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Sample records for laevis morphogenetic factor

  1. Growth factor signaling in lung morphogenetic centers: automaticity, stereotypy and symmetry

    PubMed Central

    Warburton, David; Bellusci, Saverio; Del Moral, Pierre-Marie; Kaartinen, Vesa; Lee, Matt; Tefft, Denise; Shi, Wei

    2003-01-01

    Lung morphogenesis is stereotypic, both for lobation and for the first several generations of airways, implying mechanistic control by a well conserved, genetically hardwired developmental program. This program is not only directed by transcriptional factors and peptide growth factor signaling, but also co-opts and is modulated by physical forces. Peptide growth factors signal within repeating epithelial-mesenchymal temporospatial patterns that constitute morphogenetic centers, automatically directing millions of repetitive events during both stereotypic branching and nonstereotypic branching as well as alveolar surface expansion phases of lung development. Transduction of peptide growth factor signaling within these centers is finely regulated at multiple levels. These may include ligand expression, proteolytic activation of latent ligand, ligand bioavailability, ligand binding proteins and receptor affinity and presentation, receptor complex assembly and kinase activation, phosphorylation and activation of adapter and messenger protein complexes as well as downstream events and cross-talk both inside and outside the nucleus. Herein we review the critical Sonic Hedgehog, Fibroblast Growth Factor, Bone Morphogenetic Protein, Vascular Endothelial Growth Factor and Transforming Growth Factorβ signaling pathways and propose how they may be functionally coordinated within compound, highly regulated morphogenetic gradients that drive first stereotypic and then non-stereotypic, automatically repetitive, symmetrical as well as asymmetrical branching events in the lung. PMID:12818006

  2. 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.

  3. 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

  4. 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

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

    SciTech Connect

    Bogenhagen, D.F.; Insdorf, N.F.

    1988-07-01

    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/sup Phe/ and the displacement loop.

  6. 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

  7. Overexpression of the secreted factor Mig30 expressed in the Spemann organizer impairs morphogenetic movements during Xenopus gastrulation.

    PubMed

    Hayata, Tadayoshi; Tanegashima, Kosuke; Takahashi, Shuji; Sogame, Asako; Asashima, Makoto

    2002-03-01

    The Spemann organizer secretes several antagonists of growth factors during gastrulation. We describe a novel secreted protein, Mig30, which is expressed in the anterior endomesoderm of the Spemann organizer. Mixer-inducible gene 30 (Mig30) was isolated as a target of Mixer, a homeobox gene required for endoderm development. The Mig30 gene encodes a secreted protein containing a cysteine-rich domain and an immunoglobulin-like domain that belongs to the insulin-like growth factor-binding protein family. Overexpression of Mig30 in the dorsal region results in the retardation of morphogenetic movements during gastrulation and leads to microcephalic embryos. Overexpression of Mig30 also inhibits activin-induced elongation of ectodermal explants without affecting gene expression patterns in mesoderm and endoderm. These results suggest that Mig30 is involved in the regulation of morphogenetic movements during gastrulation in the extracellular space of the Spemann organizer. PMID:11850177

  8. Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells

    PubMed Central

    Boswell, Bruce A.; Musil, Linda S.

    2015-01-01

    Fibroblast growth factors (FGFs) play a central role in two processes essential for lens transparency—fiber cell differentiation and gap junction–mediated intercellular communication (GJIC). Using serum-free primary cultures of chick lens epithelial cells (DCDMLs), we investigated how the FGF and bone morphogenetic protein (BMP) signaling pathways positively cooperate to regulate lens development and function. We found that culturing DCDMLs for 6 d with the BMP blocker noggin inhibits the canonical FGF-to-ERK pathway upstream of FRS2 activation and also prevents FGF from stimulating FRS2- and ERK-independent gene expression, indicating that BMP signaling is required at the level of FGF receptors. Other experiments revealed a second type of BMP/FGF interaction by which FGF promotes expression of BMP target genes as well as of BMP4. Together these studies reveal a novel mode of cooperation between the FGF and BMP pathways in which BMP keeps lens cells in an optimally FGF-responsive state and, reciprocally, FGF enhances BMP-mediated gene expression. This interaction provides a mechanistic explanation for why disruption of either FGF or BMP signaling in the lens leads to defects in lens development and function. PMID:25947138

  9. Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro.

    PubMed

    Muñoz-Félix, José M; Cuesta, Cristina; Perretta-Tejedor, Nuria; Subileau, Mariela; López-Hernández, Francisco J; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-09-01

    Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors. PMID:27208502

  10. 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

  11. 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. PMID:25618295

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. Distinct patterns of endosulfatase gene expression during Xenopus laevis limb development and regeneration

    PubMed Central

    Wang, Yi‐Hsuan

    2015-01-01

    Abstract The heparan sulfate 6‐O‐endosulfatases sulf1 and sulf2 regulate multiple cellular processes and organ development. Sulfs modulate a range of heparan‐sulfate‐dependent extracellular pathways, including the fibroblast growth factor, bone morphogenetic protein, and wingless/wnt signaling pathways. Known patterns of sulf transcript expression together with functional experiments have implicated the sulfs in chondrogenesis and muscle regeneration in mammals. Here, we describe the expression patterns of Xenopus laevis sulf1 and sulf2 in developing forelimbs and hindlimbs and demonstrate novel expression of the sulf transcripts in the regenerating hindlimbs, with prominent sulf2 expression in the proliferating blastema and transient expression of sulf1 in the redeveloping apical epidermal ridge. These findings further suggest involvement of the sulfs in successful limb regeneration in amphibians.

  19. Bone Morphogenetic Proteins.

    PubMed

    Katagiri, Takenobu; Watabe, Tetsuro

    2016-01-01

    Bone morphogenetic proteins (BMPs), originally identified as osteoinductive components in extracts derived from bone, are now known to play important roles in a wide array of processes during formation and maintenance of various organs including bone, cartilage, muscle, kidney, and blood vessels. BMPs and the related "growth and differentiation factors" (GDFs) are members of the transforming growth factor β (TGF-β) family, and transduce their signals through type I and type II serine-threonine kinase receptors and their intracellular downstream effectors, including Smad proteins. Furthermore, BMP signals are finely tuned by various agonists and antagonists. Because deregulation of the BMP activity at multiple steps in signal transduction is linked to a wide variety of human diseases, therapeutic use of activators and inhibitors of BMP signaling will provide potential avenues for the treatment of the human disorders that are caused by hypo- and hyperactivation of BMP signals, respectively. PMID:27252362

  20. 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

  1. 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. PMID:26954112

  2. 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. PMID:25857705

  3. Adenovirus-mediated expression of vascular endothelial growth factor-a potentiates bone morphogenetic protein9-induced osteogenic differentiation and bone formation.

