpeng; Li, Xiangxin; Shao, Hua; Ng, Jack C; Peng, Cheng

    2016-09-01

    As a widely used herbicide, atrazine (AZ) has been extensively studied for its adverse effects on the reproductive system, especially feminization in male animals. However, the relationship of gene expression changes and associated toxicological endpoints remains unclear. In this study, developing Xenopus laevis tadpoles were exposed to concentration of AZ at 0.1, 1, 10 or 100 μg/L continuously. Compared with froglets in the control group, there were no significant differences in body length, body weight, liver weight and hepatosomatic index (HSI) of males in groups treated with AZ for 90 d. At 100 μg/L AZ treatment caused a significant reduction of gonad weight and gonadosomatic index (GSI) of males (p < 0.01). In addition, AZ at all dose levels caused testicular degeneration, especially in froglets from the groups with 0.1 and 100 μg/L which exhibited U-shaped dose-response trend. We further investigated the gene expression changes associated with the testicular degeneration induced by AZ. We found that the expression of 1165 genes was significantly altered with 616 upregulated and 549 downregulated compared to the expression profile of the control animals. KEGG analysis showed that genes which were significantly affected by AZ are mainly involved in arginine and proline metabolism, cell cycle, riboflavin metabolism, spliceosome, base excision repair and progesterone-mediated oocyte maturation pathway. Our results show that AZ may affect reproductive and immune systems by interference with the related gene expression changes during the male X. laevis development. The findings may help to clarify the feminization mechanisms of AZ in male X. laevis. PMID:27288644

  13. Production of transgenic Xenopus laevis by restriction enzyme mediated integration and nuclear transplantation.

    PubMed

    Amaya, Enrique; Kroll, Kristen

    2010-01-01

    Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes. PMID:20811326

  14. Effects of perfluorooctanesulfonate and perfluorobutanesulfonate on the growth and sexual development of Xenopus laevis.

    PubMed

    Lou, Qin-Qin; Zhang, Yin-Feng; Zhou, Zhen; Shi, Ya-Li; Ge, Ya-Nan; Ren, Dong-Kai; Xu, Hai-Ming; Zhao, Ya-Xian; Wei, Wu-Ji; Qin, Zhan-Fen

    2013-09-01

    Perfluorobutanesulfonate (PFBS), as a substitute for perfluorooctanesulfonate (PFOS), is widespread in the environment and biotic samples as well as PFOS. To investigate effects of PFOS and PFBS on the growth and sexual development of amphibians, we exposed Xenopus laevis tadpoles at a series of concentrations of PFOS and PFBS (0.1; 1; 100; 1,000 μg/l) as well as 17-beta-estradiol (E2, 100 ng/l) and 5 alpha-androstan-17-beta-ol-3-one (DHT, 100 ng/l) from stage 46/47 to 2 months postmetamorphosis. We found that neither PFOS nor PFBS had a significant effect on the survival and growth. However, they caused hepatohistological impairment at higher concentrations (100; 1,000 μg/l). Unlike E2, PFOS at all concentrations did not alter the sex ratio and induce intersex, but caused degeneration of spermatogonia in testes except for the lowest concentration. PFBS had no effect on the sex ratio and gonadal histology. PFOS and PFBS promoted expression of estrogen receptor (ER) and androgen receptor (AR), but not affected aromatase expression in the brain. The increase in expression of ER and AR suggests an increase in the responsiveness to the corresponding sex hormone and potential effects on sexual development. Our results show that PFBS as well as PFOS have adverse effects on hepato-histology and sexual development on X. laevis. Also, PFOS- and PFBS-induced increase in ER and AR expression highlights the need to further study effects of PFOS and PFBS on subsequently gonadal development, sexual dimorphism, and secondary sex characteristics in X. laevis. It is debatable that PFBS is widely used as a substitute of PFOS.

  15. [Effects of phosphatidylinositol-3 kinase/protein kinase b/bone morphogenetic protein-15 pathway on the follicular development in the mammalian ovary].

    PubMed

    Wu, Yan-qing; Chen, Li-yun; Zhang, Zheng-hong; wang, Zheng-chao

    2013-04-01

    In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.

  16. Removal of 2',3'-dideoxynucleotide residues from injected DNA in Xenopus laevis oocytes.

    PubMed

    Legerski, R J; Penkala, J E; Peterson, C A; Wright, D A

    1990-07-01

    Dideoxynucleotides have proved to be potent differential inhibitors of DNA polymerases in vitro and in vivo. Used extensively in studies of DNA repair and replication, they have more recently been used as antiviral agents particularly in treating patients for acquired immunodeficiency syndrome (AIDS). Once incorporated, these sugar-modified analogues prevent the further extension of the polynucleotide chain because of the absence of a 3'-hydroxyl group. We demonstrated that, upon injection into Xenopus laevis oocytes, 2',3'-dideoxynucleotides are efficiently removed from plasmid DNA preterminated with these analogues allowing subsequent closure by ligation. The removal process is not sensitive to aphidicolin but is quantitatively inhibited by novobiocin. PMID:2366792

  17. Accelerated Gene Evolution and Subfunctionalization in thePseudotetraploid Frog Xenopus Laevis

    SciTech Connect

    Hellsten, Uffe; Khokha, Mustafa K.; Grammar, Timothy C.; Harland,Richard M.; Richardson, Paul; Rokhsar, Daniel S.

    2007-03-01

    Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization {approx} 40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication.

  18. Mechanical Force Alters Morphogenetic Movements and Segmental Gene Expression Patterns during Drosophila Embryogenesis

    PubMed Central

    Kumar, Abhishek; Shivashankar, G. V.

    2012-01-01

    The development of an organism is accompanied by various cellular morphogenetic movements, changes in cellular as well as nuclear morphology and transcription programs. Recent evidence suggests that intra and inter-cellular connections mediated by various adhesion proteins contribute to defining nuclear morphology. In addition, three dimensional organization of the cell nucleus regulate the transcription programs. However the link between cellular morphogenetic movements and its coupling to nuclear function in a developmental context is poorly understood. In this paper we use a point perturbation by tissue level laser ablation and sheet perturbation by application of force using magnetic tweezers to alter cellular morphogenetic movements and probe its impact on nuclear morphology and segmental gene expression patterns. Mechanical perturbations during blastoderm stage in a developing Drosophila embryo resulted in localized alterations in nuclear morphology and cellular movement. In addition, global defects in germ-band (GB) extension and retraction are observed when external force is applied during morphogenetic movements, suggesting a long-range physical coupling within the GB layer of cells. Further local application of force resulted in redistribution of non muscle myosin-II in the GB layer. Finally these perturbations lead to altered segmental gene (engrailed) expression patterns later during the development. Our observations suggest that there exists a tight regulation between nuclear morphology and cellular adhesive connections during morphogenetic movement of cells in the embryo. The observed spatial changes in patterning genes, with perturbation, highlight the importance of nuclear integrity to cellular movement in establishing gene expression program in a developmental system. PMID:22470437

  19. Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically releases active bone morphogenetic protein-2 for induction of osteogenic differentiation.

    PubMed

    Hulsart-Billström, Gry; Yuen, Pik Kwan; Marsell, Richard; Hilborn, Jöns; Larsson, Sune; Ossipov, Dmitri

    2013-09-01

    Regeneration of bone by delivery of bone morphogenetic proteins (BMPs) from implantable scaffolds is a promising alternative to the existing autologous bone grafting procedures. Hydrogels are used extensively in biomaterials as delivery systems for different growth factors. However, a controlled release of the growth factors is necessary to induce bone formation, which can be accomplished by various chemical functionalities. Herein we demonstrate that functionalization of a hyaluronan (HA) hydrogel with covalently linked bisphosphonate (BP) ligands provides efficient sequestering of BMP-2 in the resulting HA-BP hydrogel. The HA-BP hydrogel was investigated in comparison with its analogue lacking BP groups (HA hydrogel). While HA hydrogel released 100% of BMP-2 over two weeks, less than 10% of BMP-2 was released from the HA-BP hydrogel for the same time. We demonstrate that the sequestered growth factor can still be released by enzymatic degradation of the HA-BP hydrogel. Most importantly, entrapment of BMP-2 in HA-BP hydrogel preserves the growth factor bioactivity, which was confirmed by induction of osteogenic differentiation of mesenchymal stem cells (MSCs) after the cells incubation with the enzymatic digest of the hydrogel. At the same time, the hydrogels degradation products were not toxic to MSCs and osteoblasts. Furthermore, BP-functionalization of HA hydrogels promotes adhesion of the cells to the surface of HA hydrogel. Altogether, the present findings indicate that covalent grafting of HA hydrogel with BP groups can alter the clinical effects of BMPs in bone tissue regeneration.

  20. The B-subdomain of the Xenopus laevis XFIN KRAB-AB domain is responsible for its weaker transcriptional repressor activity compared to human ZNF10/Kox1.

    PubMed

    Born, Nadine; Thiesen, Hans-Jürgen; Lorenz, Peter

    2014-01-01

    The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

  1. The thyroid hormone receptor gene (c-erbA alpha) is expressed in advance of thyroid gland maturation during the early embryonic development of Xenopus laevis.

    PubMed Central

    Banker, D E; Bigler, J; Eisenman, R N

    1991-01-01

    The c-erbA proto-oncogene encodes the thyroid hormone receptor, a ligand-dependent transcription factor which plays an important role in vertebrate growth and development. To define the role of the thyroid hormone receptor in developmental processes, we have begun studying c-erbA gene expression during the ontogeny of Xenopus laevis, an organism in which thyroid hormone has well-documented effects on morphogenesis. Using polymerase chain reactions (PCR) as a sensitive assay of specific gene expression, we found that polyadenylated erbA alpha RNA is present in Xenopus cells at early developmental stages, including the fertilized egg, blastula, gastrula, and neurula. By performing erbA alpha-specific PCR on reverse-transcribed RNAs from high-density sucrose gradient fractions prepared from early-stage embryos, we have demonstrated that these erbA transcripts are recruited to polysomes. Therefore, erbA is expressed in Xenopus development prior to the appearance of the thyroid gland anlage in tailbud-stage embryos. This implies that erbA alpha/thyroid hormone receptors may play ligand-independent roles during the early development of X. laevis. Quantitative PCR revealed a greater than 25-fold range in the steady-state levels of polyadenylated erbA alpha RNA across early stages of development, as expressed relative to equimolar amounts of total embryonic RNA. Substantial increases in the levels of erbA alpha RNA were noted at stages well after the onset of zygotic transcription at the mid-blastula transition, with accumulation of erbA alpha transcripts reaching a relative maximum in advance of metamorphosis. We also show that erbA alpha RNAs are expressed unequally across Xenopus neural tube embryos. This differential expression continues through later stages of development, including metamorphosis. This finding suggests that erbA alpha/thyroid hormone receptors may play roles in tissue-specific processes across all of Xenopus development. Images PMID:1656222

  2. Effect of Light on Expression of Clock Genes in Xenopus laevis Melanophores.

    PubMed

    de Carvalho Magalhães Moraes, Maria Nathália; de Oliveira Poletini, Maristela; Ramos, Bruno Cesar Ribeiro; de Lima, Leonardo Henrique Ribeiro Graciani; de Lauro Castrucci, Ana Maria

    2013-12-26

    Light-dark cycles are considered important cues to entrain biological clocks. A feedback loop of clock gene transcription and translation is the molecular basis underlying the mechanism of both central and peripheral clocks. Xenopus laevis embryonic melanophores respond to light with melanin granule dispersion, response possibly mediated by the photopigment melanopsin. In order to test whether light modulates clock gene expression in Xenopus melanophores, we used qPCR to evaluate the relative mRNA levels of Per1, Per2, Clock and Bmal1 in cultured melanophores exposed to light-dark (LD) cycle or constant darkness (DD). LD cycles elicited temporal changes in the expression of Per1, Per2 and Bmal1. A 10-min pulse of blue light was able to increase the expression of Per1 and Per2. Red light had no effect on the expression of these clock genes. These data suggest the participation of a blue-wavelength sensitive pigment in the light-dark cycle-mediated oscillation of the endogenous clock. Our results add an important contribution to the emerging field of peripheral clocks, which in non-mammalian vertebrates have been mostly studied in Drosophila and Danio rerio. Within this context, we show that Xenopus laevis melanophores, which have already led to melanopsin discovery, represent an ideal model to understanding circadian rhythms. This article is protected by copyright. All rights reserved.

  3. Effect of light on expression of clock genes in Xenopus laevis melanophores.

    PubMed

    Magalhães Moraes, Maria Nathália de Carvalho; de Oliveira Poletini, Maristela; Ribeiro Ramos, Bruno Cesar; de Lima, Leonardo Henrique Ribeiro Graciani; de Lauro Castrucci, Ana Maria

    2014-01-01

    Light-dark cycles are considered important cues to entrain biological clocks. A feedback loop of clock gene transcription and translation is the molecular basis underlying the mechanism of both central and peripheral clocks. Xenopus laevis embryonic melanophores respond to light with melanin granule dispersion, response possibly mediated by the photopigment melanopsin. To test whether light modulates clock gene expression in Xenopus melanophores, we used qPCR to evaluate the relative mRNA levels of Per1, Per2, Clock and Bmal1 in cultured melanophores exposed to light-dark (LD) cycle or constant darkness (DD). LD cycles elicited temporal changes in the expression of Per1, Per2 and Bmal1. A 10-min pulse of blue light was able to increases the expression of Per1 and Per2. Red light had no effect on the expression of these clock genes. These data suggest the participation of a blue-wavelength sensitive pigment in the light-dark cycle-mediated oscillation of the endogenous clock. Our results add an important contribution to the emerging field of peripheral clocks, which in nonmammalian vertebrates have been mostly studied in Drosophila and Danio rerio. Within this context, we show that X. laevis melanophores, which have already led to melanopsin discovery, represent an ideal model to understanding circadian rhythms.

  4. Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

    PubMed Central

    Voglmeir, Josef; Laurent, Nicolas; Flitsch, Sabine L.; Oelgeschläger, Michael; Wilson, Iain B.H.

    2015-01-01

    The biosynthesis of mucin-type O-linked glycans in animals is initiated by members of the large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), which play important roles in embryogenesis, organogenesis, adult tissue homeostasis and carcinogenesis. Until now, the mammalian forms of these enzymes have been the best characterized. However, two N-acetylgalactosaminyltransferases (xGalNAc-T6 and xGalNAc-T16) from the African clawed frog (Xenopus laevis), which are most homologous to those encoded by the human GALNT6 and GALNT16 (GALNTL1) genes, were shown to have contrasting roles in TGF-β/BMP signaling in embryogenesis. In this study we have examined these two enzymes further and show differences in their in vivo function during X. laevis embyrogenesis as evidenced by in situ hybridization and overexpression experiments. In terms of enzymatic activity, both enzymes were found to be active towards the EA2 peptide, but display differential activity towards a peptide based on the sequence of ActR-IIB, a receptor relevant to TGF-β/BMP signaling. In summary, these data demonstrate that these two enzymes from different branches of the N-acetylgalactosaminyltransferase do not only display differential substrate specificities, but also specific and distinct expression pattern and biological activities in vivo. PMID:25447273

  5. CHROMATIN ASSEMBLY AND TRANSCRIPTIONAL CROSS-TALK IN XENOPUS LAEVIS OOCYTE AND EGG EXTRACTS

    PubMed Central

    Wang, Wei-Lin; Shechter, David

    2016-01-01

    Chromatin, primarily a complex of DNA and histone proteins, is the physiological form of the genome. Chromatin is generally repressive for transcription and other information transactions that occur on DNA. A wealth of post-translational modifications on canonical histones and histone variants encode regulatory information to recruit or repel effector proteins on chromatin, promoting and further repressing transcription and thereby form the basis of epigenetic information. During metazoan oogenesis, large quantities of histone proteins are synthesized and stored in preparation for the rapid early cell cycles of development and to elicit maternal control of chromatin assembly pathways. Oocyte and egg cell-free extracts of the frog Xenopus laevis are a compelling model system for the study of chromatin assembly and transcription precisely because they exist in an extreme state primed for rapid chromatin assembly or for transcriptional activity. We show that chromatin assembly rates are slower in X. laevis oocyte than in egg extracts, while conversely only oocyte extracts transcribe template plasmids. We demonstrate that rapid chromatin assembly in egg extracts represses RNA Polymerase II dependent transcription, while pre-binding of TATA-Binding Protein (TBP) to a template plasmid promotes transcription. Our experimental evidence presented here supports a model in which chromatin assembly and transcription are in competition and that the onset of zygotic genomic activation may be in part due to stable transcriptional complex assembly. PMID:27759158

  6. In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration.

    PubMed

    Suzuki, Miyuki; Takagi, Chiyo; Miura, Shinichirou; Sakane, Yuto; Suzuki, Makoto; Sakuma, Tetsushi; Sakamoto, Naoaki; Endo, Tetsuya; Kamei, Yasuhiro; Sato, Yuko; Kimura, Hiroshi; Yamamoto, Takashi; Ueno, Naoto; Suzuki, Ken-ichi T

    2016-04-01

    Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration. PMID:26914410

  7. Effect of copper contaminated food on the life cycle and secondary production of Daphnia laevis.

    PubMed

    Rocha, Giseli S; Tonietto, Alessandra E; Lombardi, Ana T; Melão, Maria da G G

    2016-11-01

    In aquatic environments, copper (Cu) plays important physiological roles in planktonic food chain, such as electron transfer in photosynthesis and constituting proteins that transport oxygen in some arthropods, while at higher concentrations it is toxic on these organisms and higher trophic levels. The combined effects of natural (e.g. volcanic activity) and anthropogenic sources (e.g. mining waste) contribute to the increase in copper pollution in different ecosystems and regions around the world. In the present study, we evaluated the bioaccumulation and effect of Cu on Raphidocelis subcapitata (freshwater algae), and the influence of Cu-contaminated food (algae) on Daphnia laevis (tropical cladoceran). The amount of copper accumulated in microalgae and cladoceran was quantified, and life-history parameters of D. laevis such as growth, reproduction and longevity were measured. The cell density of Cu exposed R. subcapitata declined, and cladoceran fed with contaminated food had lower longevity, production of eggs and neonates, and reduced secondary production. A concentration dependent increase in Cu accumulation was observed in the microalgae, while the opposite occurred in the animal, indicating a cellular metal regulatory mechanism in the latter. However, this regulation seems not to be sufficient to avoid metal induced damages in the cladoceran such as decreased longevity and reproduction. We conclude that diet is an important metal exposure route to this cladoceran, and the assessment of chronic contamination during the complete life cycle of cladoceran provides results that are similar to those observed in natural environments, especially when native organisms are investigated. PMID:27472028

  8. Entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos

    SciTech Connect

    Kao, K.R.; Elinson, R.P.

    1988-05-01

    The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland. In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted into ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise.

  9. Cloning and functional characterization of a novel aquaporin from Xenopus laevis oocytes.

    PubMed

    Virkki, Leila V; Franke, Christina; Somieski, Petra; Boron, Walter F

    2002-10-25

    We have cloned a novel aquaporin (AQP) from Xenopus laevis oocytes, which we have provisionally named AQPxlo. The predicted protein showed highest homology (39-50%) to aquaglyceroporins. Northern blot analysis showed strong hybridization to an approximately 1.4-kb transcript in X. laevis fat body and oocytes, whereas a weaker signal was obtained in kidney. We injected in vitro transcribed cRNA encoding AQPxlo into Xenopus oocytes for functional characterization. AQPxlo expression increased osmotic water permeability (P(f)), as well as the uptake of glycerol and urea. However, AQPxlo excluded larger polyols and thiourea. An alkaline extracellular pH (pH(o)) increased P(f) and to a lesser extent urea uptake but not glycerol uptake. Remarkably, low HgCl(2) concentrations (0.3-10 microm) reduced P(f) and urea uptake, whereas high concentrations (300-1000 microm) reversed the inhibition. We propose that AQPxlo is a new AQP paralogue unknown in mammals.