    PubMed

    Pi, Chang-Jun; Liang, Kai-Lu; Ke, Zhen-Yong; Chen, Fu; Cheng, Yun; Yin, Liang-Jun; Deng, Zhong-Liang; He, Bai-Cheng; Chen, Liang

    2016-08-01

    Mesenchymal stem cells (MSCs) are suitable seed cells for bone tissue engineering because they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Vascular endothelial growth factor-a (VEGF-a), an angiogenic factor, is also involved in osteogenesis and bone repair. However, the effects of VEGF-a on osteogenic MSCs differentiation remain unknown. It was previously reported that bone morphogenetic protein9 (BMP9) is one of the most important osteogenic BMPs. Here, we investigated the effects of VEGF-a on BMP9-induced osteogenesis with mouse embryo fibroblasts (MEFs). We found that endogenous VEGF-a expression was undetectable in MSCs. Adenovirus-mediated expression of VEGF-a in MEFs potentiated BMP9-induced early and late osteogenic markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In stem cell implantation assays, VEGF-a augmented BMP9-induced ectopic bone formation. VEGF-a in combination with BMP9 effectively increased the bone volume and osteogenic activity. However, the synergistic effect was efficiently abolished by the phosphoinositide 3-kinase (PI3K)/AKT inhibitor LY294002. These results demonstrated that BMP9 may crosstalk with VEGF-a through the PI3K/AKT signaling pathway to induce osteogenic differentiation in MEFs. Thus, our findings demonstrate the effects of VEGF-a on BMP9-induced bone formation and provide a new potential strategy for treating nonunion fractures, large segmental bony defects, and/or osteoporotic fractures. PMID:27003241

  4. 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

  5. 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

  6. Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells.

    PubMed

    Luo, Qing; Kang, Quan; Si, Weike; Jiang, Wei; Park, Jong Kyung; Peng, Ying; Li, Xinmin; Luu, Hue H; Luo, Jeffrey; Montag, Anthony G; Haydon, Rex C; He, Tong-Chuan

    2004-12-31

    Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was beta-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9- and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells. PMID:15496414

  7. 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

  8. 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

  9. 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. PMID:26928288

  10. Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling.

    PubMed

    Mandal, Chandi C; Das, Falguni; Ganapathy, Suthakar; Harris, Stephen E; Choudhury, Goutam Ghosh; Ghosh-Choudhury, Nandini

    2016-01-15

    Bone remodeling is controlled by dual actions of osteoclasts (OCs) and osteoblasts (OBs). The calcium-sensitive nuclear factor of activated T cells (NFAT) c1 transcription factor, as an OC signature gene, regulates differentiation of OCs downstream of bone morphogenetic protein-2 (BMP-2)-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1, we analyzed bones from OB-specific BMP-2 knock-out mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OBs. We showed that BMP-2, via the receptor-specific Smad pathway, regulates expression of NFATc1 in OBs. Phosphatidylinositol 3-kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under the basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. 1,2Bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester, an inhibitor of intracellular calcium abundance, blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity, resulting in NFATc1 nuclear translocation. Cyclosporin A, which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription, confirming its autoregulation. Together, these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk among Smad, phosphatidylinositol 3-kinase/Akt, and Ca(2+) signaling for BMP-2-induced NFATc1 expression through

  11. 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

  12. Role of growth differentiation factor-5 and bone morphogenetic protein type II receptor in the development of lumbar intervertebral disc degeneration

    PubMed Central

    Li, Yi-Fan; Tang, Xian-Zhong; Liang, Chao-Ge; Hui, Yao-Ming; Ji, Yun-Han; Xu, Wei; Qiu, WenJun; Cheng, Li-Ming

    2015-01-01

    The present study was designed to evaluate the role of growth differentiation factor-5 (GDF-5) and bone morphogenetic protein type II receptor (BMPR-II) in the development of lumbar intervertebral disc degeneration (IDD). A total of 24 patients with lumbar IDD (experiment group) and 6 patients with lumbar vertebral fracture (control group) were enrolled in the study. Tissue samples of IVD from the experiment group and control group were obtained during lumbar fusion operation, respectively. Fixation and decalcification of IVD tissue were performed, and then HE staining was carried out to observe the morphological changes of the lumbar IVD tissues. The expression of GDF-5 and BMPRII in human lumbar IVD was detected by immunohistochemical staining. HE staining results showed that non- and minimal degeneration was found in 11 cases (score range, 0-3), moderate degeneration in 12 cases (score range, 4-8), and severe degeneration in 7 cases (score range, 9-12). According to the immunohistochemical results, the positive expression rates of GDF-5 and BMPRII in NP were higher than those in AF of the non- and minimal degeneration group, moderate degeneration group and severe degeneration group (all P < 0.05). However, no significant difference in GDF-5 or BMPRII positive expression was observed among the normal, non- and minimal, moderate and severe degeneration groups in neither NP area nor AF area (all P > 0.05). In conclusion, our results showed that GDF-5 and BMPRII expressed both in normal and degenerated IVD tissues, and GDF-5 might have an inhibition effect on degenerated lumbar IVD, suggesting that gene therapy may be a useful approach in producing physiological effects during early- and late-phase of lumbar IDD. PMID:25755766

  13. Alterations in bone morphogenetic protein 15, growth differentiation factor 9, and gene expression in granulosa cells in preovulatory follicles of dairy cows given porcine LH.