  10. Distribution of single wall carbon nanotubes in the Xenopus laevis embryo after microinjection.

    PubMed

    Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

    2016-04-01

    Single wall carbon nanotubes (SWCNTs) are advanced materials with the potential for a myriad of diverse applications, including biological technologies and large-scale usage with the potential for environmental impacts. SWCNTs have been exposed to developing organisms to determine their effects on embryogenesis, and results have been inconsistent arising, in part, from differing material quality, dispersion status, material size, impurity from catalysts and stability. For this study, we utilized highly purified SWCNT samples with short, uniform lengths (145 ± 17 nm) well dispersed in solution. To test high exposure doses, we microinjected > 500 µg ml(-1) SWCNT concentrations into the well-established embryogenesis model, Xenopus laevis, and determined embryo compatibility and subcellular localization during development. SWCNTs localized within cellular progeny of the microinjected cells, but were heterogeneously distributed throughout the target-injected tissue. Co-registering unique Raman spectral intensity of SWCNTs with images of fluorescently labeled subcellular compartments demonstrated that even at regions of highest SWCNT concentration, there were no gross alterations to subcellular microstructures, including filamentous actin, endoplasmic reticulum and vesicles. Furthermore, SWCNTs did not aggregate and localized to the perinuclear subcellular region. Combined, these results suggest that purified and dispersed SWCNTs are not toxic to X. laevis animal cap ectoderm and may be suitable candidate materials for biological applications. PMID:26510384

  11. The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos.

    PubMed

    Kao, K R; Elinson, R P

    1988-05-01

    The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland (K.R. Kao, Y. Masui, and R.P. Elinson, 1986, Nature (London) 322, 371-373). In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted into ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise. PMID:3282938

  12. Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis

    PubMed Central

    Nagasawa, Kazumichi; Tanizaki, Yuta; Okui, Takehito; Watarai, Atsuko; Ueda, Shinobu; Kato, Takashi

    2013-01-01

    Summary The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism. PMID:24167716

  13. Effect of copper contaminated food on the life cycle and secondary production of Daphnia laevis.

    PubMed

    Rocha, Giseli S; Tonietto, Alessandra E; Lombardi, Ana T; Melão, Maria da G G

    2016-11-01

    In aquatic environments, copper (Cu) plays important physiological roles in planktonic food chain, such as electron transfer in photosynthesis and constituting proteins that transport oxygen in some arthropods, while at higher concentrations it is toxic on these organisms and higher trophic levels. The combined effects of natural (e.g. volcanic activity) and anthropogenic sources (e.g. mining waste) contribute to the increase in copper pollution in different ecosystems and regions around the world. In the present study, we evaluated the bioaccumulation and effect of Cu on Raphidocelis subcapitata (freshwater algae), and the influence of Cu-contaminated food (algae) on Daphnia laevis (tropical cladoceran). The amount of copper accumulated in microalgae and cladoceran was quantified, and life-history parameters of D. laevis such as growth, reproduction and longevity were measured. The cell density of Cu exposed R. subcapitata declined, and cladoceran fed with contaminated food had lower longevity, production of eggs and neonates, and reduced secondary production. A concentration dependent increase in Cu accumulation was observed in the microalgae, while the opposite occurred in the animal, indicating a cellular metal regulatory mechanism in the latter. However, this regulation seems not to be sufficient to avoid metal induced damages in the cladoceran such as decreased longevity and reproduction. We conclude that diet is an important metal exposure route to this cladoceran, and the assessment of chronic contamination during the complete life cycle of cladoceran provides results that are similar to those observed in natural environments, especially when native organisms are investigated.

  14. Proteomics analysis of Xenopus laevis gonad tissue following chronic exposure to atrazine.

    PubMed

    Chen, Xiuping; Wang, Jiamei; Zhu, Haojun; Ding, Jiatong; Peng, Yufa

    2015-08-01

    Atrazine is the most commonly detected pesticide contaminant in ground and surface water. Previous studies have shown that atrazine is an endocrine disruptor owing to its adverse effects on the male reproductive system in several vertebrates, but very few molecular mechanisms for these effects have been revealed. In the present study, Xenopus laevis were exposed to 100 ppb of atrazine for 120 d, and then the isobaric tags for relative and absolute quantitation (iTRAQ) technique was used to detect global changes in protein profiles of the testes and ovaries. The results showed that 100 ppb of atrazine exposure adversely affected the growth of X. laevis and did not induce hermaphroditism but delayed or prevented the development of male seminiferous tubules. Proteomic analysis showed that atrazine altered expression of 143 and 121 proteins in the testes and ovaries, respectively, and most of them are involved in cellular and metabolic processes and biological regulation based on their biological processes. In addition, apoptosis, tight junctions, and metabolic pathways were significantly altered in the atrazine-treated gonads. Based on the above results, it is postulated that the reproductive toxicity of atrazine may be the result of disruption of tight junctions and metabolic signaling pathways and/or induction of apoptosis in germ cells.

  15. Effects of agricultural pesticides on the immune system of Xenopus laevis and Rana pipiens.

    PubMed

    Christin, M S; Ménard, L; Gendron, A D; Ruby, S; Cyr, D; Marcogliese, D J; Rollins-Smith, L; Fournier, M

    2004-03-30

    Over the last 30 years, there have been mass declines in diverse geographic locations among amphibian populations. Multiple causes have been suggested to explain this decline. Among these, environmental pollution is gaining attention. Indeed, some chemicals of environmental concern are known to alter the immune system. Given that amphibians are frequently exposed to agricultural pesticides, it is possible that these pollutants alter their immune system and render them more susceptible to different pathogens. In this study, we exposed two frog species, Xenopus laevis and Rana pipiens, for a short period of time to a mixture of pesticides (atrazine, metribuzine, endosulfan, lindane, aldicarb and dieldrin) representative in terms of composition and concentrations to what it is found in the environment of the southwest region of the province of Quebec. The pesticides were known to be present in surface water of many tributaries of the St. Lawrence River (Quebec, Canada). Our results demonstrate that the mixture of pesticides could alter the cellularity and phagocytic activity of X. laevis and the lymphocyte proliferation of R. pipiens. Taken together, these results indicate that agricultural pesticides can alter some aspects of the immune response in frogs and could contribute to their global decline by rendering them more susceptible to certain infections.

  16. Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis.

    PubMed

    Nagasawa, Kazumichi; Tanizaki, Yuta; Okui, Takehito; Watarai, Atsuko; Ueda, Shinobu; Kato, Takashi

    2013-01-01

    The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

  17. Increased bone morphogenetic protein 7 signalling in the kidneys of dogs affected with a congenital portosystemic shunt.

    PubMed

    van Dongen, Astrid M; Heuving, Susanne M; Tryfonidou, Marianna A; van Steenbeek, Frank G; Rothuizen, Jan; Penning, Louis C

    2015-05-01

    Dogs with a congenital portosystemic shunt (CPSS) often have enlarged and hyper-filtrating kidneys. Although expression of different growth factors has been well-described in the livers of dogs affected with a CPSS, their expression in the kidneys has yet to be determined. Bone morphogenetic protein 7 (BMP-7), hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β have been implicated in renal development (BMP-7, HGF) or the onset of renal fibrosis (TGF-β). Moreover, BMP-7 and HGF have protective properties in renal fibrosis. In this study, the expression and activity of BMP-7 were investigated in renal biopsies obtained from 13 dogs affected with a CPSS and compared to similar samples from age-matched healthy control dogs. Both quantitative reverse-transcriptase PCR and Western blotting showed up-regulated BMP-7 signalling in kidneys of CPPS-affected dogs. These research findings may help to explain the renal pathology/dysfunction in dogs affected with a CPSS.

  18. Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles

    PubMed Central

    Tellier, Liane E.; Miller, Tobias; McDevitt, Todd C.; Temenoff, Johnna S.

    2015-01-01

    Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.

  19. Coating with a Modular Bone Morphogenetic Peptide Promotes Healing of a Bone-Implant Gap in an Ovine Model

    PubMed Central

    Lu, Yan; Lee, Jae Sung; Nemke, Brett; Graf, Ben K.; Royalty, Kevin; Illgen, Richard; Vanderby, Ray; Markel, Mark D.; Murphy, William L.

    2012-01-01

    Despite the potential for growth factor delivery strategies to promote orthopedic implant healing, there is a need for growth factor delivery methods that are controllable and amenable to clinical translation. We have developed a modular bone growth factor, herein termed “modular bone morphogenetic peptide (mBMP)”, which was designed to efficiently bind to the surface of orthopedic implants and also stimulate new bone formation. The purpose of this study was to coat a hydroxyapatite-titanium implant with mBMP and evaluate bone healing across a bone-implant gap in the sheep femoral condyle. The mBMP molecules efficiently bound to a hydroxyapatite-titanium implant and 64% of the initially bound mBMP molecules were released in a sustained manner over 28 days. The results demonstrated that the mBMP-coated implant group had significantly more mineralized bone filling in the implant-bone gap than the control group in C-arm computed tomography (DynaCT) scanning (25% more), histological (35% more) and microradiographic images (50% more). Push-out stiffness of the mBMP group was nearly 40% greater than that of control group whereas peak force did not show a significant difference. The results of this study demonstrated that mBMP coated on a hydroxyapatite-titanium implant stimulates new bone formation and may be useful to improve implant fixation in total joint arthroplasty applications. PMID:23185610

  20. Bone Morphogenetic Protein-9 Effectively Induces Osteo/Odontoblastic Differentiation of the Reversibly Immortalized Stem Cells of Dental Apical Papilla

    PubMed Central

    Wang, Jinhua; Zhang, Hongmei; Zhang, Wenwen; Huang, Enyi; Wang, Ning; Wu, Ningning; Wen, Sheng; Chen, Xian; Liao, Zhan; Deng, Fang; Yin, Liangjun; Zhang, Junhui; Zhang, Qian; Yan, Zhengjian; Liu, Wei; Zhang, Zhonglin; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.

    2014-01-01

    Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPARγ2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration. PMID:24517722

  1. Inverse Effects on Growth and Development Rates by Means of Endocrine Disruptors in African Clawed Frog Tadpoles ("Xenopus Laevis")

    ERIC Educational Resources Information Center

    Hackney, Zachary Carl

    2007-01-01

    Previous work on fish, frogs, and salamanders, showed the ability for estrogen (EE2) and anthropogenic endocrine disruptors to skew sex ratios and cause hermaphrodism. This study addressed the effects of estrogens on growth and development rates of African clawed frog tadpoles ("Xenopus laevis") during their gender determination stages. The…

  2. Plasma sex steroid concentrations and gonadal aromatase activities in African clawed frogs (Xenopus laevis) from South Africa.

    PubMed

    Hecker, Markus; Giesy, John P; Jones, Paul D; Jooste, Alarik M; Carr, James A; Solomon, Keith R; Smith, Ernest E; Van der Kraak, Glen; Kendall, Ronald J; Du Preez, Louis

    2004-08-01

    Adult African clawed frogs (Xenopus laevis) were collected from a corn-growing region (CGR) and a non-corn-growing region (NCGR) with different exposure profiles for atrazine and related triazines. Physical, chemical, and biological parameters from the catchment areas were also measured. Frogs were surveyed for possible effects of exposure to triazine herbicides on plasma testosterone (T) and estradiol (E2) titers, gonadal aromatase activity, and gonad growth (GSI). Concentrations of both T and E2 varied among locations and were correlated to some accessory factors, such as pH, several ions, and metals. Greatest median plasma T concentrations (males: 19 ng/ml; females: 16 ng/ml) occurred in frogs inhabiting NCGR as compared to those from the CGR (males: 4 ng/ml; females: 1 ng/ml). Median E2 concentrations were also greater in frogs collected from the NCGR (males: 3 ng/ml; females: 28 ng/ml) than those in frogs from the CGR (males: 2 ng/ml; females: 5 ng/ml). Because some exposure to agricultural chemicals at both regions occurred, as did simultaneous exposures to multiple chemicals, a regression analysis was employed. Negative correlations were observed between plasma T concentrations and concentrations of atrazine, deisopropylatrazine, deethylatrazine, and tertbuthylazine in females and between T and diaminochlorotriazine in males. Estradiol in females exhibited a significant negative correlation with atrazine and deethylatrazine. No correlations were observed between gonadal aromatase activity or GSI and any of the agricultural chemicals measured. Median aromatase activities in ovaries varied among sampling sites ranging from 7 to >3000 times greater than those in males when measurable. Testicular aromatase activity was below the detection limit of the assay in male frogs at most of the sites. Although exposure to agricultural inputs did not affect aromatase activities, effects of atrazine or coapplied pesticides on sex steroid homeostasis cannot be excluded at

  3. Protein Expression Profiling in the African Clawed Frog Xenopus laevis Tadpoles Exposed to the Polychlorinated Biphenyl Mixture Aroclor 1254S⃞

    PubMed Central

    Gillardin, Virginie; Silvestre, Frédéric; Dieu, Marc; Delaive, Edouard; Raes, Martine; Thomé, Jean-Pierre; Kestemont, Patrick

    2009-01-01

    Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into account to partly explain the worldwide decline of amphibians. PCBs induce deleterious effects on developing amphibians including deformities and delays in metamorphosis. However, the molecular mechanisms by which they express their toxicity during the development of tadpoles are still largely unknown. A proteomics analysis was performed on developing Xenopus laevis tadpoles exposed from 2 to 5 days postfertilization to either 0.1 or 1 ppm Aroclor 1254, a PCB mixture. Two-dimensional DIGE with a minimal labeling method coupled to nanoflow liquid chromatography-tandem mass spectrometry was used to detect and identify proteins differentially expressed under PCBs conditions. Results showed that 59 spots from the 0.1 ppm Aroclor 1254 condition and 57 spots from the 1 ppm Aroclor 1254 condition displayed a significant increase or decrease of abundance compared with the control. In total, 28 proteins were identified. The results suggest that PCBs induce mechanisms against oxidative stress (peroxiredoxins 1 and 2), adaptative changes in the energetic metabolism (enolase 1, glycerol-3-phosphate dehydrogenase, and creatine kinase muscle and brain types), and the implication of the unfolded protein response system (glucose-regulated protein, 58 kDa). They also affect, at least at the highest concentration tested, the synthesis of proteins involved in normal cytogenesis (α-tropomyosin, myosin heavy chain, and α-actin). For the first time, proteins such as aldehyde dehydrogenase 7A1, CArG binding factor-A, prolyl 4-hydroxylase β, and nuclear matrix protein 200 were also shown to be up-regulated by PCBs in developing amphibians. These data argue that protein expression reorganization should be taken into account while estimating the toxicological hazard of wild amphibian populations exposed to PCBs. PMID:19011258

  4. De novo Transcriptome Assemblies of Rana (Lithobates) catesbeiana and Xenopus laevis Tadpole Livers for Comparative Genomics without Reference Genomes.

    PubMed

    Birol, Inanc; Behsaz, Bahar; Hammond, S Austin; Kucuk, Erdi; Veldhoen, Nik; Helbing, Caren C

    2015-01-01

    In this work we studied the liver transcriptomes of two frog species, the American bullfrog (Rana (Lithobates) catesbeiana) and the African clawed frog (Xenopus laevis). We used high throughput RNA sequencing (RNA-seq) data to assemble and annotate these transcriptomes, and compared how their baseline expression profiles change when tadpoles of the two species are exposed to thyroid hormone. We generated more than 1.5 billion RNA-seq reads in total for the two species under two conditions as treatment/control pairs. We de novo assembled these reads using Trans-ABySS to reconstruct reference transcriptomes, obtaining over 350,000 and 130,000 putative transcripts for R. catesbeiana and X. laevis, respectively. Using available genomics resources for X. laevis, we annotated over 97% of our X. laevis transcriptome contigs, demonstrating the utility and efficacy of our methodology. Leveraging this validated analysis pipeline, we also annotated the assembled R. catesbeiana transcriptome. We used the expression profiles of the annotated genes of the two species to examine the similarities and differences between the tadpole liver transcriptomes. We also compared the gene ontology terms of expressed genes to measure how the animals react to a challenge by thyroid hormone. Our study reports three main conclusions. First, de novo assembly of RNA-seq data is a powerful method for annotating and establishing transcriptomes of non-model organisms. Second, the liver transcriptomes of the two frog species, R. catesbeiana and X. laevis, show many common features, and the distribution of their gene ontology profiles are statistically indistinguishable. Third, although they broadly respond the same way to the presence of thyroid hormone in their environment, their receptor/signal transduction pathways display marked differences. PMID:26121473

  5. De novo Transcriptome Assemblies of Rana (Lithobates) catesbeiana and Xenopus laevis Tadpole Livers for Comparative Genomics without Reference Genomes

    PubMed Central

    Birol, Inanc; Behsaz, Bahar; Hammond, S. Austin; Kucuk, Erdi; Veldhoen, Nik; Helbing, Caren C.

    2015-01-01

    In this work we studied the liver transcriptomes of two frog species, the American bullfrog (Rana (Lithobates) catesbeiana) and the African clawed frog (Xenopus laevis). We used high throughput RNA sequencing (RNA-seq) data to assemble and annotate these transcriptomes, and compared how their baseline expression profiles change when tadpoles of the two species are exposed to thyroid hormone. We generated more than 1.5 billion RNA-seq reads in total for the two species under two conditions as treatment/control pairs. We de novo assembled these reads using Trans-ABySS to reconstruct reference transcriptomes, obtaining over 350,000 and 130,000 putative transcripts for R. catesbeiana and X. laevis, respectively. Using available genomics resources for X. laevis, we annotated over 97% of our X. laevis transcriptome contigs, demonstrating the utility and efficacy of our methodology. Leveraging this validated analysis pipeline, we also annotated the assembled R. catesbeiana transcriptome. We used the expression profiles of the annotated genes of the two species to examine the similarities and differences between the tadpole liver transcriptomes. We also compared the gene ontology terms of expressed genes to measure how the animals react to a challenge by thyroid hormone. Our study reports three main conclusions. First, de novo assembly of RNA-seq data is a powerful method for annotating and establishing transcriptomes of non-model organisms. Second, the liver transcriptomes of the two frog species, R. catesbeiana and X. laevis, show many common features, and the distribution of their gene ontology profiles are statistically indistinguishable. Third, although they broadly respond the same way to the presence of thyroid hormone in their environment, their receptor/signal transduction pathways display marked differences. PMID:26121473

  6. De novo Transcriptome Assemblies of Rana (Lithobates) catesbeiana and Xenopus laevis Tadpole Livers for Comparative Genomics without Reference Genomes.