    PubMed

    Behrouzi, Amir; Colazo, Marcos Germán; Ambrose, Divakar Justus

    2016-04-15

    In a previous work, using porcine LH (pLH) in lieu of GnRH for synchronizing ovulation in dairy cows improved pregnancy rates without increasing plasma progesterone concentrations after ovulation. The LH profile is known to remain elevated above basal concentrations (≥1 ng/mL) for up to 20 hours in pLH-treated cows compared to less than 6 hours in GnRH-treated cows. Because LH triggers a cascade of signaling networks in the preovulatory follicle to promote final maturation and support oocyte competence, we hypothesized that dissimilar LH profiles will differentially regulate the intrafollicular factors and expression of downstream genes associated with improved oocyte competence. Specific objectives were to determine differences in the abundance of oocyte-secreted factors in the preovulatory follicular fluid and target genes in granulosa cells associated with oocyte competence, in response to exogenous porcine LH or GnRH-induced endogenous bovine LH exposure, in dairy cows. Follicular contents were aspirated by a transvaginal ultrasound-guided procedure from the preovulatory follicle of cyclic, nonlactating Holstein cows 21 ± 1 hour after administration of either pLH (25-mg) or GnRH (100-μg). Mature forms of bone morphogenetic protein 15, growth differentiation factor 9, and transforming growth factorβ1 were approximately 2-fold more abundant in pLH-treated cows which were exposed to an extended, low LH profile, than in GnRH-treated cows that had a short, high LH profile. The relative abundance of messenger RNA for cyclooxygenase-2, LH receptor, and progesterone receptor in granulosa cells, was about two-, eight-, and two-fold higher, respectively, in cows subjected to pLH than GnRH treatment. We infer that the improved pregnancy rate after pLH-induced ovulation reported previously, occurred through greater activation of intrafollicular transforming growth factor-β1 superfamily members, as these proteins promote cumulus expansion and oocyte competence

  14. The Chordin Morphogenetic Pathway.

    PubMed

    De Robertis, Edward M; Moriyama, Yuki

    2016-01-01

    The ancestral Chordin/bone morphogenetic protein (BMP) signaling pathway that establishes dorsal-ventral (D-V) patterning in animal development is one of the best understood morphogenetic gradients, and is established by multiple proteins that interact with each other in the extracellular space-including several BMPs, Chordin, Tolloid, Ont-1, Crossveinless-2, and Sizzled. The D-V gradient is adjusted redundantly by regulating the synthesis of its components, by direct protein-protein interactions between morphogens, and by long-range diffusion. The entire embryo participates in maintaining the D-V BMP gradient, so that for each action in the dorsal side there is a reaction in the ventral side. A gradient of Chordin is formed in the extracellular matrix that separates ectoderm from endomesoderm, called Brachet's cleft in Xenopus. The Chordin/BMP pathway is self-organizing and able to scale pattern in the dorsal half of bisected embryos or in Spemann dorsal lip transplantation experiments. PMID:26970622

  15. 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

  16. 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

  17. Menin is required for bone morphogenetic protein 2- and transforming growth factor beta-regulated osteoblastic differentiation through interaction with Smads and Runx2.

    PubMed

    Sowa, Hideaki; Kaji, Hiroshi; Hendy, Geoffrey N; Canaff, Lucie; Komori, Toshihisa; Sugimoto, Toshitsugu; Chihara, Kazuo

    2004-09-24

    Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. Biol. Chem. 278, 21058-21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA (AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late

  18. 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

  19. 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

  20. 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

  1. Demineralized Bone Matrix Combined Bone Marrow Mesenchymal Stem Cells, Bone Morphogenetic Protein-2 and Transforming Growth Factor-β3 Gene Promoted Pig Cartilage Defect Repair

    PubMed Central

    Wang, Xin; Li, Yanlin; Han, Rui; He, Chuan; Wang, Guoliang; Wang, Jianwei; Zheng, Jiali; Pei, Mei; Wei, Lei

    2014-01-01

    Objectives To investigate whether a combination of demineralized bone matrix (DBM) and bone marrow mesenchymal stem cells (BMSCs) infected with adenovirus-mediated- bone morphogenetic protein (Ad-BMP-2) and transforming growth factor-β3 (Ad-TGF-β3) promotes the repair of the full-thickness cartilage lesions in pig model. Methods BMSCs isolated from pig were cultured and infected with Ad-BMP-2(B group), Ad-TGF-β3 (T group), Ad-BMP-2 + Ad-TGF-β3(BT group), cells infected with empty Ad served as a negative group(N group), the expression of the BMP-2 and TGF-β3 were confirmed by immunofluorescence, PCR, and ELISA, the expression of SOX-9, type II collagen(COL-2A), aggrecan (ACAN) in each group were evaluated by real-time PCR at 1w, 2w, 3w, respectively. The chondrogenic differentiation of BMSCs was evaluated by type II collagen at 21d with immunohistochemical staining. The third-passage BMSCs infected with Ad-BMP-2 and Ad-TGF-β3 were suspended and cultured with DBM for 6 days to construct a new type of tissue engineering scaffold to repair full-thickness cartilage lesions in the femur condyles of pig knee, the regenerated tissue was evaluated at 1,2 and 3 months after surgery by gross appearance, H&E, safranin O staining and O'driscoll score. Results Ad-BMP-2 and Ad-TGF-β3 (BT group) infected cells acquired strong type II collagen staining compared with Ad-BMP-2 (B group) and Ad-TGF-β3 (T group) along. The Ad-BMP-2 and Ad-TGF-β3 infected BMSCs adhered and propagated well in DBM and the new type of tissue engineering scaffold produced hyaline cartilage morphology containing a stronger type II collagen and safranin O staining, the O'driscoll score was higher than other groups. Conclusions The DBM compound with Ad-BMP-2 and Ad-TGF-β3 infected BMSCs scaffold has a good biocompatibility and could well induce cartilage regeneration to repair the defects of joint cartilage. This technology may be efficiently employed for cartilage lesions repair in vivo. PMID

  2. The unique myelopoiesis strategy of the amphibian Xenopus laevis.

    PubMed

    Yaparla, Amulya; Wendel, Emily S; Grayfer, Leon

    2016-10-01

    Myeloid progenitors reside within specific hematopoietic organs and commit to progenitor lineages bearing megakaryocyte/erythrocyte (MEP) or granulocyte/macrophage potentials (GMP) within these sites. Unlike other vertebrates, the amphibian Xenopus laevis committed macrophage precursors are absent from the hematopoietic subcapsular liver and instead reside within their bone marrow. Presently, we demonstrate that while these frogs' liver-derived cells are unresponsive to recombinant forms of principal X. laevis macrophage (colony-stimulating factor-1; CSF-1) and granulocyte (CSF-3) growth factors, bone marrow cells cultured with CSF-1 and CSF-3 exhibit respectively archetypal macrophage and granulocyte morphology, gene expression and functionalities. Moreover, we demonstrate that liver, but not bone marrow cells possess erythropoietic capacities when stimulated with a X. laevis erythropoietin. Together, our findings indicate that X. laevis retain their MEP within the hematopoietic liver while sequestering their GMP to the bone marrow, thus marking a very novel myelopoietic strategy as compared to those seen in other jawed vertebrate species. PMID:27234705

  3. 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

  4. 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

  5. In X. laevis embryos high levels of the anti-apoptotic factor p27BBP/eIF6 are stage-dependently found in BrdU and TUNEL-reactive territories.

    PubMed

    De Marco, N; Campanella, C; Carotenuto, R

    2011-05-01

    p27BBP/eIF6 (β4 binding protein/eukaryotic initiation factor 6) is a highly conserved protein necessary for cell life. In adult eIF6 mice, a 50% decrease in the protein levels in all tissues is accompanied by a reduction in cell proliferation only in the liver, fat cells and cultured fibroblasts. During X. laevis embryogenesis expression of p27BBP/eIF6 is abundant in high proliferative territories. However, in Xenopus cell proliferation appears unaffected following p27BBP/eIF6 over-expression or down-regulation. Indeed, p27BBP/eIF6 is an anti-apoptotic factor acting upstream of Bcl2 that reduces endogenous apoptosis. We studied p27BBP/eIF6 protein localization in wild type embryos and compared it to proliferation and apoptosis. At the beginning of embryogenesis, high levels of p27BBP/eIF6, proliferation and apoptosis overlap. In later development stages high proliferation levels are present in the same regions where higher p27BBP/eIF6 expression is observed, while apoptosis does not appear specifically concentrated in the same sites. The higher presence of p27BBP/eIF6 would appear related to an increased need of apoptosis control in the regions where cell death is essential for normal development. PMID:20663234

  6. 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

  7. Metaphase protein phosphorylation in Xenopus laevis eggs.

    PubMed Central

    Lohka, M J; Kyes, J L; Maller, J L

    1987-01-01

    Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior. Images PMID:3821728

  8. 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

  9. 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

  10. 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

  11. Differential effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of collagen-modifying enzymes in human adipose tissue-derived mesenchymal stem cells.

    PubMed

    Knippenberg, Marlene; Helder, Marco N; Doulabi, Behrouz Zandieh; Bank, Ruud A; Wuisman, Paul I J M; Klein-Nulend, Jenneke

    2009-08-01

    Adipose tissue-derived mesenchymal stem cells (AT-MSCs) in combination with bone morphogenetic protein-2 (BMP-2) or transforming growth factor-beta1 (TGF-beta1) are under evaluation for bone tissue engineering. Posttranslational modification of type I collagen is essential for functional bone tissue with adequate physical and mechanical properties. We investigated whether BMP-2 (10-100 ng/mL) and/or TGF-beta1 (1-10 ng/mL) affect gene expression of alpha2(I) procollagen and collagen-modifying enzymes, that is, lysyl oxidase and lysyl hydroxylases 1, 2, and 3 (encoded by PLOD1, 2, and 3), by human AT-MSCs. BMP-2, but not TGF-beta1, increased alkaline phosphatase activity after 28 days, indicating osteogenic differentiation of AT-MSCs. At day 4, both BMP-2 and TGF-beta1 upregulated alpha2(I) procollagen and PLOD1, which was downregulated at day 28. TGF-beta1, but not BMP-2, downregulated PLOD3 at day 28. Lysyl oxidase was upregulated by TGF-beta1 at day 4 and by BMP-2 at day 7. Neither BMP-2 nor TGF-beta1 affected PLOD2. In conclusion, these results suggest that AT-MSCs differentially respond to BMP-2 and TGF-beta1 with changes in gene expression of collagen-modifying enzymes. AT-MSCs may thus be able to appropriately modify type I collagen to form a functional bone extracellular matrix for tissue engineering, dependent on the growth factor added. PMID:19231972

  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. Probing the Xenopus laevis inner ear transcriptome for biological function

    PubMed Central

    2012-01-01

    Background The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. Results Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip® Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. Conclusions We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the

  14. Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.

    PubMed

    Sapkota, Gopal; Knockaert, Marie; Alarcón, Claudio; Montalvo, Ermelinda; Brivanlou, Ali H; Massagué, Joan

    2006-12-29

    Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state. PMID:17085434

  15. Protein kinase signalling pathways involved in the up-regulation of the rat alpha1(I) collagen gene by transforming growth factor beta1 and bone morphogenetic protein 2 in osteoblastic cells.

    PubMed Central

    Palcy, S; Goltzman, D

    1999-01-01

    Transforming growth factor beta (TGFbeta) family members are known for their important role in bone physiology. TGFbeta(1) and, to a smaller extent, bone morphogenetic protein 2 (BMP-2) have been reported to regulate the gene expression of different osteoblast markers in vitro. However, little is known about the molecular mechanisms involved in these actions. Here we report that BMP-2, like TGFbeta(1), up-regulated alpha1(I) collagen mRNA expression in ROS 17/2.8 osteoblastic cells. This was mediated through an increase in the transcriptional rate of the gene rather than through the stabilization of alpha1(I) collagen mRNA, and required new protein synthesis. In addition, TGFbeta(1)- and BMP-2-induced increases in alpha1(I) collagen mRNA levels were both dependent on protein kinase C and protein tyrosine kinase activities. Furthermore, the mitogen-activated protein kinase (MAPK) [MAPK/extracellular signal-regulated protein kinase kinase 1/extracellular signal-regulated protein kinase (MEK-1/ERK)] pathway participated in the up-regulation of alpha1(I) collagen gene expression by TGFbeta(1) and BMP-2. In response to either TGFbeta(1) or BMP-2, the stimulation of alpha1(I) collagen mRNA levels was paralleled by an early increase in extracellular signal-regulated kinase protein activity. Moreover, the effects of both TGFbeta(1) and BMP-2 on alpha1(I) collagen gene expression were markedly decreased in transfected ROS 17/2.8 cells expressing a dominant-negative MEK-1. Our findings therefore show that TGFbeta(1) and BMP-2, which signal through discrete cell-surface receptors, are able to trigger analogous, if not identical, protein-phosphorylation-transducing cascades leading to comparable actions on the transcription of the alpha1(I) collagen gene in osteoblastic cells. PMID:10493907

  16. Synergistic induction of early stage of bone formation by combination of recombinant human bone morphogenetic protein-2 and epidermal growth factor

    PubMed Central

    Lee, Jae Hyup; Jang, Soo-Jeong; Baek, Hae-Ri; Lee, Kyung Mee; Chang, Bong-Soon; Lee, Choon-Ki