    PubMed

    Birol, Inanc; Behsaz, Bahar; Hammond, S Austin; Kucuk, Erdi; Veldhoen, Nik; Helbing, Caren C

    2015-01-01

    In this work we studied the liver transcriptomes of two frog species, the American bullfrog (Rana (Lithobates) catesbeiana) and the African clawed frog (Xenopus laevis). We used high throughput RNA sequencing (RNA-seq) data to assemble and annotate these transcriptomes, and compared how their baseline expression profiles change when tadpoles of the two species are exposed to thyroid hormone. We generated more than 1.5 billion RNA-seq reads in total for the two species under two conditions as treatment/control pairs. We de novo assembled these reads using Trans-ABySS to reconstruct reference transcriptomes, obtaining over 350,000 and 130,000 putative transcripts for R. catesbeiana and X. laevis, respectively. Using available genomics resources for X. laevis, we annotated over 97% of our X. laevis transcriptome contigs, demonstrating the utility and efficacy of our methodology. Leveraging this validated analysis pipeline, we also annotated the assembled R. catesbeiana transcriptome. We used the expression profiles of the annotated genes of the two species to examine the similarities and differences between the tadpole liver transcriptomes. We also compared the gene ontology terms of expressed genes to measure how the animals react to a challenge by thyroid hormone. Our study reports three main conclusions. First, de novo assembly of RNA-seq data is a powerful method for annotating and establishing transcriptomes of non-model organisms. Second, the liver transcriptomes of the two frog species, R. catesbeiana and X. laevis, show many common features, and the distribution of their gene ontology profiles are statistically indistinguishable. Third, although they broadly respond the same way to the presence of thyroid hormone in their environment, their receptor/signal transduction pathways display marked differences.

  7. Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector.

    PubMed

    Sugiyama, Shin-Ichiro; Miyoshi, Hiroyuki; Yamauchi, Kiyoshi

    2005-12-01

    We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3'-l-triiodothyronine (T(3)) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T(3)-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T(3) that significantly induced the luciferase gene expression was 10(-11)M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10(-6)M, had a weak T(3)-agonist activity, whereas trichlorobisphenol A, at 10(-8) - 10(-6)M had a weak T(3)-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T(3)-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals. PMID:16102758

  8. Fine mapping of the human bone morphogenetic protein-4 gene (BMP4) to chromosome 14q22-q23 by in situ hybridization

    SciTech Connect

    Wijngaard, A. van den; Boersma, C.J.C.; Olijve, W.

    1995-06-10

    Bone morphogenetic protein-4 (BMP-4) is a member of the transforming growth factor-{beta} (TGF-{beta}) superfamily and is involved in morphogenesis and bone cell differentiation. Recombinant BMP-4 can induce ectopic cartilage and bone formation when implanted subcutaneously or intramuscularly in rodents. This ectopic bone formation process resembles the process of bone formation during embryogenesis and fracture healing. A cosmid clone containing the complete human bone morphogenetic protein-4 gene (BMP4) was isolated (details to be published elsewhere) and used as a probe to determine the precise chromosomal localization of the human BMP4 gene. This cosmid clone was labeled with biotin-14-dATP and hybridized in situ to chromosomal preparations of metaphase cells as described previously. In 20 metaphase preparations, an intense and specific fluorescence signal (FITC) was detected on the q arm of chromosome 14. The DAPI-counterstained chromosomes were computer-converted into GTG-like banding patterns, allowing the regional localization of BMP4 within 14q22-q23. 10 refs., 1 fig.

  9. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells

    PubMed Central

    Suzuki, Atsushi; Raya, Ángel; Kawakami, Yasuhiko; Morita, Masanobu; Matsui, Takaaki; Nakashima, Kinichi; Gage, Fred H.; Rodríguez-Esteban, Concepción; Izpisúa Belmonte, Juan Carlos

    2006-01-01

    ES cells represent a valuable model for investigating early embryo development and hold promise for future regenerative medicine strategies. The self-renewal of pluripotent mouse ES cells has been shown to require extrinsic stimulation by the bone morphogenetic protein (BMP) and leukemia inhibitory factor signaling pathways and the expression of the transcription factors Oct4 and Nanog. However, the network of interactions among extrinsic and intrinsic determinants of ES cell pluripotency is currently poorly understood. Here, we show that Nanog expression is up-regulated in mouse ES cells by the binding of T (Brachyury) and STAT3 to an enhancer element in the mouse Nanog gene. We further show that Nanog blocks BMP-induced mesoderm differentiation of ES cells by physically interacting with Smad1 and interfering with the recruitment of coactivators to the active Smad transcriptional complexes. Taken together, our findings illustrate the existence of ES cell-specific regulatory networks that underlie the maintenance of ES cell pluripotency and provide mechanistic insights into the role of Nanog in this process. PMID:16801560

  10. Oocyte expression, secretion and somatic cell interaction of mouse bone morphogenetic protein 15 during the peri-ovulatory period.

    PubMed

    Mester, Brigitta; Ritter, Lesley J; Pitman, Janet L; Bibby, Adrian H; Gilchrist, Robert B; McNatty, Kenneth P; Juengel, Jennifer L; McIntosh, C Joy

    2015-06-01

    Bone morphogenetic protein 15 (BMP15) is a key intraovarian growth factor regulating mammalian fertility, yet expression and localisation of different BMP15 protein forms within ovarian follicles around the time of the preovulatory LH surge remains unclear. Using immunoblotting and immunocytochemistry, the present study identified that post-translationally processed BMP15 proregion and mature proteins are increasingly expressed and localised with cumulus and granulosa cells from mice treated with pregnant mare's serum gonadotropin (PMSG) + human chorionic gonadotrophin (hCG). However, this increased expression was absent in cumulus-oocyte complexes matured in vitro. Pull-down assays further revealed that the recombinant BMP15 proregion is capable of specific interaction with isolated granulosa cells. To verify an oocyte, and not somatic cell, origin of Bmp15 mRNA and coregulated growth differentiation factor 9 (Gdf9), in situ hybridisation and quantitative polymerase chain reaction results confirmed the exclusive oocyte localisation of Bmp15 and Gdf9, regardless of treatment or assay method. Relative oocyte expression levels of Bmp15 and Gdf9 decreased significantly after PMSG + hCG treatment; nevertheless, throughout all treatments, the Bmp15:Gdf9 mRNA expression ratio remained unchanged. Together, these data provide evidence that the preovulatory LH surge leads to upregulation of several forms of BMP15 protein secreted by the oocyte for putative sequestration and/or interaction with ovarian follicular somatic cells.

  11. Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

    PubMed

    Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

    2015-11-01

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells.

  12. Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

    PubMed

    Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

    2015-11-01

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells. PMID:26475222

  13. Estrogen Modulates Bone Morphogenetic Protein-Induced Sclerostin Expression Through the Wnt Signaling Pathway.

    PubMed

    Kim, Ri Youn; Yang, Hoon Joo; Song, Yun Mi; Kim, In Sook; Hwang, Soon Jung

    2015-07-01

    Clinical data show that estrogen levels are inversely associated with the production of sclerostin, a Wnt antagonist that recently attracted great attention over the use of its antibody in the anabolic treatment of osteoporotic conditions. However, the molecular link between sclerostin expression and estrogen signaling is not yet known. We investigated the mechanisms by which estrogen modulates sclerostin (SOST) gene expression at the cellular level in human osteoblast cells in association with bone morphogenetic protein (BMP)2 signaling given that BMP2 is a potential inducer of SOST in human mesenchymal stromal cells (hMSCs). 17β-Estradiol (E2) alone had no effect on SOST expression, which was significantly induced by treatment with BMP2 in hMSCs and osteoblasts derived from the mandibles of female donors. However, E2 suppressed the induction of SOST and other BMP2 target genes by BMP2 in hMSCs. E2 signaling was independent of the Smad pathway, which plays a critical role in SOST induction mediated by BMP2. Instead, E2 increased the transcriptional expression of β-catenin and levels of its activated form. Silencing of the gene encoding estrogen receptor (ER)α decreased E2 activity in β-catenin activation and the suppression of SOST induction by BMP2, but had no influence on BMP2-mediated SOST induction in the same conditions. Similar results were obtained after treatment with ERα antagonist as a Wnt inhibitor. In human osteoblasts, the effect of E2 on SOST expression was either suppressive or absent, depending on the cell donor. Interestingly, the SOST expression pattern after treatment with BMP2 or BMP2/E2 in human osteoblasts showing a pattern of E2 suppression on SOST induction by BMP2 correlated with the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoprotegerin (OPG) expression. These results demonstrate that estrogen signaling in osteoblasts negatively regulates SOST expression in an indirect manner through interaction with

  14. Stimulation of circus movement by activin, bFGF and TGF-beta 2 in isolated animal cap cells of Xenopus laevis.

    PubMed

    Minoura, I; Nakamura, H; Tashiro, K; Shiokawa, K

    1995-01-01

    Lobopodium is a hyaline cytoplasmic protrusion which rotates circumferencially around a cell. This movement is called circus movement, which is seen in dissociated cells of amphibian embryos. Relative abundance of the lobopodia-forming cells changes temporally and spatially within Xenopus embryos, reflecting stage-dependent difference of morphogenetic movements. The lobopodia-forming activity of dissociated animal cap cells was stimulated strongly by activin and bFGF, and weakly by TGF-beta 2. In addition, activin A was found to stimulate cellular attachment to the substratum when the cultivation lasted long. Thus, mesoderm-inducing growth factors stimulate lobopodia formation and cellular movements which may be necessary for gastrulation and neurulation in Xenopus early embryos.

  15. Influence of contrast morphogenetic features of urban constructed soils on the functioning of Moscow green lawn urban ecosystems: analysis based on the field model experiment

    NASA Astrophysics Data System (ADS)

    Epikhina, Anna; Vizirskaya, Mariya; Mazirov, Ilya; Vasenev, Vyacheslav; Vasenev, Ivan; Valentini, Riccardo

    2014-05-01

    Green lawns are the key element of the urban environment. They occupy a considerable part of the city area and locate in different urban functional zones. Urban constructed soils under green lawns have a unique spatial variability in chemical and morphogenetic features. So far, there is lack of information on the influence of morphogenetic features of urban soils on the functioning of the green lawn ecosystems especially in Moscow - the biggest megalopolis in Europe. Urban lawns perform a number of principal functions including both aesthetic and environmental. The role of the green lawn ecosystems in global carbon cycle is one of their main environmental functions. It is traditionally assessed through carbon stocks and fluxes in the basic ecosystem components. So far, such a data for the urban lawn ecosystems of the Moscow megapolis is lacking. In addition to environmental functions, green lawns perform an important ornamental role, which is also a critical criterion of their optimal functioning. Considering the variability of driving factors, influencing green lawns in urban environment, we carry out the model experiment in order to analyze "pure" effect of soil morphogenetic features. The current study aimed to analyze the influence of contrast morphogenetic features of urban constructed soils on the environmental and aesthetic functions of lawn ecosystems in Moscow megapolis basing in the model experiment. We carry out the model experiment located at the experimental field of the Russian State Agrarian University. Special transparent containers developed for the experiment, provided an option to observe soil morphogenetic features dynamics, including the depth and material of the organic transformation. At the same soil body inside the containers was united with the outside environment through the system of holes in the bottom and walls. The set of urban constructed soils includ four contrast types of the top soil (turf (T), turf-sand (TSa), turf-soil (TSo) and

  16. GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

    PubMed Central

    Strittmatter, S M; Cannon, S C; Ross, E M; Higashijima, T; Fishman, M C

    1993-01-01

    The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction. Images Fig. 1 Fig. 2 PMID:7685122

  17. Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts.

    PubMed

    Hara, Yuki; Merten, Christoph A

    2015-06-01

    Nuclear size changes dynamically during development and has long been observed to correlate with the space surrounding the nucleus, as well as with the volume of the cell. Here we combine an in vitro cell-free system of Xenopus laevis egg extract with microfluidic devices to systematically analyze the effect of spatial constraints. The speed of nuclear expansion depended on the available space surrounding the nucleus up to a threshold volume in the nanoliter range, herein referred to as the nuclear domain. Under spatial constraints smaller than this nuclear domain, the size of microtubule-occupied space surrounding the nucleus turned out to be limiting for the accumulation of membranes around the nucleus via the motor protein dynein, therefore determining the speed of nuclear expansion. This mechanism explains how spatial information surrounding the nucleus, such as the positioning of the nucleus inside the cell, can control nuclear expansion.

  18. Planar induction of anteroposterior pattern in the developing central nervous system of Xenopus laevis

    NASA Technical Reports Server (NTRS)

    Doniach, T.; Phillips, C. R.; Gerhart, J. C.

    1992-01-01

    It has long been thought that anteroposterior (A-P) pattern in the vertebrate central nervous system is induced in the embryo's dorsal ectoderm exclusively by signals passing vertically from underlying, patterned dorsal mesoderm. Explants from early gastrulae of the frog Xenopus laevis were prepared in which vertical contact between dorsal ectoderm and mesoderm was prevented but planar contact was maintained. In these, four position-specific neural markers (engrailed-2, Krox-20, XlHbox 1, and XlHbox 6) were expressed in the ectoderm in the same A-P order as in the embryo. Thus, planar signals alone, following a path available in the normal embryo, can induce A-P neural pattern.

  19. Waterborne infectivity of the Ranavirus frog virus 3 in Xenopus laevis.

    PubMed

    Robert, Jacques; George, Erica; De Jesús Andino, Francisco; Chen, Guangchun

    2011-09-01

    Ranaviruses like frog virus 3 (FV3) are responsible for emerging infectious diseases spreading worldwide to fish, amphibian and reptilian species. We have developed, in Xenopus laevis, an experimental model to investigate viral transmission. We show that FV3 released in water by immunocompromised infected adults can infect adult and larval stages of Xenopus within 3h of exposure. Time course of virus load and viral transcription in different tissues suggests that early waterborne FV3 infection through the digestive tract leads to dissemination in the kidney. Finally, a fraction of adult macrophages becomes infected following exposure to waterborne FV3 as visualized by fluorescence microscopy using macrophage- and FV3-specific antibodies. Little cytopathicity and apoptosis were detected in infected macrophages, which is consistent with our proposition that macrophages are permissive to FV3. These data highlight the efficiency of FV3 infectivity by the water route and the ability of FV3 to adapt to its hosts.

  20. Purification and Fluorescent Labeling of Tubulin from Xenopus laevis Egg Extracts.

    PubMed

    Groen, Aaron C; Mitchison, Timothy J

    2016-01-01

    For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog egg tubulin incorporates into meiotic spindles assembled in egg extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus egg extract experiments. Frog egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro. PMID:27193841

  1. Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

    PubMed

    Rakshit, Tatini; Senapati, Subhadip; Sinha, Satyabrata; Whited, A M; Park, Paul S-H

    2015-01-01

    Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates. PMID:26492040

  2. A kinetic model of the cyclin E/Cdk2 developmental timer in Xenopus laevis embryos.

    PubMed

    Ciliberto, Andrea; Petrus, Matthew J; Tyson, John J; Sible, Jill C

    2003-07-01

    Early cell cycles of Xenopus laevis embryos are characterized by rapid oscillations in the activity of two cyclin-dependent kinases. Cdk1 activity peaks at mitosis, driven by periodic degradation of cyclins A and B. In contrast, Cdk2 activity oscillates twice per cell cycle, despite a constant level of its partner, cyclin E. Cyclin E degrades at a fixed time after fertilization, normally corresponding to the midblastula transition. Based on published data and new experiments, we constructed a mathematical model in which: (1) oscillations in Cdk2 activity depend upon changes in phosphorylation, (2) Cdk2 participates in a negative feedback loop with the inhibitory kinase Wee1; (3) cyclin E is cooperatively removed from the oscillatory system; and (4) removed cyclin E is degraded by a pathway activated by cyclin E/Cdk2 itself. The model's predictions about embryos injected with Xic1, a stoichiometric inhibitor of cyclin E/Cdk2, were experimentally validated. PMID:12914904

  3. Subchronic and chronic developmental effects of copper oxide (CuO) nanoparticles on Xenopus laevis.

    PubMed

    Nations, Shawna; Long, Monique; Wages, Mike; Maul, Jonathan D; Theodorakis, Christopher W; Cobb, George P

    2015-09-01

    Metal oxide nanoparticles, such as copper oxide (CuO), are mass produced for use in a variety of products like coatings and ceramics. Acute exposure to CuO nanoparticles has caused toxicity to many aquatic organisms, yet there is no information on the effect of prolonged CuO nanomaterial exposures. This study examined effects of chronic exposure to CuO nanoparticles on Xenopus laevis growth and development. Experiments included a 14 d subchronic exposure and a 47 d chronic exposure throughout metamorphosis. The subchronic exposure caused mortality in all tested CuO concentrations, and significant growth effects occurred after exposure to 2.5 mg L(-1) CuO. Chronic exposure to 0.3 mg L(-1) CuO elicited significant mortality and affected the rate of metamorphosis. Exposure to lower concentrations of CuO stimulated metamorphosis and growth, indicating that low dose exposure can have hormetic effects. PMID:25950410

  4. D-Amino acid oxidase and presence of D-proline in Xenopus laevis.

    PubMed

    Soma, Hiroki; Furuya, Ryuji; Kaneko, Ryo; Tsukamoto, Ayaka; Shirasu, Kazumitsu; Tanigawa, Minoru; Nagata, Yoko

    2013-10-01

    We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

  5. Optogenetic Control of Apoptosis in Targeted Tissues of Xenopus laevis Embryos

    PubMed Central

    Jewhurst, Kyle; Levin, Michael; McLaughlin, Kelly A

    2014-01-01

    KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering apoptosis in a KR-expressing cell. This property allows for the use of KR as a means of killing cells in an organism with great temporal and spatial specificity, while minimizing the nonspecific effects that can result from mechanical or chemical exposure damage techniques. Such optogenetic control of cell death, and the resulting ability to induce the targeted death of specific tissues, is invaluable for regeneration/repair studies—particularly in Xenopus laevis, where apoptosis plays a key role in regeneration and repair. We here describe a method by which membrane-bound KR, introduced to Xenopus embryos by mRNA microinjection, can be activated with green light to induce apoptosis in specific organs and tissues, with a focus on the developing eye and pronephric kidney. PMID:25374461

  6. Light conditions affect the roll-induced vestibuloocular reflex in Xenopus laevis tadpoles

    NASA Astrophysics Data System (ADS)

    El-Yamany, Nabil A.

    2008-12-01

    In Xenopus laevis tadpoles, effects of asymmetrical light conditions on the roll-induced vestibuloocular reflex (rVOR) were tested for the developmental period between stage 47 and 49. For comparison, the rVOR was tested in dim- and high-symmetrical light environments. Test parameters were the rVOR gain and rVOR amplitude. Under all light conditions, the rVOR increased from tadpole stage 47 to 49. For all stages, the asymmetrical light field induced the strongest response, the dim light field the weakest one. The response for the left and right eye was identical, even if the tadpoles were tested under asymmetrical light conditions. The experiments can be considered as hints (1) for an age-dependent light sensitivity of vestibular neurons, and (2) for the existence of control systems for coordinated eye movements that has its origin in the proprioceptors of the extraocular eye muscles.

  7. The morphology and attachment of Protopolystoma xenopodis (Monogenea: Polystomatidae) infecting the African clawed frog Xenopus laevis

    PubMed Central

    Theunissen, Maxine; Tiedt, Louwrens; Du Preez, Louis H.