    2015-01-01

    This study evaluates whether the combination of the rhBMP-2 and various types of growth factors including EGF, FGF, PDGF and VEGF increases osteoinductivity compared to the single use of rhBMP-2 through in vitro and in vivo study. Cultured human MSCs were treated with rhBMP-2 only or in combination with growth factors. For in vivo evaluation, rhBMP-2 only or with growth factors was implanted into the calvarial defect made on SD rats. Both EGF and PDGF significantly increased both ALP activity and expression level in hMSCs when treated in combination with rhBMP-2 at 3 and 7 days of differentiation and significantly raised the accumulation of the calcium at day 14. Furthermore, micro-CT scanning revealed that the EGF an FGF groups show significantly increased new bone surface ratio compared to the rhBMP-2 only group and, the EGF treatment significantly up regulated percent bone volume and trabecular number at two weeks after the surgery. VEGF treatment also significantly raised trabecular number and FGF treatment significantly increased the trabecular thickness. Histological examination revealed that the EGF combination group showed enhanced bone regeneration than the rhBMP-2 only group two weeks after the implantation. Even though the treatment of rhBMP-2 with PDGF and FGF failed to show enhanced osteogenesis in vitro and in vivo simultaneously, these results suggest that the positive effect of the combination of EGF and rhBMP-2 is expected to induce the bone formation earlier compared to the single use of rhBMP-2 in vitro and in vivo. © 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd. PMID:24764222

  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. Replication of ribosomal DNA in Xenopus laevis.

    PubMed

    Bozzoni, I; Baldari, C T; Amaldi, F; Buongiorno-Nardelli, M

    1981-09-01

    The study of the localization of the replication origins of rDNA in Xenopus laevis has been approached by two different methods. 1. The DNA of X. laevis larvae was fractionated by CsCl gradient centrifugation in bulk and ribosomal DNA and examined in the electron microscope. In bulk DNA, clusters of microbubbles, which are related with the origins of replication, appear to be spaced along the DNA molecules at intervals comparable with the size of the 'average' replicon of X. laevis. In ribosomal DNA, the distance between adjacent clusters is much shorter and corresponds to the size of the rDNA repeating unit. When ribosomal DNA was submitted to digestion with restriction enzymes (Eco RI and HindIII) the microbubbles are observed in the non-transcribed spacer-containing fragment. 2. Cultured cells of X. laevis were synchronized by mitotic selection and incubated with 5-fluoro-2-deoxyuridine for a time longer than the G1 phase. This treatment synchronizes the replicons and allows them to start replicating very slowly. It was thus possible to obtain a preferential labelling of the regions containing the origins. The analysis by gel electrophoresis of the Eco Ri-digested rDNA showed that the radioactivity was preferentially incorporated in the fragments which contain the non-transcribed spacer. The results of these two approaches indicate that the rRNA gene cluster consists of multiple units of replication, possibly one per gene unit. Furthermore they show that the origins of replication are localized into the non-transcribed spacer. PMID:7297565

  19. Is metal accumulation in Pomphorhynchus laevis dependent on parasite sex or infrapopulation size?

    PubMed

    Nachev, M; Zimmermann, S; Rigaud, T; Sures, B

    2010-07-01

    Concentrations of the elements As, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, Sn, V, Zn were analysed by inductively coupled plasma mass spectrometry (ICP-MS) in the acanthocephalan Pomphorhynchus laevis and its fish host Barbus barbus. A total of 27 barbels were collected from the Danube River in autumn 2006 close to the town Kozloduy (685 river kilometer) on the Bulgarian river bank. Fish were divided into 3 groups. According to their P. laevis infrapopulation size hosts were considered as heavily infected (>100 worms per fish) and lightly infected (<20 worms per fish). The third group was used to compare heavy metal concentrations between male and female P. laevis. The 5 elements As, Cd, Cu, Pb and Zn were detected in significantly higher concentrations in parasites compared to host tissues (muscle, intestine, liver). According to the calculated mean bioconcentration factors, 3 more elements (Co, Mn, V) showed usually higher concentrations in P. laevis. Comparisons between heavily and lightly infected fish revealed significant differences only for V with higher concentrations for the heavily infected group. Concerning sex-specific metal accumulation V and Zn showed significant differences (V, at P<0.05; Zn, at P=0.05), with higher levels of both metals in females of P. laevis. Our results suggest that - for the metals analysed - the size of the parasite infrapopulation plays no role in the degree of metal accumulation. Similarly, parasite sex seems not to be a crucial factor for metal accumulation in the parasites. Thus, for metal monitoring purposes there is no need to take these aspects into account, which makes the use of parasites as bioindicators more applicable. PMID:20380766

  20. [Bone morphogenetic proteins (BMP): clinical application for reconstruction of bone defects].

    PubMed

    Sierra-García, Gerardo Daniel; Castro-Ríos, Rocío; Gónzalez-Horta, Azucena; Lara-Arias, Jorge; Chávez-Montes, Abelardo

    2016-01-01

    Since the introduction of bone morphogenetic proteins, their use has become an invaluable ally for the treatment of bone defects. These proteins are potent growth factors, related to angiogenic and osteogenic activity. The osteoinductive capacity of recombinant bone morphogenetic protein (rhBMP) in the formation of bone and cartilage has been confirmed in in vitro studies and evaluated in clinical trials. To obtain a therapeutic effect, administration is systemic, by injection over the physiological dose. Among the disadvantages, ectopic bone formation or high morbidity in cases of spinal fusion is observed. In this review, the roles of bone morphogenetic proteins in bone repair and clinical applications are analyzed. These findings represent advances in the study of bone regeneration and application of growth factors for more predictable results. PMID:27335195

  1. Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation.

    PubMed Central

    Aoufouchi, S; Shall, S

    1997-01-01

    Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation. PMID:9230139

  2. 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

  3. 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

  4. 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

  5. α1-adrenergic receptor signaling in osteoblasts regulates clock genes and bone morphogenetic protein 4 expression through up-regulation of the transcriptional factor nuclear factor IL-3 (Nfil3)/E4 promoter-binding protein 4 (E4BP4).