    2014-01-01

    The African clawed frog Xenopus laevis (Anura: Pipidae) is host to more than 25 parasite genera encompassing most of the parasitic invertebrate groups. Protopolystoma xenopodis Price, 1943 (Monogenea: Polystomatidae) is one of two monogeneans infecting X. laevis. This study focussed on the external morphology of different developmental stages using scanning electron microscopy, histology and light microscopy. Eggs are released continuously and are washed out when the frog urinates. After successful development, an active swimming oncomiracidium leaves the egg capsule and locates a potential post-metamorphic clawed frog. The oncomiracidium migrates to the kidney where it attaches and starts to feed on blood. The parasite then migrates to the urinary bladder where it reaches maturity. Eggs are fusiform, about 300 μm long, with a smooth surface and are operculated. Oncomiracidia are elongated and cylindrical in shape, with an oval posterior cup-shaped haptor that bears a total of 20 sclerites; 16 marginal hooklets used for attachment to the kidney of the host and two pairs of hamulus primordia. Cilia from the 64 ciliated cells enable the oncomiracidium to swim for up to 24 h when the cilia subsequently curl up, become non-functional and are shed from the body. The tegument between the ciliated cells bears a series of sensory papillae. The body of the mature parasite is elongated and pyriform and possesses an opisthaptor armed with three pairs of suckers and two pairs of falciform hooks to ensure a firm grip on the flexible internal surface of the urinary bladder. PMID:24823278

  8. Teratogenicity of Ni2+ in Xenopus laevis, assayed by the FETAX procedure.

    PubMed

    Hopfer, S M; Plowman, M C; Sweeney, K R; Bantle, J A; Sunderman, F W

    1991-06-01

    The teratogenicity of Ni2+ was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In seven assays, beginning at 5 h postfertilization, groups of Xenopus embryos were incubated for 96 h in media that contained Ni2+ (added as NiCl2) at concentrations ranging from 1 x 10(-7) to 3 x 10(-3) mol/L; control groups were incubated in the same medium without added NiCl2. At 101 h postfertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to determine their developmental stages, malformations, and head-to-tail lengths. In control embryos, survival was greater than or equal to 95% and malformations were less than or equal to 7%. Malformations were found in greater than 95% of embryos exposed to Ni2+ concentrations greater than or equal to 5.6 mumol/L. The most frequent malformations in Ni(2+)-exposed embryos were ocular, skeletal, and intestinal deformities; less common malformations included facial, cardiac, and integumentary deformities. Other abnormalities, not categorized as malformations, included stunted growth, dermal hypopigmentation, and coelomic effusions or hemorrhages. The median embryolethal concentration (LC50) of Ni2+ was 365 (SE +/- 9) mumol/L; the median teratogenic concentration (EC50) was 2.5 (SE +/- 0.1) mumol/L; the Teratogenic Index (TI = LC50/EC50) was 147 (SE +/- 5), indicating that Ni2+ is a potent teratogen for Xenopus laevis. Experiments in which Ni(2+)-exposures were limited to specific 24 h periods showed that Xenopus embryos were most susceptible to Ni(2+)-induced malformations on the second and third days of life, during the most active period of organogenesis.

  9. Exposure to butachlor causes thyroid endocrine disruption and promotion of metamorphosis in Xenopus laevis.

    PubMed

    Li, Shuying; Li, Meng; Wang, Qiangwei; Gui, Wenjun; Zhu, Guonian

    2016-06-01

    Butachlor is extensively applied in rice paddy ecosystem in china, and has been widespread contaminant in the aquatic environment. Here, Xenopus laevis was used for the evaluation of teratogenesis developmental toxicity, and disruption of thyroid system when exposure to different concentrations of butachlor by window phase exposure. Acute toxicity investigation shown that 96 h-LC50 value of butachlor was 1.424 mg L(-1) and 0.962 mg L(-1) for tadpoles (starting from stages 46/47) and embryos (starting from stages 8/9), respectively. Exposure to butachlor caused malformation, including abnormal eye, pericardial edema, enlarged proctodaeum and bent tail. Window phase exposure test indicated that butachlor significantly promote the contents of whole-body thyroid hormones (THs, T3 and T4) at higher levels, indicating thyroid endocrine disruption. At 7 days, exposure to butachlor up-regulated the mRNA expression of genes involved in THs synthesis and metabolism (tshα, tg, tpo and dio1) and THs receptors (trα and trβ). At 14 days, up-regulation of the mRNA expression of genes related to THs synthesis and metabolism (tshα, tshβ, tg, tpo, dio1, dio2 and ttr) and THs receptors (trβ) were also observed after the exposure to butachlor. At 21 days, butachlor up-regulated the mRNA expression of tshα, tg, tpo genes and down-regulated the mRNA expression of tshβ, tg, dio1, ttr and trα genes. These results showed that butachlor could change the mRNA expression of genes involved in the HPT axis and increase whole-body thyroid hormones levels of X. laevis tadpoles in a dose- and time-dependent manner, causing thyroid endocrine disruption and developmental toxicity.

  10. Effects of Transgenic cry1Ca Rice on the Development of Xenopus laevis

    PubMed Central

    Zhu, Haojun; Li, Yunhe; Ding, Jiatong; Peng, Yufa

    2015-01-01

    In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 μg L–1 or 10 μg L–1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development. PMID:26695426

  11. Heterogeneous nuclear ribonucleoprotein complexes from Xenopus laevis oocytes and somatic cells.

    PubMed

    Marcu, A; Bassit, B; Perez, R; Piñol-Roma, S

    2001-09-01

    HnRNP proteins have been implicated in most stages of cellular mRNA metabolism, including processing, nucleocytoplasmic transport, stability, and localization. Several hnRNP proteins are also known to participate in key early developmental decisions. In order to facilitate functional studies of these pre-mRNA- and mRNA-binding proteins in a vertebrate organism amenable to developmental studies and experimental manipulation, we identified and purified the major hnRNP proteins and isolated the hnRNP complex from Xenopus laevis oocytes and somatic cells. Using affinity chromatography and immunological methods, we isolated a family of >15 abundant single-stranded nucleic acid-binding proteins, which range in apparent molecular weight from approximately 20 kDa to >150 kDa, and with isoelectric points from <5 to >8. Monoclonal antibodies revealed that a subset of these proteins are major hnRNP proteins in both oocytes and somatic cells in culture, and include proteins related to human hnRNP A2/B1/B2 and hnRNP K. UV crosslinking in living cells demonstrated that these proteins bind poly(A)+ RNA in vivo. Immunopurification using a monoclonal antibodyto X. aevishnRNPA2 resulted in the isolation of RNP complexes that contain a specific subset of single-stranded nucleic acid-binding proteins. The protein composition of complexes isolated from somatic cells and from oocyte germinal vesicles was similar, suggesting that the overall properties and functions of hnRNP proteins in these two cell types are comparable. These findings, together with the novel probes generated here, will also facilitate studies of the function of vertebrate RNA-binding proteins using the well characterized X. laevis oocyte and early embryo as experimental systems. PMID:11669376

  12. A role for biliverdin IXalpha in dorsal axis development of Xenopus laevis embryos.

    PubMed

    Falchuk, Kenneth H; Contin, Jennifer M; Dziedzic, T Scott; Feng, Zhongling; French, Thayer C; Heffron, Gregory J; Montorzi, Marcelo

    2002-01-01

    The determinants of Xenopus laevis embryos that act before their first cell division are mandatory for the formation of mRNas required to establish the dorsal axis. Although their chemical identities are unknown, a number of their properties have long been recognized. One of the determinants is present in the cytoplasm and is sensitive to UV light. Thus, exposing stage 1 embryos to either standard 254-nm or, as shown here, to 366-nm UV light during the 0.3-0.4 time fraction of their first cycle inactivates the cytoplasmic determinant. As a consequence, both types of irradiated embryos fail to express dorsal markers, e.g., goosecoid and chordin, without affecting formation of ventral markers, e.g., Vent-1. The developmental outcome is dorsal axis-deficient morphology. We report here that biliverdin IXalpha, a normal constituent of cytoplasmic yolk platelets, is photo-transformed by irradiation with either 254- or 366-nm UV light and that the transformation triggers the dorsal axis deficiency. When the 254- or 366-nm UV-irradiated embryos, fated to dorsal axis deficiency, are incubated solely with microM amounts of biliverdin, they recover and form the axis. In contrast, incubation with either in vitro photo-transformed biliverdin or biliverdin IXalpha dimethyl ester does not induce recovery. The results define an approach to produce dorsal axis-deficient embryos by photo-transforming its biliverdin by irradiation with 366-nm UV light and identify an unsuspected role for biliverdin IXalpha in X. laevis embryogenesis. PMID:11782548

  13. Effects of 17 beta-estradiol exposure on Xenopus laevis gonadal histopathology.

    PubMed

    Wolf, Jeffrey C; Lutz, Ilka; Kloas, Werner; Springer, Timothy A; Holden, Larry R; Krueger, Henry O; Hosmer, Alan J

    2010-05-01

    The natural estrogen 17 beta-estradiol (E2) is a potential environmental contaminant commonly employed as a positive control substance in bioassays involving estrogenic effects. The aquatic anuran Xenopus laevis is a frequent subject of reproductive endocrine disruptor research; however, histopathological investigations have tended to be less than comprehensive. Consequently, a study was designed to characterize gross and microscopic changes in the gonads of X. laevis as a result of E2 exposure. Additional goals of this study, which consisted of three separate experiments, included the standardization of diagnostic terminology and criteria, the validation of statistical methodology, and the establishment of a half maximal effective concentration (EC50) for E2 as defined by an approximately 50% conversion of presumptive genotypic males to phenotypic females. In the first experiment, frogs were exposed to nominal concentrations of 0, 0.2, 1.5, or 6.0 microg/L E2. From these experimental results and those of a subsequent range finding trial, the EC50 for E2 was determined to be approximately 0.2 microg/L. This E2 concentration was utilized in the other two experiments, which were performed at different facilities to confirm the reproducibility of results. Experiments were conducted according to Good Laboratory Practice guidelines, and the histopathologic evaluations were peer reviewed by an independent pathologist. Among the three trials, the histopathological findings that were strongly associated with E2-exposure (p<0.001 to 0.0001) included an increase in the proportion of phenotypic females, mixed sex, dilated testis tubules, dividing gonocytes in the testis, and dilated ovarian cavities in phenotypic ovaries. A comparison of the gross and microscopic evaluations suggested that some morphologic changes in the gonads may potentially be missed if studies rely entirely on macroscopic assessment.

  14. Functional and Structural Effects of Amyloid-β Aggregate on Xenopus laevis Oocytes

    PubMed Central

    Parodi, Jorge; la Paz, Lenin Ochoa-de; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2012-01-01

    Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of “spontaneous” blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca2+ was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca2+-dependent Cl− currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (Tout) and the serum-activated, oscillatory Cl− currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca2+-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication. PMID:23104436

  15. Identification of a novel dehydration responsive gene, drp10, from the African clawed frog, Xenopus laevis.

    PubMed

    Biggar, Kyle K; Biggar, Yulia; Storey, Kenneth B

    2015-07-01

    During periods of environmental stress a number of different anuran species employ adaptive strategies to promote survival. Our study found that in response to dehydration (i.e., loss of total body water content), the African clawed frog (Xenopus laevis) increased the expression of a novel gene (drp10) that encodes a structural homolog of the freeze-responsive FR10 protein found in wood frogs. Similar to FR10, the DRP10 protein was found to also contain a highly conserved N-terminal cleavable signal peptide. Furthermore, DRP10 was found to have high structural homology to the available crystal structures of type A and E apolipoproteins in Homo sapiens, and a type IV LS-12 anti-freeze protein in the longhorn sculpin, Myoxocephalus octodecemspinosis. In response to dehydration, the transcript expression of drp10 was found to increase 1.52 ± 0.16-fold and 1.97 ± 0.11-fold in response to medium (15%) and high (30%) dehydration stresses in the liver tissue of X. laevis, respectively, while drp10 expression increased 2.12 ± 0.12-fold and 1.46 ± 0.16-fold in kidney tissue. Although the molecular function of both dehydration-responsive DRP10 and the freeze-responsive FR10 have just begun to be elucidated, it is likely that both are frog-specific proteins that likely share a similar purpose during water-related stresses. PMID:25866033

  16. Effects of Transgenic cry1Ca Rice on the Development of Xenopus laevis.

    PubMed

    Chen, Xiuping; Wang, Jiamei; Zhu, Haojun; Li, Yunhe; Ding, Jiatong; Peng, Yufa

    2015-01-01

    In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 μg L-1 or 10 μg L-1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development. PMID:26695426

  17. Evaluation of Presurgical Skin Preparation Agents in African Clawed Frogs (Xenopus laevis)

    PubMed Central

    Philips, Blythe H; Crim, Marcus J; Hankenson, F Claire; Steffen, Earl K; Klein, Peter S; Brice, Angela K; Carty, Anthony J

    2015-01-01

    Despite the routine collection of oocytes from African clawed frogs (Xenopus laevis) for use in research, few studies have evaluated methods for preparing their skin for surgery. We evaluated 3 skin preparatory agents by examining their antibacterial efficacy and the gross and microscopic appearance of Xenopus skin after exposure. Frogs (n = 14) were sedated and treated (contact time, 10 min) with 0.9% sterile NaCl on one-half of the ventrum and with 0.5% povidone–iodine or 0.75% chlorhexidine on the other half. Bacterial cultures were obtained before and after skin treatment; bacteria were identified by mass spectrometry. To assess inflammation and degenerative changes, the incision sites were photographed and biopsied at 0, 1, and 7 d after surgery. We isolated at least 22 genera of bacteria from the skin of our frog population (mean ± SE, 5.21 ± 0.82 genera per frog). Iodine (2.00 ± 0.44 genera) and chlorhexidine (0.29 ± 0.76 genera) both had greater antimicrobial activity than did saline. Skin erythema did not correlate with treatment group. Histologic evidence of epidermal degeneration and necrosis was greater on days 1 and 7 after chlorhexidine treatment than after iodine or saline. In addition, frogs treated with chlorhexidine had a higher incidence of clinical illness associated with the exposure site. In summary, although chlorhexidine has adequate antimicrobial activity against organisms on X. laevis skin, it leads to skin damage and subsequent clinical complications. We therefore do not recommend chlorhexidine as a preoperative preparation agent in Xenopus. PMID:26632790

  18. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes

    PubMed Central

    Mulsant, Philippe; Lecerf, Frédéric; Fabre, Stéphane; Schibler, Laurent; Monget, Philippe; Lanneluc, Isabelle; Pisselet, Claudine; Riquet, Juliette; Monniaux, Danielle; Callebaut, Isabelle; Cribiu, Edmond; Thimonier, Jacques; Teyssier, Jacques; Bodin, Loys; Cognié, Yves; Chitour, Nour; Elsen, Jean-Michel

    2001-01-01

    Ewes from the Booroola strain of Australian Mérino sheep are characterized by high ovulation rate and litter size. This phenotype is due to the action of the FecBB allele of a major gene named FecB, as determined by statistical analysis of phenotypic data. By genetic analysis of 31 informative half-sib families from heterozygous sires, we showed that the FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22–23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the transforming growth factor-β (TGF-β) receptor family. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was found to be associated fully with the hyperprolificacy phenotype of Booroola ewes. In vitro, ovarian granulosa cells from FecBB/FecBB ewes were less responsive than granulosa cells from FecB+/FecB+ ewes to the inhibitory effect on steroidogenesis of GDF-5 and BMP-4, natural ligands of BMPR-IB. It is suggested that in FecBB/FecBB ewes, BMPR-IB would be inactivated partially, leading to an advanced differentiation of granulosa cells and an advanced maturation of ovulatory follicles. PMID:11320249

  19. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    SciTech Connect

    Jiang Fangxu . E-mail: jiang@wehi.edu.au; Harrison, Leonard C.

    2005-08-01

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its {alpha}{sub 6} integrin and {alpha}-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells.

  20. Molecular and cellular mechanisms of bone morphogenetic proteins and activins in the skin: potential benefits for wound healing.

    PubMed

    Moura, J; da Silva, L; Cruz, M T; Carvalho, E

    2013-09-01

    Bone morphogenetic proteins (BMPs) and activins are phylogenetically conserved proteins, belonging to the transforming growth factor-β superfamily, that signal through the phosphorylation of receptor-regulated Smad proteins, activating different cell responses. They are involved in various steps of skin morphogenesis and wound repair, as can be evidenced by the fact that their expression is increased in skin injuries. BMPs play not only a role in bone regeneration but are also involved in cartilage, tendon-like tissue and epithelial regeneration, maintain vascular integrity, capillary sprouting, proliferation/migration of endothelial cells and angiogenesis, promote neuron and dendrite formation, alter neuropeptide levels and are involved in immune response modulation, at least in animal models. On the other hand, activins are involved in wound repair through the regulation of skin and immune cell migration and differentiation, re-epithelialization and granulation tissue formation, and also promote the expression of collagens by fibroblasts and modulate scar formation. This review aims at enunciating the effects of BMPs and activins in the skin, namely in skin development, as well as in crucial phases of skin wound healing, such as inflammation, angiogenesis and repair, and will focus on the effects of these proteins on skin cells and their signaling pathways, exploring the potential therapeutic approach of the application of BMP-2, BMP-6 and activin A in chronic wounds, particularly diabetic foot ulcerations.

  1. Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

    PubMed Central

    Chen, Fenfang; Lin, Xia; Xu, Pinglong; Zhang, Zhengmao; Chen, Yanzhen; Wang, Chao; Han, Jiahuai; Zhao, Bin; Xiao, Mu

    2015-01-01

    Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation. PMID:25755279

  2. Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion.

    PubMed

    Chiang, Chih-Wei; Chen, Wei-Chuan; Liu, Hsia-Wei; Wang, I-Chun; Chen, Chih-Hwa

    2015-01-01

    Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated and then characterized by flow cytometry, morphological observation, and immunofluorescence staining. The LF cells were also cultivated in polyethylene (glycol) diacrylate (PEGDA) hydrogels that incorporated bone morphogenetic protein-2 (BMP-2) growth factor, to evaluate their proliferation and secretion of ECM and differentiation in vitro. The experimental results thus obtained that the proliferation, ECM secretion, and differentiation of the PEGDA-BMP-2 group exceeded those of the PEGDA group during the period of cultivation. The mineralization and histological staining results differed similarly. A nude mice model was utilized to prove that LF cells on hydrogels could undergo osteogenic differentiation in vivo. These experimental results also revealed that the PEGDA-BMP-2 group had better osteogenic effects than the PEGDA group following a 12 weeks after transplantation. According to all of these experimental results, LF cells are a source of cells for spinal fusion and PEGDA-BMP-2 hydrogel is a candidate biomaterial for spinal fusion by tissue engineering. PMID:26426006

  3. Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion

    PubMed Central

    Chiang, Chih-Wei; Chen, Wei-Chuan; Liu, Hsia-Wei; Wang, I-Chun; Chen, Chih-Hwa

    2015-01-01

    Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated and then characterized by flow cytometry, morphological observation, and immunofluorescence staining. The LF cells were also cultivated in polyethylene (glycol) diacrylate (PEGDA) hydrogels that incorporated bone morphogenetic protein-2 (BMP-2) growth factor, to evaluate their proliferation and secretion of ECM and differentiation in vitro. The experimental results thus obtained that the proliferation, ECM secretion, and differentiation of the PEGDA-BMP-2 group exceeded those of the PEGDA group during the period of cultivation. The mineralization and histological staining results differed similarly. A nude mice model was utilized to prove that LF cells on hydrogels could undergo osteogenic differentiation in vivo. These experimental results also revealed that the PEGDA-BMP-2 group had better osteogenic effects than the PEGDA group following a 12 weeks after transplantation. According to all of these experimental results, LF cells are a source of cells for spinal fusion and PEGDA-BMP-2 hydrogel is a candidate biomaterial for spinal fusion by tissue engineering. PMID:26426006

  4. Pomphorhynchus laevis (Acanthocephala) from the Sava River basin: New insights into strain formation, mtDNA-like sequences and dynamics of infection.