    PubMed

    Hirai, Takao; Tanaka, Kenjiro; Togari, Akifumi

    2014-06-13

    Several studies have demonstrated that the α1-adrenergic receptor (AR) plays an important role in regulating cell growth and function in osteoblasts. However, the physiological role of α1-AR signaling in bone metabolism is largely unknown. In this study, the stimulation of phenylephrine (PHE), a nonspecific α1-AR agonist, increased the transcriptional factor Nfil3/E4BP4 and led to the rhythmic expression of bone morphogenetic protein 4 (Bmp4) in MC3T3-E1 osteoblastic cells. We also showed that Bmp4 mRNA expression peaked in bone near zeitgeber time 8 in a 24-h rhythm. Furthermore, the expression of Nfil3 and Bmp4 displayed a circadian pattern with opposing phases, which suggested that Nfil3 repressed the expression of the Bmp4 gene during a circadian cycle. On a molecular level, both loss-of-function and gain-of-function experiments demonstrated that Nfil3/E4BP4 negatively regulated Bmp4 expression in osteoblasts. Furthermore, the systemic administration of PHE increased the expression of Nfil3 mRNA in bone, whereas it decreased that of Bmp4 mRNA. The expression of Bmp4 mRNA was decreased significantly by exposure to PHE, and this was concomitant with the increase in Nfil3 binding to the D-box-containing Bmp4 promoter region in MC3T3-E1 cells, which indicates that the expression of Nfil3 by α1-AR signaling can bind directly to the Bmp4 promoter and inhibit Bmp4 expression in osteoblasts. Our results suggest that α1-AR signaling regulates clock genes and Bmp4 expression in osteoblasts. Moreover, α1-AR signaling negatively regulated Bmp4 expression by up-regulating the transcriptional factor Nfil3/E4BP4 in osteoblasts. PMID:24794868

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Occludin and hydromineral balance in Xenopus laevis.

    PubMed

    Chasiotis, Helen; Kelly, Scott P

    2009-01-01

    To investigate the response of the tight junction (TJ) protein occludin to environmental change in an anuran amphibian, we examined occludin tissue distribution, immunolocalization and alterations in mRNA expression in African clawed frogs (Xenopus laevis) acclimated to brackish water (BW) conditions (from freshwater to 2 per thousand, 5 per thousand or 10 per thousand salt water). Occludin mRNA is widely expressed in Xenopus and is abundant in tissues involved in regulating salt and water balance, such as the gastrointestinal (GI) tract, kidney and urinary bladder. Immunohistochemical analyses revealed strong occludin immunolabelling in the apicolateral region of epithelia lining the GI tract and mRNA expression increased along the longitudinal axis of the gut. In kidney tissue, occludin was differentially expressed on the luminal side of the nephron tubule, appearing in the distal tubules and collecting ducts only. In response to BW acclimation, Xenopus exhibited a significant loss of tissue water as well as salinity-dependent elevations in serum osmolality as a result of increased urea levels followed by elevated serum Na(+) and Cl(-) levels. Tissue-specific alterations in the ionomotive enzyme Na(+),K(+)-ATPase were also observed in Xenopus in response to BW acclimation. Most notably, Na(+),K(+)-ATPase activity in the rectum increased in response to elevated environmental salt concentrations while renal activity decreased. Furthermore, acclimation to BW caused tissue-specific and salinity-dependent alterations in occludin mRNA expression within select Xenopus osmoregulatory organs. Taken together, these studies suggest that alterations in occludin, in conjunction with active transport processes, may contribute to amphibian hydromineral homeostasis during environmental change. PMID:19112148

  12. 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. PMID:12673597

  13. Bone morphogenetic protein 4 mediates human embryonic germ cell derivation.

    PubMed

    Hiller, Marc; Liu, Cyndi; Blumenthal, Paul D; Gearhart, John D; Kerr, Candace L

    2011-02-01

    Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and/or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)-polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency. PMID:20486775

  14. A transgenic Xenopus laevis reporter model to study lymphangiogenesis.

    PubMed

    Ny, Annelii; Vandevelde, Wouter; Hohensinner, Philipp; Beerens, Manu; Geudens, Ilse; Diez-Juan, Antonio; Brepoels, Katleen; Plaisance, Stéphane; Krieg, Paul A; Langenberg, Tobias; Vinckier, Stefan; Luttun, Aernout; Carmeliet, Peter; Dewerchin, Mieke

    2013-01-01

    The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening. PMID:24143274

  15. 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

  16. 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

  17. Bone morphogenetic protein-2: a potential regulator in scleral remodeling

    PubMed Central

    Hu, Jianmin; Cui, Dongmei; Yang, Xiao; Wang, Shaowei; Hu, Shoulong; Li, Chuanxu

    2008-01-01

    Purpose Bone morphogenetic protein 2 (BMP-2) is a member of the main subgroup of bone morphogenetic proteins within the transforming growth factor-β superfamily. BMP-2 is involved in numerous cellular functions including development, cell proliferation, apoptosis, and extracellular matrix synthesis. We examined BMP-2 expression in human scleral fibroblasts (HSF) and assessed the effects of recombinant human BMP-2 (rhBMP-2) on HSF proliferation, matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Methods We used confocal fluorescence microscopy (CFM) to study BMP-2 distribution in HSF cells and frozen human scleral sections. The influence of rhBMP-2 on cell proliferation at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml) was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The effects of rhBMP-2 on the cell cycle were investigated with flow cytometric analysis. Reverse transcription polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine MMP-2 and TIMP-2 mRNAs and secreted proteins in HSF that were incubated with rhBMP-2. Results BMP-2 protein expression from human sclera was confirmed by CFM. Cell proliferation was significantly increased with 100 ng/ml rhBMP-2 in a time-dependent manner (p<0.05). The HSF cell cycle moved to the S and S+G2M phases after rhBMP-2 stimulation at 100 ng/ml compared to normal cells (p<0.05). TIMP-2 mRNA levels were significantly increased in HSF incubated for 24 h with 100 ng/ml rhBMP-2 (p<0.01). A 48 h incubation with 10 ng/ml or 100 ng/ml rhBMP-2 resulted in significantly increased TIMP-2 mRNA and protein expression and significantly decreased MMP-2 mRNA expression (p<0.01) while MMP-2 protein expression significantly decreased at 100 ng/ml rhBMP-2 (p<0.01). Conclusions Human sclera fibroblasts expressed BMP-2, which promoted cell proliferation, and elicited changes in MMP-2 and TIMP-2

  18. The role of Sdf-1α signaling in Xenopus laevis somite morphogenesis

    PubMed Central

    Leal, Marisa A.; Fickel, Sarah R.; Sabillo, Armbien; Ramirez, Julio; Vergara, Hernando Martínez; Nave, Ceazar; Saw, Daniel; Domingo, Carmen R.

    2014-01-01

    Background Stromal derived factor-1α (sdf-1α), a chemoattractant chemokine, plays a major role in tumor growth, angiogenesis, metastasis and in embryogenesis. The sdf-1α signaling pathway has also been shown to be important for somite rotation in zebrafish (Hollway, et al 2007). Given the known similarities and differences between zebrafish and Xenopus laevis somitogenesis, we sought to determine whether the role of sdf-1α is conserved in Xenopus laevis. Results Using a morpholino approach, we demonstrate that knockdown of sdf-1α or its receptor, cxcr4, leads to a significant disruption in somite rotation and myotome alignment. We further show that depletion of sdf-1α or cxcr4 leads to the near absence of β-dystroglycan and laminin expression at the intersomitic boundaries. Finally, knockdown of sdf-1α decreases the level of activated RhoA, a small GTPase known to regulate cell shape and movement. Conclusion Our results show that sdf-1α signaling regulates somite cell migration, rotation and myotome alignment by directly or indirectly regulating dystroglycan expression and RhoA activation. These findings support the conservation of sdf-1α signaling in vertebrate somite morphogenesis; however, the precise mechanism by which this signaling pathway influences somite morphogenesis is different between the fish and the frog. PMID:24357195

  19. 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

  20. 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

  1. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

    PubMed

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

    2016-06-10

    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

  2. Role of bone morphogenetic protein-7 in renal fibrosis

    PubMed Central

    Li, Rui Xi; Yiu, Wai Han; Tang, Sydney C. W.

    2015-01-01

    Renal fibrosis is final common pathway of end stage renal disease. Irrespective of the primary cause, renal fibrogenesis is a dynamic process which involves a large network of cellular and molecular interaction, including pro-inflammatory cell infiltration and activation, matrix-producing cell accumulation and activation, and secretion of profibrogenic factors that modulate extracellular matrix (ECM) formation and cell-cell interaction. Bone morphogenetic protein-7 is a protein of the TGF-β super family and increasingly regarded as a counteracting molecule against TGF-β. A large variety of evidence shows an anti-fibrotic role of BMP-7 in chronic kidney disease, and this effect is largely mediated via counterbalancing the profibrotic effect of TGF-β. Besides, BMP-7 reduced ECM formation by inactivating matrix-producing cells and promoting mesenchymal-to-epithelial transition (MET). BMP-7 also increased ECM degradation. Despite these observations, the anti-fibrotic effect of BMP-7 is still controversial such that fine regulation of BMP-7 expression in vivo might be a great challenge for its ultimate clinical application. PMID:25954203

  3. 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

  4. Morphogenetic action through flux-limited spreading

    NASA Astrophysics Data System (ADS)

    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

  5. Endocannabinoid system in Xenopus laevis development: CB1 receptor dynamics.

    PubMed

    Migliarini, Beatrice; Beatrice, Migliarini; Marucci, Gabriella; Gabriella, Marucci; Ghelfi, Francesca; Francesca, Ghelfi; Carnevali, Oliana; Oliana, Carnevali

    2006-04-01

    This study investigates for the first time the dynamics of endocannabinoid system appearance during low vertebrate Xenopus laevis development. We observed that the CB1 gene started to be expressed during the organogenesis period (+/- 1 dpf, st. 28) and expression persisted throughout the three further stages analyzed. Attention was focused on the localization of the CB1 messenger that was found both at the central level (in romboencephalon and in olfactory placods) and at the peripheral level (in the gastrointestinal tract) at +/- 3 dpf (st. 41), +/- 4 dpf (st. 46) and +/- 12 dpf (st. 49). We also considered the synthesis of CB1 protein that occurred from st. 41 onwards and, from this stage, we tested the receptor functionality in response to anandamide using cytosensor microphysiometry. CB1 functionality increased with development at both central and peripheral level. These data provide sufficient evidence to encourage further analysis on endocannabinoid physiological roles during embryonic and larval X. laevis growth. PMID:16519888

  6. Bone morphogenetic protein 2 (bmp2) and krüppel-like factor 9 (klf9) cross-regulation in uterine stromal cells promotes timing of uterine endometrial receptivity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our laboratory has identified a novel progesterone receptor (PGR) co-activator protein, designated Krüppel-like Factor 9 (KLF9), whose absence in mice is associated with subfertility with decreased number of implanting embryos due to altered patterns of proliferation, apoptosis and aberrant P-respon...

  7. 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.

  8. Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

    PubMed Central

    Dominguez, I; Diaz-Meco, M T; Municio, M M; Berra, E; García de Herreros, A; Cornet, M E; Sanz, L; Moscat, J

    1992-01-01

    A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways. Images PMID:1508183

  9. 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.

  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. 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...

  12. 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.

  13. Purification and characterization of Xenopus laevis topoisomerase II.

    PubMed Central

    Benedetti, P; Baldi, M I; Mattoccia, E; Tocchini-Valentini, G P

    1983-01-01

    We have purified to apparent homogeneity a type II DNA topoisomerase from Xenopus laevis oocyte nuclei (germinal vesicles, or GV). The most pure preparations contain a single polypeptide of 175,000 daltons as determined by SDS-gel electrophoresis. The enzyme changes the linking number of DNA circles in steps of two and reversibly knots or catenates DNA rings. No gyrase activity is detectable and ATP is required. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:10872324

  14. Antifungal and antibacterial naphthoquinones from Newbouldia laevis roots.

    PubMed

    Gafner, S; Wolfender, J L; Nianga, M; Stoeckli-Evans, H; Hostettmann, K

    1996-07-01

    From a dichloromethane extract of Newbouldia laevis roots, four new (6-hydroxydehydroiso-alpha-lapachone, 7-hydroxydehydroiso-alpha-lapachone, 5,7-dihydroxydehydroiso-alpha-lapachone and 3-hydroxy-5-methoxydehydroiso-alpha-lapachone) and six known naphthoquinones have been isolated. Their structures were established by spectroscopic methods (UV, EI mass spectrometry, 1H and 13C NMR) and that of 7-hydroxydehydroiso-alpha-lapachone was confirmed by X-ray crystallography. All naphthoquinones showed antifungal activity against Cladosporium cucumerinum and Candida albicans, and activity against the bacteria Bacillus subtilis and Escherichia coli. PMID:9397206

  15. 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

  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. The EGF receptor and notch signaling pathways control the initiation of the morphogenetic furrow during Drosophila eye development.