    PubMed

    Vardić Smrzlić, Irena; Valić, Damir; Kapetanović, Damir; Filipović Marijić, Vlatka; Gjurčević, Emil; Teskeredžić, Emin

    2015-10-01

    Here we report the genetic variability and presence of mtDNA-like sequences of Pomphorhynchus laevis from the chub, Squalius cephalus, caught at the sampling sites along the Sava River and its tributary the Sutla River in Croatia. Sequences of the cytochrome c oxidase subunit 1 (COI) gene of the recovered P. laevis specimens were used for haplotype network construction and phylogenetic analysis. These analyses showed that some specimens contained mitochondrial-like sequences, and they uncovered the existence of a Sava River basin strain different from known strains of P. laevis. This is the first time that P. laevis has been shown to contain mtDNA-like sequences, suggesting the need to exercise caution during COI analyses of P. laevis using universal primers. Highly conserved sequences of two nuclear markers, the ITS region and 18S rRNA, were not helpful for understanding genetic variability or differentiating strains. Furthermore, analysis of the dynamics of P. laevis infections in S. cephalus from the Sava and Sutla Rivers showed decreased prevalence and abundance at sites with inferior water quality, positive association of parasite abundance with fish size, and no clear association of parasite abundance with fish condition index or sex. PMID:25728305

  5. Pomphorhynchus laevis (Acanthocephala) from the Sava River basin: New insights into strain formation, mtDNA-like sequences and dynamics of infection.

    PubMed

    Vardić Smrzlić, Irena; Valić, Damir; Kapetanović, Damir; Filipović Marijić, Vlatka; Gjurčević, Emil; Teskeredžić, Emin

    2015-10-01

    Here we report the genetic variability and presence of mtDNA-like sequences of Pomphorhynchus laevis from the chub, Squalius cephalus, caught at the sampling sites along the Sava River and its tributary the Sutla River in Croatia. Sequences of the cytochrome c oxidase subunit 1 (COI) gene of the recovered P. laevis specimens were used for haplotype network construction and phylogenetic analysis. These analyses showed that some specimens contained mitochondrial-like sequences, and they uncovered the existence of a Sava River basin strain different from known strains of P. laevis. This is the first time that P. laevis has been shown to contain mtDNA-like sequences, suggesting the need to exercise caution during COI analyses of P. laevis using universal primers. Highly conserved sequences of two nuclear markers, the ITS region and 18S rRNA, were not helpful for understanding genetic variability or differentiating strains. Furthermore, analysis of the dynamics of P. laevis infections in S. cephalus from the Sava and Sutla Rivers showed decreased prevalence and abundance at sites with inferior water quality, positive association of parasite abundance with fish size, and no clear association of parasite abundance with fish condition index or sex.

  6. Sequence variation in transcription factor IIIA.

    PubMed Central

    Gaskins, C J; Hanas, J S

    1990-01-01

    Previous studies characterized macromolecular differences between Xenopus and Rana transcription factor IIIA (TFIIIA) (Gaskins et al., 1989, Nucl. Acids Res. 17, 781-794). In the present study, cDNAs for TFIIIA from Xenopus borealis and Rana catesbeiana (American bullfrog) were cloned and sequenced in order to gain molecular insight into the structure, function, and species variation of TFIIIA and the TFIIIA-type zinc finger. X. borealis and R. catesbeiana TFIIIAs have 339 and 335 amino acids respectively, 5 and 9 fewer than X. laevis TFIIIA. X. borealis TFIIIA exhibited 84% sequence homology (55 amino acid differences) with X. laevis TFIIIA and R. catesbeiana TFIIIA exhibited 63% homology (128 amino acid changes) with X. laevis TFIIIA. This sequence variation is not random; the C-terminal halves of these TFIIIAs contain substantially more non-conservative changes than the N-terminal halves. In particular, the N-terminal region of TFIIIA (that region forming strong DNA contacts) is the most conserved and the C-terminal tail (that region involved in transcription promotion) the most divergent. Hydropathy analyses of these sequences revealed zinc finger periodicity in the N-terminal halves, extreme hydrophilicity in the C-terminal halves, and a different C-terminal tail hydropathy for R. catesbeiana TFIIIA. Although considerable sequence variation exists in these TFIIIA zinc fingers, the Cys/His, Tyr/Phe and Leu residues are strictly conserved between X. laevis and X. borealis. Strict conservation of only the Cys/His motif is observed between X. laevis and R. catesbeiana TFIIIA. Overall, Cys/His zinc fingers in TFIIIA are much less conserved than Cys/Cys fingers in erythroid transcription factor (Eryf 1) and also less conserved than homeo box domains in segmentation genes. The collective evidence indicates that TFIIIA evolved from a common precursor containing up to 12 finger domains which subsequently evolved at different rates. Images PMID:2110661

  7. LaeA, a Regulator of Morphogenetic Fungal Virulence Factors†

    PubMed Central

    Bok, Jin Woo; Balajee, S. Arunmozhi; Marr, Kieren A.; Andes, David; Nielsen, Kristian Fog; Frisvad, Jens C.; Keller, Nancy P.

    2005-01-01

    Opportunistic animal and plant pathogens, well represented by the genus Aspergillus, have evolved unique mechanisms to adapt to and avoid host defenses. Aspergillus fumigatus, an increasingly serious pathogen owing to expanding numbers of immunocompromised patients, causes the majority of human infections; however, an inability to identify bona fide virulence factors has impeded therapeutic advances. We show that an A. fumigatus mutation in a developmentally expressed transcriptional regulator (ΔlaeA) coordinating morphological and chemical differentiation reduces virulence in a murine model; impaired virulence is associated with decreased levels of pulmonary gliotoxin and multiple changes in conidial and hyphal susceptibility to host phagocytes ex vivo. LaeA, a conserved protein in filamentous fungi, is a developmental regulator of virulence genes and, possibly, the first antimicrobial target specific to filamentous fungi that are pathogenic to plants and animals. PMID:16151250

  8. The amphibians Xenopus laevis and Silurana tropicalis possess a family of activating KIR-related Immunoglobulin-like receptors

    PubMed Central

    Guselnikov, Sergey V; Reshetnikova, Evdokiya S; Najakshin, Alexander M; Mechetina, Ludmila V; Robert, Jacques; Taranin, Alexander V

    2009-01-01

    In this study, we searched the amphibian species Xenopus laevis and Silurana (Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3–4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcR subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory. PMID:19896971

  9. A preliminary investigation into the effect of thyroid hormones on the metamorphic changes in Meckel's cartilage in Xenopus laevis.

    PubMed Central

    Thomson, D A

    1989-01-01

    The effects of short-term dosage with thyroid powder on the metamorphic changes in Meckel's cartilage in Xenopus laevis are described. The Lag phase of development appears to be by-passed, and the usual sequential arrangement of the Division and Synthesis phases is disrupted, the processes of normal metamorphic changes being considerably accelerated. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:2808112

  10. The Role of TGF-β2 and Bone Morphogenetic Proteins in the Trabecular Meshwork and Glaucoma

    PubMed Central

    Sharma, Tasneem; Clark, Abbot F.

    2014-01-01

    Abstract Primary open-angle glaucoma (POAG) is the second leading cause of blindness worldwide. Elevated intraocular pressure (IOP) is a primary risk factor associated with POAG. Increased aqueous humor (AH) outflow resistance through the trabecular meshwork (TM) results in elevated IOP in POAG patients. Resistance to AH outflow is associated with increased accumulation of extracellular matrix (ECM) proteins in the TM. In addition, levels of transforming growth factor-beta2 (TGF-β2) are elevated in the AH and TM tissue of POAG patients. Elevated levels of TGF-β2 in other tissues have been associated with fibrosis and increased tissue stiffness. However, locally produced effectors that maintain homeostatic relationships must also be present. Bone morphogenetic proteins (BMPs) serve this purpose in the TM as they inhibit TGF-β2-induced ECM changes in TM cells. This review article first describes the TGF-β superfamily of growth factors including BMPs and their canonical and noncanonical signaling pathways. The article then addresses the role of TGF-β2 in the pathophysiology of POAG as related to the ECM and ECM crosslinking enzymes. This is followed by a discussion of potential homeostatic control mechanisms of TGF-β2 signaling in the TM including the inhibitory role of BMP-4 and BMP-7. We then describe the relationship of TGF-β2 and BMPs in TM fibrosis including the role of antagonists. Lastly, in future directions, we identify potential future studies that explore new and unique cellular interactions within the TM for potential therapeutic interventions. PMID:24517218

  11. Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals.

    PubMed

    Nishimori, Hikaru; Ehata, Shogo; Suzuki, Hiroshi I; Katsuno, Yoko; Miyazono, Kohei

    2012-06-01

    Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.

  12. Expression of bone morphogenetic proteins and receptors in porcine cumulus-oocyte complexes during in vitro maturation.

    PubMed

    Zhu, Guiyu; Guo, Bingran; Pan, Dengke; Mu, Yulian; Feng, Shutang

    2008-03-01

    In vitro oocyte growth is the essential technology which enables oocytes to achieve maturation and acquire the competence for subsequent manipulation. There is increasing evidence that members of the transforming growth factor-beta (TGF-beta) superfamily are expressed in a variety of cell types within the ovary in a developmental stage-related manner and function as crucial factors in oocyte growth and follicular development. However, the expression of TGF-beta family members has been studied extensively in follicular compartment cells in the ovaries while poorly explored in the cumulus-oocytes complex (COC) within culture systems. Using semi-quantitative RT-PCR, we investigated the temporal and spatial expression patterns of several bone morphogenetic proteins (BMP-4, BMP-6, BMP-15 and GDF-9), as well as BMP receptors (BMPRIA, BMPRIB, BMPRII and ActRII), in porcine COCs throughout in vitro maturation (IVM). In oocytes, the transcription of BMP-6, BMP-15, GDF-9 and BMPRII were down-regulated, while BMP-4, BMPRIA and BMPRIB remained unchanged during IVM. In cumulus cells, BMP-4 mRNA expression increased significantly, while BMP-6 and ActRII was down-regulated during IVM. Nevertheless, mRNAs of BMPRIA, BMPRIB and BMPRII were constantly expressed in cumulus cells in the process. However, BMP-15 was absent in cumulus cells and ActRII was not detected in oocytes. In addition, hardly any transcription of BMP-2, BMP-5, BMP-7, ActRIA was found in porcine COCs throughout IVM. These data demonstrate a complex BMP-signaling system for member gene expression within porcine COCs during IVM and indicate the need for further functional characterization of these factors during oocyte maturation.

  13. Immune defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, in the South African clawed frog, Xenopus laevis.

    PubMed

    Ramsey, Jeremy P; Reinert, Laura K; Harper, Laura K; Woodhams, Douglas C; Rollins-Smith, Louise A

    2010-09-01

    Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.

  14. Root iron uptake efficiency of Ulmus laevis and U. minor and their distribution in soils of the Iberian Peninsula.

    PubMed

    Venturas, Martin; Fernández, Victoria; Nadal, Paloma; Guzmán, Paula; Lucena, Juan J; Gil, Luis

    2014-01-01

    The calcifuge and calcicole character of wild plants has been related to nutrient availability shortages, including iron (Fe)-deficiency. Surprisingly, just a few studies examined the relation between root Fe uptake and plant distribution in different soil types. We assessed the root Fe acquisition efficiency of two Ulmus species with calcareous (Ulmus minor) and siliceous (U. laevis) soil distribution patterns in the Iberian Peninsula. Seedlings of both elm species were grown hydroponically with different Fe concentrations during 6 weeks. Plant physiological responses to Fe-limiting conditions were evaluated as were the ferric reductase activity and proton (H(+)) extrusion capacity of the roots. Iron deprived elm seedlings of both species were stunted and suffered severe Fe-chlorosis symptoms. After Fe re-supply leaf chlorophyll concentrations rose according to species-dependent patterns. While U. minor leaves and seedlings re-greened evenly, U. laevis did so along the nerves of new growing leaves. U. minor had a higher root ferric reductase activity and H(+)-extrusion capability than U. laevis and maintained a better nutrient balance when grown under Fe-limiting conditions. The two elm species were found to have different Fe acquisition efficiencies which may be related to their natural distribution in calcareous and siliceous soils of the Iberian Peninsula. PMID:24723927

  15. Serum clinical biochemical and hematologic reference ranges of laboratory-reared and wild-caught Xenopus laevis.

    PubMed

    Wilson, Sabrina; Felt, Stephen; Torreilles, Stéphanie; Howard, Antwain; Behan, Colleen; Moorhead, Roberta; Green, Sherril

    2011-09-01

    The South African clawed frogs Xenopus laevis and X. tropicalis are fully aquatic amphibians and well-established animal models. Because genetically engineered laboratory Xenopus are now being produced, the establishment of normal reference ranges for serum biochemical and hematologic parameters is essential for phenotyping and as a diagnostic aide. We determined normal reference ranges for hematologic values from 3 populations of X. laevis: wild-caught frogs (n = 43) and frogs from 2 commercial sources (A, n = 166; B, n = 109). For serum biochemistry, we determined normal reference ranges for frogs from source A and wild-caught frogs divided by sex and season. Significant differences across populations were found in WBC and RBC counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume. Among serum biochemical analytes, significant differences were found for albumin:globulin ratio, anion gap, and concentrations of albumin, globulin, total protein, lipase, alanine transaminase, γ-glutamyl transpeptidase; creatine phosphokinase; indirect, direct, and total bilirubin; cholesterol, low-density lipoprotein lipase, carbon dioxide, glucose, lactacte dehydrogenase, calcium, chloride, and sodium. We hypothesize that these differences can be attributed to differences in water quality, habitat, ambient temperature, diet, sex, recent transport or shipment, and genetic background. However, testing that hypothesis is beyond the scope of the current study. In addition, clinical chemistry and hematologic reference range values Xenopus laevis are quite distinct from those for other species and are most consistent with the only values published for another fully aquatic amphibian, the Eastern hellbender (Cryptobranchus alleganiensis). PMID:22330708

  16. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

    PubMed Central

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-01-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

  17. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    PubMed

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

  18. Serum Clinical Biochemical and Hematologic Reference Ranges of Laboratory-Reared and Wild-Caught Xenopus laevis

    PubMed Central

    Wilson, Sabrina; Felt, Stephen; Torreilles, Stéphanie; Howard, Antwain; Behan, Colleen; Moorhead, Roberta; Green, Sherril

    2011-01-01

    The South African clawed frogs Xenopus laevis and X. tropicalis are fully aquatic amphibians and well-established animal models. Because genetically engineered laboratory Xenopus are now being produced, the establishment of normal reference ranges for serum biochemical and hematologic parameters is essential for phenotyping and as a diagnostic aide. We determined normal reference ranges for hematologic values from 3 populations of X. laevis: wild-caught frogs (n = 43) and frogs from 2 commercial sources (A, n = 166; B, n = 109). For serum biochemistry, we determined normal reference ranges for frogs from source A and wild-caught frogs divided by sex and season. Significant differences across populations were found in WBC and RBC counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume. Among serum biochemical analytes, significant differences were found for albumin:globulin ratio, anion gap, and concentrations of albumin, globulin, total protein, lipase, alanine transaminase, γ-glutamyl transpeptidase; creatine phosphokinase; indirect, direct, and total bilirubin; cholesterol, low-density lipoprotein lipase, carbon dioxide, glucose, lactacte dehydrogenase, calcium, chloride, and sodium. We hypothesize that these differences can be attributed to differences in water quality, habitat, ambient temperature, diet, sex, recent transport or shipment, and genetic background. However, testing that hypothesis is beyond the scope of the current study. In addition, clinical chemistry and hematologic reference range values Xenopus laevis are quite distinct from those for other species and are most consistent with the only values published for another fully aquatic amphibian, the Eastern hellbender (Cryptobranchus alleganiensis). PMID:22330708

  19. Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes.

    PubMed Central

    Hagenbuch, B; Scharschmidt, B F; Meier, P J

    1996-01-01

    A Na(+)-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na(+)-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na(+)-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na(+)-dependent taurocholate uptake, but it blocked Na(+)-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na(+)-dependent and Na(+)-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver. PMID:8670169

  20. A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation.

    PubMed

    Edens, Lisa J; Levy, Daniel L

    2016-08-08

    A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

  1. Anxa4 Genes are Expressed in Distinct Organ Systems in Xenopus laevis and tropicalis But are Functionally Conserved

    PubMed Central

    Massé, Karine L; Collins, Robert J; Bhamra, Surinder; Seville, Rachel A

    2007-01-01

    Anxa4 belongs to the multigenic annexin family of proteins which are characterized by their ability to interact with membranes in a calcium-dependent manner. Defined as a marker for polarized epithelial cells, Anxa4 is believed to be involved in many cellular processes but its functions in vivo are still poorly understood. Previously, we cloned Xanx4 in Xenopus laevis (now referred to as anxa4a) and demonstrated its role during organogenesis of the pronephros, providing the first evidence of a specific function for this protein during the development of a vertebrate. Here, we describe the strict conservation of protein sequence and functional domains of anxa4 during vertebrate evolution. We also identify the paralog of anxa4a, anxa4b and show its specific temporal and spatial expression pattern is different from anxa4a. We show that anxa4 orthologs in X. laevis and tropicalis display expression domains in different organ systems. Whilst the anxa4a gene is mainly expressed in the kidney, Xt anxa4 is expressed in the liver. Finally, we demonstrate Xt anxa4 and anxa4a can display conserved function during kidney organogenesis, despite the fact that Xt anxa4 transcripts are not expressed in this domain. This study highlights the divergence of expression of homologous genes during Xenopus evolution and raises the potential problems of using X. tropicalis promoters in X. laevis. PMID:19279706

  2. Continued Studies on the Effects of Simazine on the Liver Histological Structure and Metamorphosis in the Developing Xenopus laevis.

    PubMed

    Sai, Linlin; Qu, Binpeng; Li, Yan; Jia, Qiang; Bo, Cunxiang; Liu, Yanzhong; Yu, Gongchang; Xie, Lin; Li, Ling; Ng, Jack C; Peng, Cheng

    2016-10-01

    This study continued our previous work (Sai et al. in Bull Environ Contam Toxicol 95:157-163, 2015a) by analysing the effects of simazine on the liver histological structure and metamorphosis in the developing Xenopus laevis. Tadpoles (Nieuwkoop-Faber stage 46) were exposed to simazine at 0.1, 1.2, 11.0 and 100.9 μg/L for 100 days. When tadpoles were exposed to simazine at 11.0 and 100.9 µg/L, an increased mortality and damaged liver tissues were observed together with significant inhibition of percent of X. laevis completing metamorphosis on days 80 and 90 and prolonged time of completing metamorphosis. On the other hand, we found that simazine has no significant effects on liver weight and altered hepatosomatic index. Results of this study may be considered to inform risk assessment of the effects of simazine on the development of X. laevis. PMID:27485009

  3. Root iron uptake efficiency of Ulmus laevis and U. minor and their distribution in soils of the Iberian Peninsula

    PubMed Central

    Venturas, Martin; Fernández, Victoria; Nadal, Paloma; Guzmán, Paula; Lucena, Juan J.; Gil, Luis

    2014-01-01

    The calcifuge and calcicole character of wild plants has been related to nutrient availability shortages, including iron (Fe)-deficiency. Surprisingly, just a few studies examined the relation between root Fe uptake and plant distribution in different soil types. We assessed the root Fe acquisition efficiency of two Ulmus species with calcareous (Ulmus minor) and siliceous (U. laevis) soil distribution patterns in the Iberian Peninsula. Seedlings of both elm species were grown hydroponically with different Fe concentrations during 6 weeks. Plant physiological responses to Fe-limiting conditions were evaluated as were the ferric reductase activity and proton (H+) extrusion capacity of the roots. Iron deprived elm seedlings of both species were stunted and suffered severe Fe-chlorosis symptoms. After Fe re-supply leaf chlorophyll concentrations rose according to species-dependent patterns. While U. minor leaves and seedlings re-greened evenly, U. laevis did so along the nerves of new growing leaves. U. minor had a higher root ferric reductase activity and H+-extrusion capability than U. laevis and maintained a better nutrient balance when grown under Fe-limiting conditions. The two elm species were found to have different Fe acquisition efficiencies which may be related to their natural distribution in calcareous and siliceous soils of the Iberian Peninsula. PMID:24723927

  4. A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation.

    PubMed

    Edens, Lisa J; Levy, Daniel L

    2016-01-01

    A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies. PMID:27584618

  5. Morphogenetic changes occurring in the regenerating newt tail under changed gravity conditions

    NASA Astrophysics Data System (ADS)

    Radugina, Elena A.; Grigoryan, Eleonora N.; Dvorochkin, Natasha; Almeida, Eduardo

    2012-07-01

    It is widely accepted that gravity greatly affects animal physiology, development, and alters gene expression. Recently it became apparent that it can also affect tissue morphogenesis. In our work, we developed special laboratory conditions that allow us to produce the gravity-dependent alterations in tail regenerates of the newt Pleurodeles waltl. We examined the dynamic morphogenetic changes during 50-day tail regeneration using computer morphometric analysis. Changes that we observed under these conditions were comparable with those found earlier in our spaceflight experiments. The newts kept in aquarium deep water (low g) after 1/3 tail amputation developed normal lanceolate regenerates. In contrast, the animals that stayed on the moist mat (1g) developed tail regenerates curved ventrally, with tips almost touching the mat. The similar results were obtained with a 12-day centrifugation at 2g. The study of the regenerate morphology in low g, 1g, and 2g animal groups allowed us to determine the stage at which the morphological changes in regenerates become apparent, and to detect the main morphological events associated with the development of tail curve, such as bending of ependymal tube and reorientation of the forming cartilage. We describe cellular processes foregoing observed tissue morphogenetic changes, such as cell migration, condensation in cell population, and unequal proliferation in different areas of epidermis and blastema. Cell proliferation in epidermis and blastema of tails regenerated under the conditions of different gravitational load was evaluated by BrdU assay. In 1g newts, cell proliferation increased within the dorso-apical region of the regenerates compared with that in low g group. These results provide us with a valuable insight into the regenerative tissue homostasis that involves cell division, cell death, and migration in the newt regenerating tail. In addition, these findings could provide us with better understanding of the

  6. Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos

    NASA Astrophysics Data System (ADS)

    Débarre, Delphine; Supatto, Willy; Farge, Emmanuel; Moulia, Bruno; Schanne-Klein, Marie-Claire; Beaurepaire, Emmanuel

    2004-12-01

    We demonstrate the association of third-harmonic generation (THG) microscopy and particle image velocimetry (PIV) analysis as a novel functional imaging technique for automated micrometer-scale characterization of morphogenetic movements in developing embryos. Using a combined two-photon-excited fluorescence and THG microscope, we characterize the optical properties of Drosophila embryos and show that sustained THG imaging does not perturb sensitive developmental dynamics. Velocimetric THG imaging provides a quantitative description of the dynamics of internal structures in unstained wild-type and mutant embryos.

  7. Bone morphogenetic protein signaling in vertebrate motor neurons and neuromuscular communication

    PubMed Central

    Osses, Nelson; Henríquez, Juan P.

    2015-01-01

    An accurate communication between motor neurons and skeletal muscle fibers is required for the proper assembly, growth and maintenance of neuromuscular junctions (NMJs). Several signaling and extracellular matrix molecules play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in Drosophila have revealed crucial functions for early morphogens, such as members of the Wnt and Bone Morphogenetic Proteins (BMP) signaling pathways, during the assembly and maturation of the NMJ. Here, we bring together recent findings that led us to propose that BMPs also work in vertebrate organisms as diffusible cues to communicate motor neurons and skeletal muscles. PMID:25674047

  8. Maxillary anterior ridge augmentation with recombinant human bone morphogenetic protein 2.

    PubMed

    Edmunds, Ryan K; Mealey, Brian L; Mills, Michael P; Thoma, Daniel S; Schoolfield, John; Cochran, David L; Mellonig, Jim

    2014-01-01

    No human studies exist on the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS) as a sole graft material for lateral ridge augmentation in large ridge defect sites. This series evaluates the treatment outcome of maxillary anterior lateral ridge augmentation with rhBMP-2/ACS. Twenty patients were treated with rhBMP-2/ACS and fixation screws for space maintenance. Cone beam volumetric tomography measurements were used to determine gain in ridge width, and a bone core biopsy was obtained. The mean horizontal ridge gain was 1.2 mm across sites, and every site gained width. PMID:25006772

  9. Bone Morphogenetic Protein 4 Signalling in Neural Stem and Progenitor Cells during Development and after Injury

    PubMed Central

    Cole, Alistair E.; Murray, Simon S.; Xiao, Junhua

    2016-01-01

    Substantial progress has been made in identifying the extracellular signalling pathways that regulate neural stem and precursor cell biology in the central nervous system (CNS). The bone morphogenetic proteins (BMPs), in particular BMP4, are key players regulating neuronal and glial cell development from neural precursor cells in the embryonic, postnatal, and injured CNS. Here we review recent studies on BMP4 signalling in the generation of neurons, astrocytes, and oligodendroglial cells in the CNS. We also discuss putative mechanisms that BMP4 may utilise to influence glial cell development following CNS injury and highlight some questions for further research. PMID:27293450

  10. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

    PubMed

    Yu, Shuangying; Tang, Song; Mayer, Gregory D; Cobb, George P; Maul, Jonathan D

    2015-02-01

    Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides increased transcript abundance of CSA and MUTL. In addition, mRNA abundance of HSP70 and GADD45α were increased by endosulfan and mRNA abundance of XPG was increased by α-cypermethrin. XPC, HR23B, XPG, and GADD45α exhibited elevated mRNA concentrations whereas there was a reduction in MUTL transcript concentrations in UVB-alone treatments. It appeared that even

  11. Peripheral myelin of Xenopus laevis: Role of electrostatic and hydrophobic interactions in membrane compaction

    PubMed Central

    Luo, XiaoYang; Cerullo, Jana; Dawli, Tamara; Priest, Christina; Haddadin, Zaid; Kim, Angela; Inouye, Hideyo; Suffoletto, Brian P.; Avila, Robin L.; Lees, Jonathan P.B.; Sharma, Deepak; Xie, Bo; Costello, Catherine E.; Kirschner, Daniel A.

    2008-01-01

    P0 glycoprotein is the major structural protein of peripheral nerve myelin where it is thought to modulate inter-membrane adhesion at both the extracellular apposition, which is labile upon changes in pH and ionic strength, and the cytoplasmic apposition, which is resistant to such changes. Most studies on P0 have focused on structure-function correlates in higher vertebrates. Here, we focused on its role in the structure and interactions of frog (Xenopus laevis) myelin, where it exists primarily in a dimeric form. As part of our study, we deduced the full sequence of Xenopus laevis P0 (xP0) from its cDNA. The xP0 sequence was found to be similar to P0 sequences of higher vertebrates, suggesting that a common mechanism of PNS myelin compaction via P0 interaction might have emerged through evolution. As previously reported for mouse PNS myelin, a similar change of extracellular apposition in frog PNS myelin as a function of pH and ionic strength was observed, which can be explained by a conformational change of P0 due to protonation-deprotonation of His52 at P0’s putative adhesive interface. On the other hand, the cytoplasmic apposition in frog PNS myelin, like that in the mouse, remained unchanged at different pH and ionic strength. The contribution of hydrophobic interactions to stabilizing the cytoplasmic apposition was tested by incubating sciatic nerves with detergents. Dramatic expansion at the cytoplasmic apposition was observed for both frog and mouse, indicating a common hydrophobic nature at this apposition. Urea also expanded the cytoplasmic apposition of frog myelin likely owing to denaturation of P0. Removal of the fatty acids that attached to the single Cys residue in the cytoplasmic domain of P0 did not change PNS myelin structure of either frog or mouse, suggesting that the P0-attached fatty acyl chain does not play a significant role in PNS myelin compaction and stability. These results help clarify the present understanding of P0’s adhesion

  12. Determining the optimal developmental stages of Xenopus laevis for initiating exposures to chemicals for sensitively detecting their feminizing effects on gonadal differentiation.

    PubMed

    Li, Yuan-Yuan; Chen, Juan; Qin, Zhan-Fen

    2016-10-01

    Xenopus laevis is an important model for detecting feminizing effects of endocrine disrupting chemicals (EDCs) on amphibians because its genetic males can be induced to phenotypic females by estrogenic chemicals. It is crucial that chemical exposures begin at sensitive developmental stages for gonadal sex-reversal in X. laevis. To determine the optimal stages for initiating exposures, we investigated gonadal sex-reversal induced by low concentrations of 17α-ethinylestradiol (EE2) when exposures were initiated at different stages (3/4, 45/46, 48 and 50) until stage 58. We found that 0.1nM EE2 resulted in 85%, 86%, 43%, and 19% intersex, whereas 1nM EE2 caused 77%, 81%, 17%, and 8% phenotypic females, when genetic male tadpoles were exposed from stages 3/4, 45/46, 48 and 50, respectively. The data show the sensitivity of X. laevis gonads to EE2 at stages 45/46 is similar with that at stages 3/4, but the sensitivity decreases at stage 48 and stage 50, displaying a developmental stage-dependent manner. In another experiment using the offspring of another pair of frogs, we confirmed high sensitivity of X. laevis gonads at stages 45/46 to low concentrations of EE2. Considering that stages 45/46 tadpoles are easier to manipulate and have higher survival rates than earlier embryos, we propose that stages 45/46 are the optimal stages for initiating exposure for detecting feminizing effects of EDCs on gonadal differentiation in X. laevis. The developmental stages for initiating exposures we determined will guarantee the high sensitivity for detecting feminizing effects of EDCs with low estrogenic activities on gonadal differentiation in X. laevis. Also, our study suggests that gonadal differentiation in X. laevis possibly begins at stages 45/46, but not at later stages.

  13. Expression and localization of the Xenopus laevis small heat shock protein, HSPB6 (HSP20), in A6 kidney epithelial cells.

    PubMed

    Khamis, Imran; Chan, Daniel W; Shirriff, Cody S; Campbell, James H; Heikkila, John J

    2016-11-01

    Small heat shock proteins (sHSPs) are molecular chaperones that bind to unfolded protein, inhibit the formation of toxic aggregates and facilitate their refolding and/or degradation. Previously, the only sHSPs that have been studied in detail in the model frog system, Xenopus laevis, were members of the HSP30 family and HSPB1 (HSP27). We now report the analysis of X. laevis HSPB6, an ortholog of mammalian HSPB6. X. laevis HSPB6 cDNA encodes a 168 aa protein that contains an α-crystallin domain, a polar C-terminal extension and some possible phosphorylation sites. X. laevis HSPB6 shares 94% identity with a X. tropicalis HSPB6, 65% with turtle, 59% with humans, 49% with zebrafish and only 50% and 43% with X. laevis HSPB1 and HSP30C, respectively. Phylogenetic analysis revealed that X. laevis HSPB6 grouped more closely with mammalian and reptilian HSPB6s than with fish HSPB6. X. laevis recombinant HSPB6 displayed molecular chaperone properties since it had the ability to inhibit heat-induced aggregation of citrate synthase. Immunoblot analysis determined that HSPB6 was present constitutively in kidney epithelial cells and that heat shock treatment did not upregulate HSPB6 levels. While treatment with the proteasomal inhibitor, MG132, resulted in a 2-fold increase in HSPB6 levels, exposure to cadmium chloride produced a slight increase in HSPB6. These findings were in contrast to HSP70, which was enhanced in response to all three stressors. Finally, immunocytochemical analysis revealed that HSPB6 was present in the cytoplasm in the perinuclear region with some in the nucleus.

  14. Determining the optimal developmental stages of Xenopus laevis for initiating exposures to chemicals for sensitively detecting their feminizing effects on gonadal differentiation.

    PubMed

    Li, Yuan-Yuan; Chen, Juan; Qin, Zhan-Fen

    2016-10-01

    Xenopus laevis is an important model for detecting feminizing effects of endocrine disrupting chemicals (EDCs) on amphibians because its genetic males can be induced to phenotypic females by estrogenic chemicals. It is crucial that chemical exposures begin at sensitive developmental stages for gonadal sex-reversal in X. laevis. To determine the optimal stages for initiating exposures, we investigated gonadal sex-reversal induced by low concentrations of 17α-ethinylestradiol (EE2) when exposures were initiated at different stages (3/4, 45/46, 48 and 50) until stage 58. We found that 0.1nM EE2 resulted in 85%, 86%, 43%, and 19% intersex, whereas 1nM EE2 caused 77%, 81%, 17%, and 8% phenotypic females, when genetic male tadpoles were exposed from stages 3/4, 45/46, 48 and 50, respectively. The data show the sensitivity of X. laevis gonads to EE2 at stages 45/46 is similar with that at stages 3/4, but the sensitivity decreases at stage 48 and stage 50, displaying a developmental stage-dependent manner. In another experiment using the offspring of another pair of frogs, we confirmed high sensitivity of X. laevis gonads at stages 45/46 to low concentrations of EE2. Considering that stages 45/46 tadpoles are easier to manipulate and have higher survival rates than earlier embryos, we propose that stages 45/46 are the optimal stages for initiating exposure for detecting feminizing effects of EDCs on gonadal differentiation in X. laevis. The developmental stages for initiating exposures we determined will guarantee the high sensitivity for detecting feminizing effects of EDCs with low estrogenic activities on gonadal differentiation in X. laevis. Also, our study suggests that gonadal differentiation in X. laevis possibly begins at stages 45/46, but not at later stages. PMID:27611864

  15. Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

    PubMed

    Fenwick, Mark A; Mora, Jocelyn M; Mansour, Yosef T; Baithun, Christina; Franks, Stephen; Hardy, Kate

    2013-09-01

    Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are 2 closely related TGF-β ligands implicated as key regulators of follicle development and fertility. Animals harboring mutations of these factors often exhibit a blockage in follicle development beyond the primary stage and therefore little is known about the role of these ligands during subsequent (preantral) stages. Preantral follicles isolated from immature mice were cultured with combinations of BMP15, GDF9, and activin receptor-like kinase (ALK) inhibitors. Individually, GDF9 and BMP15 promoted follicle growth during the first 24 hours, whereas BMP15 subsequently (48-72 h) caused follicle shrinkage and atresia with increased granulosa cell apoptosis. Inhibition of ALK6 prevented the BMP15-induced reduction in follicle size and under basal conditions promoted a rapid increase in granulosa cell proliferation, suggesting BMP15 signals through ALK6, which in turn acts to restrain follicle growth. In the presence of GDF9, BMP15 no longer promoted atresia and in fact follicle growth was increased significantly more than with either ligand alone. This cooperative effect was accompanied by differential expression of Id1-3, Smad6-7, and Has2 and was blocked by the same ALK5 inhibitor used to block GDF9 signaling. Immunostaining for SMAD2/3 and SMAD1/5/8, representing the 2 main branches of TGF-β signaling, supported the fact that both canonical pathways have the potential to be active in growing follicles, whereas primordial follicles only express SMAD2/3. Overall results highlight differential effects of the 2 main TGF-β signaling pathways during preantral follicle growth.

  16. The Amphibian (Xenopus laevis) Type I Interferon Response to Frog Virus 3: New Insight into Ranavirus Pathogenicity

    PubMed Central

    Grayfer, Leon; De Jesús Andino, Francisco

    2014-01-01

    ABSTRACT The increasing prevalence of ranavirus (RV; Iridoviridae) infections of wild and commercially maintained aquatic species is raising considerable concerns. While Xenopus laevis is the leading model for studies of immunity to RV, amphibian antiviral interferon (IFN) responses remain largely uncharacterized. Accordingly, an X. laevis type I interferon was identified, the expression of the gene for this IFN was examined in RV (frog virus 3 [FV3])-infected tadpoles and adult frogs by quantitative PCR, and a recombinant form of this molecule (recombinant X. laevis interferon [rXlIFN]) was produced for the purpose of functional studies. This rXlIFN protected the kidney-derived A6 cell line and tadpoles against FV3 infection, decreasing the infectious viral burdens in both cases. Adult frogs are naturally resistant to FV3 and clear the infection within a few weeks, whereas tadpoles typically succumb to this virus. Hence, as predicted, virus-infected adult X. laevis frogs exhibited significantly more robust FV3-elicited IFN gene expression than tadpoles; nevertheless, they also tolerated substantially greater viral burdens following infection. Although tadpole stimulation with rXlIFN prior to FV3 challenge markedly impaired viral replication and viral burdens, it only transiently extended tadpole survival and did not prevent the eventual mortality of these animals. Furthermore, histological analysis revealed that despite rXlIFN treatment, infected tadpoles had considerable organ damage, including disrupted tissue architecture and extensive necrosis and apoptosis. Conjointly, these findings indicate a critical protective role for the amphibian type I IFN response during ranaviral infections and suggest that these viruses are more pathogenic to tadpole hosts than was previously believed, causing extensive and fatal damage to multiple organs, even at very low titers. IMPORTANCE Ranavirus infections are threatening wild and commercially maintained aquatic species. The

  17. Readaptation of the vestibuloocular reflex to 1g-Condition in immature lower vertebrates ( Xenopus laevis) after micro- or hypergravity exposure

    NASA Astrophysics Data System (ADS)

    Sebastian, C.; Horn, E.; Eβeling, K.; Neubert, J.

    The effects of altered gravitational conditions (AGC) on the development of the static vestibulo-ocular reflex (VOR) and readaptation to 1g were investigated in the amphibian Xenopus laevis. Tadpoles were exposed to microgravity (μg) during the German Space Mission D-2 for 10 days, using the STATEX closed survival system, or to 3g for 9 days during earth-bound experiments. At the beginning of AGC, the tadpoles had not yet developed the static VOR. The main results were: (i) Tadpoles with ug- or 3g-experience had a lower gain of the static VOR than the 1g-controls during the 2nd and 5th post-AGC days, (ii) Readaptation to response levels of 1g-reared controls usually occurred during the following weeks, except in slowly developing tadpoles with 3g-experience. Readaptation was less pronounced if, during the acute VOR test, tadpoles were rolled from the inclined to the normal posture than in the opposite test situation. It is postulated that (i) gravity is necessarily involved in the development of the static VOR, but only during a period including the time before onset of the first behavioural response; and (ii) readaptation which is superimposed by the processes of VOR development depends on many factors including the velocity of development, the actual excitation level of the vestibular systems and the neuroplastic properties of its specific pathways.

  18. Morphogenetic behavior of tropical marine yeast Yarrowia lipolytica in response to hydrophobic substrates.

    PubMed

    Zinjarde, Smita S; Kale, Bhagyashree V; Vishwasrao, Paresh V; Kumar, Ameeta R

    2008-09-01

    The morphogenetic behavior of a tropical marine Yarrowia lipolytica strain on hydrophobic substrates was studied. Media containing coconut oil or palm kernel oil (rich in lauric and myristic acids) prepared in distilled water or seawater at a neutral pH supported 95% of the cells to undergo a transition from the yeast form to the mycelium form. With potassium laurate, 51% of the cells were in the mycelium form, whereas with myristate, 32% were in the mycelium form. However, combinations of these two fatty acids in proportions that are present in coconut oil or palm kernel oil enhanced the mycelium formation to 65%. The culture also produced extracellular lipases during the morphogenetic change. The yeast cells were found to attach to the large droplets of the hydrophobic substrates during the transition, while the mycelia were associated with the aqueous phase. The alkane-grown yeast partitioned more efficiently in the hydrophobic phases when compared with the coconut oil-grown mycelia. A fatty acid analysis of the mycelial form revealed the presence of lauric acid in addition to the long-chain saturated and unsaturated fatty acids observed in the yeast form. The mycelia underwent a rapid transition to the yeast form with n-dodecane, a medium-chain aliphatic hydrocarbon. Thus, the fungus displayed a differential behavior towards the two types of saturated hydrophobic substrates. PMID:18852507

  19. Morphogenetic fields in embryogenesis, regeneration, and cancer: Non-local control of complex patterning

    PubMed Central

    Levin, Michael

    2012-01-01

    Establishment of shape during embryonic development, and the maintenance of shape against injury or tumorigenesis, requires constant coordination of cell behaviors toward the patterning needs of the host organism. Molecular cell biology and genetics have made great strides in understanding the mechanisms that regulate cell function. However, generalized rational control of shape is still largely beyond our current capabilities. Significant instructive signals function at long range to provide positional information and other cues to regulate organism-wide systems properties like anatomical polarity and size control. Is complex morphogenesis best understood as the emergent property of local cell interactions, or as the outcome of a computational process that is guided by a physically-encoded map or template of the final goal state? Here I review recent data and molecular mechanisms relevant to morphogenetic fields: large-scale systems of physical properties that have been proposed to store patterning information during embryogenesis, regenerative repair, and cancer suppression that ultimately controls anatomy. Placing special emphasis on the role of endogenous bioelectric signals as an important component of the morphogenetic field, I speculate on novel approaches for the computational modeling and control of these fields with applications to synthetic biology, regenerative medicine, and evolutionary developmental biology. PMID:22542702

  20. The Ess1 prolyl isomerase is required for growth and morphogenetic switching in Candida albicans.

    PubMed Central

    Devasahayam, Gina; Chaturvedi, Vishnu; Hanes, Steven D

    2002-01-01

    Prolyl-isomerases (PPIases) are found in all organisms and are important for the folding and activity of many proteins. Of the 13 PPIases in Saccharomyces cerevisiae only Ess1, a parvulin-class PPIase, is essential for growth. Ess1 is required to complete mitosis, and Ess1 and its mammalian homolog, Pin1, interact directly with RNA polymerase II. Here, we isolate the ESS1 gene from the pathogenic fungus Candida albicans and show that it is functionally homologous to the S. cerevisiae ESS1. We generate conditional-lethal (ts) alleles of C. albicans ESS1 and use these mutations to demonstrate that ESS1 is essential for growth in C. albicans. We also show that reducing the dosage or activity of ESS1 blocks morphogenetic switching from the yeast to the hyphal and pseudohyphal forms under certain conditions. Analysis of double mutants of ESS1 and TUP1 or CPH1, two genes known to be involved in morphogenetic switching, suggests that ESS1 functions in the same pathway as CPH1 and upstream of or in parallel to TUP1. Given that switching is important for virulence of C. albicans, inhibitors of Ess1 might be useful as antifungal agents. PMID:11805043

  1. Origin of the novel chemoreceptor Aesthetasc "Y" in Ostracoda: morphogenetical thresholds and evolutionary innovation.

    PubMed

    Kaji, Tomonari; Tsukagoshi, Akira

    2008-01-01

    The morphology and developmental processes of the two types of ostracod chemoreceptors, the Aesthetasc "Y" and the "Grouped setae," were compared. Cypridoidea and Pontocypridoidea, belonging to Cypridocopina, have a large baseball bat-like seta as an autapomorphic character on the second antenna, whereas most ostracod taxa with plesiomorphic characters bear "Grouped setae" consisting of multiple setae on the second antenna. Their budding positions, morphology, and ontogenetic changes were compared, and our deduction is that the Aesthetasc "Y" originated from "Grouped setae-like" organ in the Paleozoic. The morphogenetic processes in the molting period of these chemoreceptors were compared at the cellular level. The observations suggest that the "Grouped setae" are formed by hypodermal cells and share sheath cells corresponding to those of the Aesthetasc "Y" as a common constraint in the molting process of setae. We conclude that modification of the morphogenetic processes in the molting period of the "Grouped setae" gave rise to the Aesthetasc "Y" as a novel organ in the evolutionary pathway of the Ostracoda.

  2. 15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation.

    PubMed

    Sumanas, Saulius; Zhang, Bo; Dai, Rujuan; Lin, Shuo

    2005-07-01

    A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.

  3. Occurrence of immune cells in the intestinal wall of Squalius cephalus infected with Pomphorhynchus laevis.

    PubMed

    Dezfuli, Bahram S; Manera, Maurizio; Giari, Luisa; DePasquale, Joseph A; Bosi, Giampaolo

    2015-11-01

    A sub-population of 34 specimens of chub, Squalius cephalus, was sampled from the River Brenta (Northern Italy) and examined for ecto- and endo-parasites. Pomphorhynchus laevis (Acanthocephala) was the only enteric helminth encountered. Immunofluorescence and ultrastructural studies were conducted on the intestines of chub. Near the site of parasite's attachment, mucous cells, mast cells (MCs), neutrophils and rodlet cells (RCs) were found to co-occur within the intestinal epithelium. The numbers of mucous cells, MCs and neutrophils were significantly higher in infected fish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the lectin Dolichos Biflorus Agglutinin (DBA) and the macrophage-specific MAC387 monoclonal antibody, with parallel transmission electron microscopy, revealed that epithelial MCs often made intimate contact with the mucous cells. Degranulation of a large number of MCs around the site of the acanthocephalan's attachment and in proximity to mucous cells was also documented. MCs and neutrophils were abundant in the submucosa. Immune cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions are discussed.

  4. Characterization of highly and moderately repetitive 500 bp Eco RI fragments from Xenopus laevis DNA.

    PubMed Central

    Hummel, S; Meyerhof, W; Korge, E; Knöchel, W

    1984-01-01

    Three different types of repetitive Eco RI fragments, which comigrate within a visible band of approximately 500 bp at gel electrophoresis of Xenopus laevis DNA Eco RI digests have been cloned and sequenced. These sequences are designated as Repetitive Eco RI Monomers: REM 1, REM 2 and REM 3. The sequences contain direct repeats, inverted repeats and palindromic elements. Genomic organization of the most abundant sequence (REM 1; 0.4% of total DNA) is that of an interspersed sequence. REM 2 (0.08%) is partly organized as an interspersed element and partly found in tandem arrangement, whereas REM 3 (0.02%) represents the tandemly repeated monomeric unit of a satellite DNA. In situ hybridization has shown that REM 1 and REM 2 sequences are found on most chromosomes, REM 1 being preferentially located on specific chromosomal loci. REM 3 is located near the centromere region of only one chromosome pair (presumably number 1). Hybridization of Northern blots from RNAs of different developmental stages revealed that REM 1, REM 2 and REM 3 sequences are transcribed and that transcription is under developmental control. Images PMID:6330690

  5. Onset and early development of hypoxic ventilatory responses and branchial neuroepithelial cells in Xenopus laevis.

    PubMed

    Pan, Tien-Chien F; Burggren, Warren W

    2010-12-01

    Onset and ontogeny of the O₂ chemoreceptive control of ventilation was investigated in Xenopus laevis. The density and size of branchial serotonin-immunoreactive neuroepithelial cells (5-HT-IR NECs) were also determined using confocal immunofluorescent microscopy. Larvae started gill ventilation at 3 days post-fertilization (dpf), and, at this early stage, acute hypoxic exposure produced an increase in frequency from 28 ± 4 to 60 ± 2 beats x min⁻¹. Concurrent with the onset of ventilatory responses, 5-HT-IR NECs appeared in the gill filament bud. Lung ventilation began at 5 dpf and exhibited a 3-fold increase in frequency during acute hypoxia. At 10 dpf, gill ventilatory sensitivity to hypoxia increased, as did NEC density, from 15 ± 1 (5 dpf) to 29 ± 2 (10 dpf) cells x mm of filament⁻¹. Unlike ventilation frequency, gill ventilation amplitude and lung expired volume were unaltered by acute hypoxia. Chronic exposure to moderate hypoxia, at a P(O₂) of 110 mmHg, attenuated acute responses to moderate hypoxia at 10 and 14 dpf but had no effect at more severe hypoxia or at other stages. Chronic hypoxia also stimulated 5-HT-IR NECs growth at 21 dpf. Collectively, larvae at 5 dpf exhibited strong O₂-driven gill and lung ventilatory responses, and between 10 and 21 dpf, the early hypoxic responses can be shaped by the ambient P(O₂).

  6. Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis.

    PubMed

    Spadafora, C; Riccardi, P

    1985-12-20

    The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.

  7. Metamorphic remodeling of the olfactory organ of the African clawed frog, Xenopus laevis.

    PubMed

    Dittrich, Katarina; Kuttler, Josua; Hassenklöver, Thomas; Manzini, Ivan

    2016-04-01

    The amphibian olfactory system undergoes massive remodeling during metamorphosis. The transition from aquatic olfaction in larvae to semiaquatic or airborne olfaction in adults requires anatomical, cellular, and molecular modifications. These changes are particularly pronounced in Pipidae, whose adults have secondarily adapted to an aquatic life style. In the fully aquatic larvae of Xenopus laevis, the main olfactory epithelium specialized for sensing water-borne odorous substances lines the principal olfactory cavity (PC), whereas a separate olfactory epithelium lies in the vomeronasal organ (VNO). During metamorphosis, the epithelium of the PC is rearranged into the adult "air nose," whereas a new olfactory epithelium, the adult "water nose," forms in the emerging middle cavity (MC). Here we performed a stage-by-stage investigation of the anatomical changes of the Xenopus olfactory organ during metamorphosis. We quantified cell death in all olfactory epithelia and found massive cell death in the PC and the VNO, suggesting that the majority of larval sensory neurons is replaced during metamorphosis in both sensory epithelia. The moderate cell death in the MC shows that during the formation of this epithelium some cells are sorted out. Our results show that during MC formation some supporting cells, but not sensory neurons, are relocated from the PC to the MC and that they are eventually eliminated during metamorphosis. Together our findings illustrate the structural and cellular changes of the Xenopus olfactory organ during metamorphosis.

  8. Analysis of Histones and Chromatin in Xenopus laevis Egg and Oocyte Extracts

    PubMed Central

    Banaszynski, Laura A.; Allis, C. David; Shechter, David

    2010-01-01

    Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since work in recent years has solidified the importance of post-translational modification of histones in directing biological processes, the study of histones in a biochemically dissectible model such as Xenopus is crucial for the understanding of their biological significance. Here we present a rationale and methods for isolating and studying histones and chromatin in different Xenopus egg and oocyte extracts. In particular, we present protocols for the preparation of: cell-free egg and oocyte extract; nucleoplasmic extract (“NPE”); biochemical purification of maternally-deposited, stored histones in the oocyte and the egg; assembly of pronuclei in egg extract and the isolation of pronuclear chromatin and histones; and an extract chromatin assembly assay. We also demonstrate aspects of the variability of the system to be mindful of when working with extract and the importance of proper laboratory temperature in preparing quality extracts. We expect that these methods will be of use in promoting further understanding of embryonic chromatin in a unique experimental system. PMID:20051265

  9. Functional joint regeneration is achieved using reintegration mechanism in Xenopus laevis.

    PubMed

    Tsutsumi, Rio; Yamada, Shigehito; Agata, Kiyokazu

    2016-02-01

    A functional joint requires integration of multiple tissues: the apposing skeletal elements should form an interlocking structure, and muscles should insert into skeletal tissues via tendons across the joint. Whereas newts can regenerate functional joints after amputation, Xenopus laevis regenerates a cartilaginous rod without joints, a "spike." Previously we reported that the reintegration mechanism between the remaining and regenerated tissues has a significant effect on regenerating joint morphogenesis during elbow joint regeneration in newt. Based on this insight into the importance of reintegration, we amputated frogs' limbs at the elbow joint and found that frogs could regenerate a functional elbow joint between the remaining tissues and regenerated spike. During regeneration, the regenerating cartilage was partially connected to the remaining articular cartilage to reform the interlocking structure of the elbow joint at the proximal end of the spike. Furthermore, the muscles of the remaining part inserted into the regenerated spike cartilage via tendons. This study might open up an avenue for analyzing molecular and cellular mechanisms of joint regeneration using Xenopus.

  10. Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones.

    PubMed

    Stout, Alina; D'Amico, Salvatore; Enzenbacher, Tiffany; Ebbert, Patrick; Lowery, Laura Anne

    2014-01-01

    Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics(1,2). One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization(1). Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs(1-3). Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker(4), following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones(5). This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types(6-8). PMID:25225829

  11. Inositol 1,4,5-trisphosphate mass changes from fertilization through first cleavage in Xenopus laevis.

    PubMed Central

    Stith, B J; Goalstone, M; Silva, S; Jaynes, C

    1993-01-01

    After fertilization in Xenopus laevis, inositol 1,4,5-trisphosphate (IP3) mass increased from 53 to 261 fmol/cell and returned to near basal by 10 min after insemination. IP3 was also elevated over control egg levels during first mitosis and first cleavage. Because IP3 levels and the fertilization calcium wave decline at about the same time and because calcium ionophore or pricking the egg increased IP3, the fertilization calcium wave may be due to calcium-induced IP3 production. In addition, the onset of sperm motility was associated with an increase, whereas the acrosomal reaction was accompanied by a decrease in IP3 mass. Combining our published data with this report, the first chronology of the levels of IP3 from the induction of meiosis (maturation) through fertilization and cleavage in one cellular system is summarized. These data suggest an in vivo dose response for IP3 and calcium release. A small (17 fmol/cell) IP3 change during the induction of meiosis may not be associated with a calcium change. Larger IP3 changes at cleavage (40 fmol/cell) and mitosis (125 fmol/cell) are associated with localized small calcium increases, whereas the largest IP3 change (208 fmol/cell) is associated with the large calcium increase at fertilization. PMID:8507898

  12. Inflammation-Induced Reactivation of the Ranavirus Frog Virus 3 in Asymptomatic Xenopus laevis

    PubMed Central

    Robert, Jacques; Grayfer, Leon; Edholm, Eva-Stina; Ward, Brian; De Jesús Andino, Francisco

    2014-01-01

    Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals. PMID:25390636

  13. Inflammation-induced reactivation of the ranavirus Frog Virus 3 in asymptomatic Xenopus laevis.

    PubMed

    Robert, Jacques; Grayfer, Leon; Edholm, Eva-Stina; Ward, Brian; De Jesús Andino, Francisco

    2014-01-01

    Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals.

  14. A simple behavioral assay for testing visual function in Xenopus laevis.

    PubMed

    Viczian, Andrea S; Zuber, Michael E

    2014-01-01

    Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once. PMID:24962702

  15. Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

    PubMed Central

    Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

    2014-01-01

    During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

  16. In vitro maintenance of spermatogenesis in Xenopus laevis testis explants cultured in serum-free media

    SciTech Connect

    Risley, M.S.; Miller, A.; Bumcrot, D.A.

    1987-05-01

    Spermatogenesis has been maintained for extended periods in Xenopus laevis testis explants cultured in serum-free media supplemented with bovine serum albumin, insulin, transferrin, follicle-stimulating hormone, dihydrotestosterone, testosterone, retinol, ascorbate, and tocopherol. The organization of the testis fragments was maintained for 28 days, and all stages of development were present throughout the culture period. /sup 3/H-Thymidine-labeled secondary (Type B) spermatogonia developed in 28 days into spermatids at the acrosomal vesicle stage whereas labeled zygotene spermatocytes became mature spermatids in 28 days. Spermatogonial proliferation also continued in vitro for 28 days. Germ cell differentiation was not dependent upon exogenous testosterone, ascorbate, or tocopherol since /sup 3/H-labeled spermatogonia became mature spermatids in testes cultured 35 days in media lacking these supplements. Autoradiography demonstrated that 55% of the luminal sperm present in explants cultured 10 days had differentiated in vitro. Sperm from testes cultured 10-35 days were similar to sperm from freshly dissected testes with regard to motility and fecundity, and eggs fertilized with sperm from explant cultures developed normally into swimming tadpoles. The results demonstrate the feasibility of maintaining vertebrate spermatogenesis in culture and suggest that in vitro analysis of Xenopus spermatogenesis using defined media may provide important insights into the evolution of regulatory mechanisms in spermatogenesis.

  17. Gonadal development of larval male Xenopus laevis exposed to atrazine in outdoor microcosms

    USGS Publications Warehouse

    Jooste, A.M.; Du Preez, L.H.; Carr, J.A.; Giesy, J.P.; Gross, T.S.; Kendall, R.J.; Smith, E.E.; Van Der Kraak, G. L.; Solomon, K.R.

    2005-01-01

    The potential effects of atrazine on gonadal development in metamorphs and subadults of the African clawed frog (Xenopus laevis) were studied under conditions of natural photoperiod and temperatures in outdoor microcosms from August 2002 to June 2003 in South Africa. Triplicate 1100 L microcosms for each nominal concentration of 0.0, 1, 10, and 25 ??g of atrazine/L were used. Measured atrazine concentrations varied <25% throughout the study, and no atrazine was detected in the control microcosms. Tadpoles developed well at all concentrations. On the basis of histological examination of testes of recently metamorphosed stage 66 frogs, 57% of the individuals in the reference group exhibited testicular oocytes as compared with 57, 59, and 39% of the 1, 10, and 25 ??g/L atrazine groups, respectively. The average prevalence of testicular oocytes for all of the treatments including the controls was 54% in a single testis, while, in 35% of individuals, testicular oocytes were observed in both testes. The number of testicular oocytes per individual ranged from 0 to 58 with means of 9.5, 9.8, 8.5, and 11.1 for the 0.0, 1, 10, and 25 ??g of atrazine/L groups, respectively. Ten months after metamorphosis, another subset of juveniles was examined, and the maximum number of testicular oocytes observed was five in one animal. The presence of testicular oocytes was not related to exposure to atrazine and may be a natural phenomenon during ontogeny. ?? 2005 American Chemical Society.

  18. Functional joint regeneration is achieved using reintegration mechanism in Xenopus laevis

    PubMed Central

    Yamada, Shigehito

    2016-01-01

    Abstract A functional joint requires integration of multiple tissues: the apposing skeletal elements should form an interlocking structure, and muscles should insert into skeletal tissues via tendons across the joint. Whereas newts can regenerate functional joints after amputation, Xenopus laevis regenerates a cartilaginous rod without joints, a “spike.” Previously we reported that the reintegration mechanism between the remaining and regenerated tissues has a significant effect on regenerating joint morphogenesis during elbow joint regeneration in newt. Based on this insight into the importance of reintegration, we amputated frogs’ limbs at the elbow joint and found that frogs could regenerate a functional elbow joint between the remaining tissues and regenerated spike. During regeneration, the regenerating cartilage was partially connected to the remaining articular cartilage to reform the interlocking structure of the elbow joint at the proximal end of the spike. Furthermore, the muscles of the remaining part inserted into the regenerated spike cartilage via tendons. This study might open up an avenue for analyzing molecular and cellular mechanisms of joint regeneration using Xenopus. PMID:27499877

  19. Occurrence of immune cells in the intestinal wall of Squalius cephalus infected with Pomphorhynchus laevis.

    PubMed

    Dezfuli, Bahram S; Manera, Maurizio; Giari, Luisa; DePasquale, Joseph A; Bosi, Giampaolo

    2015-11-01

    A sub-population of 34 specimens of chub, Squalius cephalus, was sampled from the River Brenta (Northern Italy) and examined for ecto- and endo-parasites. Pomphorhynchus laevis (Acanthocephala) was the only enteric helminth encountered. Immunofluorescence and ultrastructural studies were conducted on the intestines of chub. Near the site of parasite's attachment, mucous cells, mast cells (MCs), neutrophils and rodlet cells (RCs) were found to co-occur within the intestinal epithelium. The numbers of mucous cells, MCs and neutrophils were significantly higher in infected fish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the lectin Dolichos Biflorus Agglutinin (DBA) and the macrophage-specific MAC387 monoclonal antibody, with parallel transmission electron microscopy, revealed that epithelial MCs often made intimate contact with the mucous cells. Degranulation of a large number of MCs around the site of the acanthocephalan's attachment and in proximity to mucous cells was also documented. MCs and neutrophils were abundant in the submucosa. Immune cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions are discussed. PMID:26434712

  20. Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

    SciTech Connect

    Mertz, L.M.; Catt, K.J. )

    1991-10-01

    The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNA in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.

  1. Tolerance maintenance depends on persistence of the tolerizing antigen: evidence from transplantation studies on Xenopus laevis.

    PubMed

    Kaye, C; Schermer, J A; Tompkins, R

    1983-01-01

    In order to assess the role of antigen persistence in the tolerant state, tolerance was induced in Xenopus laevis by the embryonic transplantation of whole eyes or tail tissue. Both types of transplants were seen to heal in and persist, with no signs of immunological incompatibility. At metamorphosis, tail resorption occurred and grafted tail tissue was lost. Eye transplants were maintained through metamorphosis in most eye grafted animals. Eye graft recipients which had maintained the transplant were observed to accept challenge skin allografts from donors of the same genotype as the eye donor in all but one case, while recipients which had lost the eye transplant at metamorphosis or had the eye transplant experimentally removed sometimes did not accept the challenge skin graft. Animals tail grafted as embryos did not accept post metamorphic skin grafts from donors of the same genotype as the tail tissue donor, but rejection was not accelerated. It is proposed that tolerance induction is dependent on the presence of appropriately presented antigen at a time when precursor thymocyte cells are migrating to the thymus, prior to their processing into alloreactive cells, and that tolerance maintenance is dependent upon the persistence of the tolerizing antigen. PMID:6357880

  2. Characterization of tweety gene (ttyh1-3) expression in Xenopus laevis during embryonic development

    PubMed Central

    Rabe, Brian A.; Huyck, Ryan W.; Williams, Cheyenne C.; Saha, Margaret S.

    2015-01-01

    The tweety family of genes encodes large-conductance chloride channels and has been implicated in a wide array of cellular processes including cell division, cell adhesion, regulation of calcium activity, and tumorigenesis, particularly in neuronal cells. However, their expression patterns during early development remain largely unknown. Here, we describe the spatial and temporal patterning of ttyh1, ttyh2, and ttyh3 in Xenopus laevis during early embryonic development. Ttyh1 and ttyh3 are initially expressed at the late neurula stage are and primarily localized to the developing nervous system; however ttyh1 and ttyh3 both show transient expression in the somites. By swimming tadpole stages, all three genes are expressed in the brain, spinal cord, eye, and cranial ganglia. While ttyh1 is restricted to proliferative, ventricular zones, ttyh3 is primarily localized to postmitotic regions of the developing nervous system. Ttyh2, however, is strongly expressed in cranial ganglia V, VII, IX and X. The differing temporal and spatial expression patterns of ttyh1, ttyh2, and ttyh3 suggest that they may play distinct roles throughout embryonic development. PMID:25541457

  3. Microfluidic platform for electrophysiological studies on Xenopus laevis oocytes under varying gravity levels.

    PubMed

    Schaffhauser, Daniel F; Andrini, Olga; Ghezzi, Chiara; Forster, Ian C; Franco-Obregón, Alfredo; Egli, Marcel; Dittrich, Petra S

    2011-10-21

    Voltage clamp measurements reveal important insights into the activity of membrane ion channels. While conventional voltage clamp systems are available for laboratory studies, these instruments are generally unsuitable for more rugged operating environments. In this study, we present a non-invasive microfluidic voltage clamp system developed for the use under varying gravity levels. The core component is a multilayer microfluidic device that provides an immobilisation site for Xenopus laevis oocytes on an intermediate layer, and fluid and electrical connections from either side of the cell. The configuration that we term the asymmetrical transoocyte voltage clamp (ATOVC) also permits electrical access to the cytosol of the oocyte without physical introduction of electrodes by permeabilisation of a large region of the oocyte membrane so that a defined membrane patch can be voltage clamped. The constant low level air pressure applied to the oocyte ensures stable immobilisation, which is essential for keeping the leak resistance constant even under varying gravitational forces. The ease of oocyte mounting and immobilisation combined with the robustness and complete enclosure of the fluidics system allow the use of the ATOVC under extreme environmental conditions, without the need for intervention by a human operator. Results for oocytes over-expressing the epithelial sodium channel (ENaC) obtained under laboratory conditions as well as under conditions of micro- and hypergravity demonstrate the high reproducibility and stability of the ATOVC system under distinct mechanical scenarios.

  4. Male-Male Clasping May Be Part of an Alternative Reproductive Tactic in Xenopus laevis

    PubMed Central

    Rhodes, Heather J.; Stevenson, Rachel J.; Ego, Courtney L.

    2014-01-01

    Male Xenopus laevis frogs have been observed to clasp other males in a sustained, amplectant position, the purpose of which is unknown. We examined three possible hypotheses for this counter-intuitive behavior: 1) clasping males fail to discriminate the sex of the frogs they clasp; 2) male-male clasping is an aggressive or dominant behavior; or 3) that males clasp other males to gain proximity to breeding events and possibly engage in sperm competition. Our data, gathered through a series of behavioral experiments in the laboratory, refute the first two hypotheses. We found that males did not clasp indiscriminately, but showed a sex preference, with most males preferentially clasping a female, but a proportion preferentially clasping another male. Males that clasped another male when there was no female present were less likely to “win” reproductive access in a male-male-female triad, indicating that they did not establish dominance through clasping. However, those males did gain proximity to oviposition by continued male-male clasping in the presence of the female. Thus, our findings are consistent with, but cannot confirm, the third hypothesis of male-male clasping as an alternative reproductive tactic. PMID:24849114

  5. Metabolic cost of osmoregulation in a hypertonic environment in the invasive African clawed frog Xenopus laevis.

    PubMed

    Peña-Villalobos, Isaac; Narváez, Cristóbal; Sabat, Pablo

    2016-01-01

    Studies of aquatic invertebrates reveal that salinity affects feeding and growth rates, reproduction, survival, and diversity. Little is known, however, about how salinity impacts the energy budget of vertebrates and amphibians in particular. The few studies focused on this topic in vertebrates suggest that the ingestion of salts and the resulting osmoregulatory activity is energetically expensive. We analyzed the effect of saline acclimation on standard metabolic rates (SMR) and the activities of metabolic enzymes of internal organs and osmoregulatory variables (plasma osmolality and urea plasma level) in females of Xenopus laevis by means of acclimating individuals to an isosmotic (235 mOsm NaCl; ISO group) and hyper-osmotic (340 mOsm NaCl; HYP group) environment for 40 days. After acclimation, we found that total and mass-specific SMR was approximately 80% higher in the HYP group than those found in the ISO group. These changes were accompanied by higher citrate synthase activities in liver and heart in the HYP group than in the ISO group. Furthermore, we found a significant and positive correlation between metabolic rates and plasma urea, and citrate synthase activity in liver and heart. These results support the notion that the cost of osmoregulation is probably common in most animal species and suggest the existence of a functional association between metabolic rates and the adjustments in osmoregulatory physiology, such as blood distribution and urea synthesis. PMID:27334694

  6. Ectopic blastema induction by nerve deviation and skin wounding: a new regeneration model in Xenopus laevis

    PubMed Central

    Mitogawa, Kazumasa; Hirata, Ayako; Moriyasu, Miyuki; Makanae, Aki; Miura, Shinichirou; Endo, Tetsuya

    2014-01-01

    Abstract Recently, the accessory limb model (ALM) has become an alternative study system for limb regeneration studies in axolotls instead of using an amputated limb. ALM progresses limb regeneration study in axolotls because of its advantages. To apply and/or to compare knowledge in axolotl ALM studies to other vertebrates is a conceivable next step. First, Xenopus laevis, an anuran amphibian, was investigated. A Xenopus frog has hypomorphic regeneration ability. Its regeneration ability has been considered intermediate between that of non‐regenerative higher vertebrates and regenerative urodele amphibians. Here, we successfully induced an accessory blastema in Xenopus by skin wounding and rerouting of brachial nerve bundles to the wound site, which is the regular ALM surgery. The induced Xenopus ALM blastemas have limited regenerative potential compared with axolotl ALM blastemas. Comparison of ALM blastemas from species with different regenerative potentials may facilitate the identification of the novel expression programs necessary for the formation of cartilage and other tissues during limb regeneration. PMID:27499859

  7. Responses of young Xenopus laevis tadpoles to light dimming: possible roles for the pineal eye.

    PubMed

    Jamieson, D; Roberts, A

    2000-06-01

    When the light is dimmed, the pineal eye of hatchling Xenopus laevis tadpoles excites the central pattern generator for swimming, but the behavioural significance of pineal excitation is unclear. We show that tadpoles spend 99 % of their time hanging from the surface meniscus or solid objects using mucus secreted by a cement gland on the head. Attachment inhibits swimming, but unattached tadpoles swim spontaneously. Provided that their pineal eye is intact, they attach closer to the water surface in the dark than in the light and attach preferentially to the underside of floating objects that cast shadows. Dimming causes tadpoles swimming horizontally to turn upwards and is very effective in initiating upward swimming in unattached tadpoles. Similar pineal-dependent responses during swimming are present up to stage 44. Pinealectomy blocks responses to dimming at all stages. Recordings from immobilised tadpoles reveal that light dimming induces faster fictive swimming and that pineal activity is increased for up to 20 min during sustained light dimming. We suggest that the increase in pineal discharge during dimming increases the probability of upward swimming and, in this way, increases the probability of tadpoles attaching to objects higher in the water column that cast shadows.

  8. Platanna (Xenopus laevis) as a test organism for determining the embryotoxic effects of environmental chemicals

    SciTech Connect

    Dumpert, K.; Zietz, E.

    1984-02-01

    It has been successfully demonstrated that platanna (Xenopus laevis) allows the artificial induction of spawning at any time during the year. The number of eggs collected from a female ranged between 500 and 2400, the fertilization rate varying between 10 and 85%. When unaffected by chemicals, the embryonic development of the larvae took between 8 and 30 weeks. Di(2-ethylhexyl) phthalate (DEHP), methylmercury chloride, and the thalidomide analog EM 12 were used for the experiments described. DEHP at a concentration of 2 ppm retarded the development of the larvae and caused reduced pigmentation of the tadpoles. Methylmercury chloride has been found to have teratogenic and embryolethal effects at a concentration as low as 0.01 ppm. The following teratogenic effects have been determined: bent tails of the larvae, retarded development of the filter system, disturbed osmotic regulation, deranged positional and spatial orientation. EM 12 has been proven to have embryolethal effects at concentrations around 100 ppm. At lower concentrations this substance has teratogenic effects, i.e., it interferes in various ways with the development of the limbs.

  9. Gonadal development of larval male Xenopus laevis exposed to atrazine in outdoor microcosms.

    PubMed

    Jooste, Alarik M; Du Preez, Louis H; Carr, James A; Giesy, John P; Gross, Timothy S; Kendall, Ronald J; Smith, Ernest E; Van der Kraak, Glen L; Solomon, Keith R

    2005-07-15

    The potential effects of atrazine on gonadal development in metamorphs and subadults of the African clawed frog (Xenopus laevis) were studied under conditions of natural photoperiod and temperatures in outdoor microcosms from August 2002 to June 2003 in South Africa. Triplicate 1100 L microcosms for each nominal concentration of 0.0, 1, 10, and 25 microg of atrazine/L were used. Measured atrazine concentrations varied <25% throughout the study, and no atrazine was detected in the control microcosms. Tadpoles developed well at all concentrations. On the basis of histological examination of testes of recently metamorphosed stage 66 frogs, 57% of the individuals in the reference group exhibited testicular oocytes as compared with 57, 59, and 39% of the 1, 10, and 25 microg/L atrazine groups, respectively. The average prevalence of testicular oocytes for all of the treatments including the controls was 54% in a single testis, while, in 35% of individuals, testicular oocytes were observed in both testes. The number of testicular oocytes per individual ranged from 0 to 58 with means of 9.5, 9.8, 8.5, and 11.1 forthe 0.0, 1, 10, and 25 microg of atrazine/L groups, respectively. Ten months after metamorphosis, another subset of juveniles was examined, and the maximum number of testicular oocytes observed was five in one animal. The presence of testicular oocytes was not related to exposure to atrazine and may be a natural phenomenon during ontogeny.

  10. Intestinal immune response of Silurus glanis and Barbus barbus naturally infected with Pomphorhynchus laevis (Acanthocephala).

    PubMed

    Dezfuli, B S; Castaldelli, G; Bo, T; Lorenzoni, M; Giari, L

    2011-02-01

    Immunopathological and ultrastructural studies were conducted on the intestine of barbel Barbus barbus and sheatfish Silurus glanis that were naturally infected with the acanthocephalan Pomphorhynchus laevis. Enteric helminths often cause inflammation of the digestive tract, inducing the recruitment of different types of immune cells at the site of infection. The results of our study clearly demonstrated that mast cells (MC) were the dominant immune cells which occur at the site of inflammation in both hosts. MC were associated with fibroblasts and were found in close proximity to, and inside, the capillaries of the intestine, thus, migration of mast cells via the bloodstream was suggested. Significant degranulation of MC was present. Immunohistochemical staining revealed met-enkephalin and serotonin (5-HT) in intestinal MC of both uninfected and infected barbel and the absence of the antimicrobial peptides piscidin 3 and piscidin 4 in both species. Data are discussed with respect to host immune response to an intestinal helminth and compared with other host-parasite systems.

  11. Effect of chronic copper and pentachlorophenol exposure to early life stages of Xenopus laevis

    SciTech Connect

    Fort, D.J.; Stover, E.L.

    1995-12-31

    An evaluation of the effects of low-level copper and pentachlorophenol exposure on various early life stages of the South African clawed frog, Xenopus laevis was performed using stage-specific and long-term continuous exposures. Stage-specific exposure experiments were conducted such that separate subsets of embryos and larvae from the same clutch were exposed to two toxicants, copper and pentachlorophenol, from 0 d to 4 d (standard Frog Embryo Teratagenesis Assay Xenopus [FETAX]), 4 d to 8 d, 8 d to 12 d, and 12 d to 16 d. Results from two separate concentration-response experiments indicated that sensitivity to either toxicant increased in each successive time period. Continuous exposure studies conducted for 60 to 75 days indicated that copper, but not pentachlorophenol induced reduction deficiency malformations of the hind limb at concentrations as low as 0.05 mg/L. Pentachlorophenol concentrations as low as 0.5/{micro}g/L inhibited tail resorption. However, copper did not adversely affect the process of tail resorption. These results indicated that studies evaluating longer-term developmental processes are important in ecological hazard evaluation.

  12. Budgett’s frog (Lepidobatrachus laevis): a new amphibian embryo for developmental biology

    PubMed Central

    Amin, Nirav M.; Womble, Mandy; Ledon-Rettig, Cris; Hull, Margaret; Dickinson, Amanda; Nascone-Yoder, Nanette

    2015-01-01

    The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett’s frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett’s frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett’s tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett’s frog model provides inimitable advantages for developmental studies—and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution. PMID:26169245

  13. Functional joint regeneration is achieved using reintegration mechanism in Xenopus laevis.

    PubMed

    Tsutsumi, Rio; Yamada, Shigehito; Agata, Kiyokazu

    2016-02-01

    A functional joint requires integration of multiple tissues: the apposing skeletal elements should form an interlocking structure, and muscles should insert into skeletal tissues via tendons across the joint. Whereas newts can regenerate functional joints after amputation, Xenopus laevis regenerates a cartilaginous rod without joints, a "spike." Previously we reported that the reintegration mechanism between the remaining and regenerated tissues has a significant effect on regenerating joint morphogenesis during elbow joint regeneration in newt. Based on this insight into the importance of reintegration, we amputated frogs' limbs at the elbow joint and found that frogs could regenerate a functional elbow joint between the remaining tissues and regenerated spike. During regeneration, the regenerating cartilage was partially connected to the remaining articular cartilage to reform the interlocking structure of the elbow joint at the proximal end of the spike. Furthermore, the muscles of the remaining part inserted into the regenerated spike cartilage via tendons. This study might open up an avenue for analyzing molecular and cellular mechanisms of joint regeneration using Xenopus. PMID:27499877

  14. Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

    PubMed

    Boggavarapu, Rajendra; Hirschi, Stephan; Harder, Daniel; Meury, Marcel; Ucurum, Zöhre; Bergeron, Marc J; Fotiadis, Dimitrios

    2016-01-01

    This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail. PMID:27485339

  15. Metabolic cost of osmoregulation in a hypertonic environment in the invasive African clawed frog Xenopus laevis

    PubMed Central

    Peña-Villalobos, Isaac; Narváez, Cristóbal

    2016-01-01

    ABSTRACT Studies of aquatic invertebrates reveal that salinity affects feeding and growth rates, reproduction, survival, and diversity. Little is known, however, about how salinity impacts the energy budget of vertebrates and amphibians in particular. The few studies focused on this topic in vertebrates suggest that the ingestion of salts and the resulting osmoregulatory activity is energetically expensive. We analyzed the effect of saline acclimation on standard metabolic rates (SMR) and the activities of metabolic enzymes of internal organs and osmoregulatory variables (plasma osmolality and urea plasma level) in females of Xenopus laevis by means of acclimating individuals to an isosmotic (235 mOsm NaCl; ISO group) and hyper-osmotic (340 mOsm NaCl; HYP group) environment for 40 days. After acclimation, we found that total and mass-specific SMR was approximately 80% higher in the HYP group than those found in the ISO group. These changes were accompanied by higher citrate synthase activities in liver and heart in the HYP group than in the ISO group. Furthermore, we found a significant and positive correlation between metabolic rates and plasma urea, and citrate synthase activity in liver and heart. These results support the notion that the cost of osmoregulation is probably common in most animal species and suggest the existence of a functional association between metabolic rates and the adjustments in osmoregulatory physiology, such as blood distribution and urea synthesis. PMID:27334694

  16. Post-translational Regulation of Hexokinase Function and Protein Stability in the Aestivating Frog Xenopus laevis.

    PubMed

    Childers, Christine L; Storey, Kenneth B

    2016-02-01

    Xenopus laevis endure substantial dehydration which can impose hypoxic stress due to impaired blood flow. Tissues may increase reliance on anaerobic glycolysis for energy production making the regulation of hexokinase (HK) important. We investigated t