    PubMed

    Kumar, J P; Moses, K

    2001-07-01

    The onset of pattern formation in the developing Drosophila retina begins with the initiation of the morphogenetic furrow, the leading edge of a wave of retinal development that transforms a uniform epithelium, the eye imaginal disc into a near crystalline array of ommatidial elements. The initiation of this wave of morphogenesis is under the control of the secreted morphogens Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). We show that the Epidermal Growth Factor Receptor and Notch signaling cascades are crucial components that are also required to initiate retinal development. We also show that the initiation of the morphogenetic furrow is the sum of two genetically separable processes: (1) the 'birth' of pattern formation at the posterior margin of the eye imaginal disc; and (2) the subsequent 'reincarnation' of retinal development across the epithelium. PMID:11526075

  18. 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.

  19. Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation

    PubMed Central

    Blanchard, Guy B.; Kabla, Alexandre J.; Schultz, Nora L.; Butler, Lucy C.; Sanson, Benedicte; Gorfinkiel, Nicole; Mahadevan, L.; Adams, Richard J.

    2016-01-01

    The dynamic reshaping of tissues during morphogenesis results from a combination of individual cell behaviours and collective cell rearrangements. However, a comprehensive framework to unambiguously measure and link cell behaviour to tissue morphogenesis is lacking. Here we introduce such a kinematic framework, bridging cell and tissue behaviours at an intermediate, mesoscopic, level of cell clusters or domains. By measuring domain deformation in terms of the relative motion of cell positions and the evolution of their shapes, we characterize the basic invariant quantities that measure fundamental classes of cell behaviour, namely tensorial rates of cell shape change and cell intercalation. In doing so we introduce an explicit definition of cell intercalation as a continuous process. We demonstrate how spatiotemporal mapping of strain rates in three models of tissue morphogenesis leads to new insight into morphogenetic mechanisms. Our quantitative approach has broad relevance for the precise characterisation and comparison of morphogenetic phenotypes. PMID:19412170

  20. Morphogenetic competence of HNF4α-deficient mouse hepatic cells✩

    PubMed Central

    Hayhurst, Graham P.; Strick-Marchand, Hélène; Mulet, Céline; Richard, Anne-Françoise; Morosan, Serban; Kremsdorf, Dina; Weiss, Mary C.

    2008-01-01

    Background/Aims To specify roles of HNF4α in mouse liver development, we have analyzed the ex vivo morphogenetic potential of HNF4α-null embryonic hepatic cells. Methods Using mice with floxed or deficiency alleles of HNF4α, hepatic cells lacking this transcription factor were explanted into primary culture and derived into cell lines. Results Contrary to behavior in vivo where HNF4α-null liver cells fail to show normal polarity and epithelialization, e18.5 hepatic cells in primary culture from mutant embryos show restoration of apical expression of tight junction protein-1 and of transcripts for E-cadherin. Clones of control and HNF4α -null cell lines were indistinguishable, even when differentiation of bile canalicular formation was induced. HNF4α-null and control cell lines showed similar potential to colonize livers of the murine ALB-uPA/SCID model of liver regeneration, but null cells formed only bile ducts and not clusters of hepatocytes. Finally, analysis of mutant embryonic livers revealed a transcriptional signature consistent with a stress response, which could underlie the morphogenetic defects observed in vivo. Conclusions We conclude that the lack of epithelialization characteristic of the HNF4α-null embryonic liver is due, at least in part, to non-cell autonomous defects, and that null cells do not suffer intrinsic defects in polarization. PMID:18617288

  1. 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

  2. Genes encoding farnesyl cysteine carboxyl methyltransferase in Schizosaccharomyces pombe and Xenopus laevis.

    PubMed Central

    Imai, Y; Davey, J; Kawagishi-Kobayashi, M; Yamamoto, M

    1997-01-01

    The mam4 mutation of Schizosaccharomyces pombe causes mating deficiency in h- cells but not in h+ cells. h- cells defective in mam4 do not secrete active mating pheromone M-factor. We cloned mam4 by complementation. The mam4 gene encodes a protein of 236 amino acids, with several potential membrane-spanning domains, which is 44% identical with farnesyl cysteine carboxyl methyltransferase encoded by STE14 and required for the modification of a-factor in Saccharomyces cerevisiae. Analysis of membrane fractions revealed that mam4 is responsible for the methyltransferase activity in S. pombe. Cells defective in mam4 produced farnesylated but unmethylated cysteine and small peptides but no intact M-factor. These observations strongly suggest that the mam4 gene product is farnesyl cysteine carboxyl methyltransferase that modifies M-factor. Furthermore, transcomplementation of S. pombe mam4 allowed us to isolate an apparent homolog of mam4 from Xenopus laevis (Xmam4). In addition to its sequence similarity to S. pombe mam4, the product of Xmam4 was shown to have a farnesyl cysteine carboxyl methyltransferase activity in S. pombe cells. The isolation of a vertebrate gene encoding farnesyl cysteine carboxyl methyltransferase opens the way to in-depth studies of the role of methylation in a large body of proteins, including Ras superfamily proteins. PMID:9032282

  3. Studying MAP Kinase pathways during early development of Xenopus laevis.

    PubMed

    Keren, Aviad; Bengal, Eyal

    2010-01-01

    The following chapter describes several methods involved in the detection of MAPK activities and phosphorylated proteins during early development of Xenopus laevis. The Xenopus embryo provides a powerful platform for biochemical studies. We describe here basic methods of embryo manipulations such as egg fertilization, embryo growth and maintenance, microinjection of capped RNA and antisense morpholino oligonucleotides (AMOs), and isolation of explants. In addition, we describe methods to detect phosphorylated proteins, to analyze kinase activity, and to interfere with signaling pathways. Immunohistochemical staining performed on whole embryos or on tissue sections is an additional method for the detection of phosphorylated proteins in the developing embryo. Approaches to activate or inhibit MAPK activities including the ectopic expression of mutated isoforms of MAPK kinase, or the incubation of embryo explants with pharmacological inhibitors are described. Finally, we describe an in vitro kinase assay specifically designed for the Xenopus embryo. PMID:20811998

  4. Sexually differentiated central pattern generators in Xenopus laevis.

    PubMed

    Zornik, Erik; Yamaguchi, Ayako

    2008-06-01

    Understanding the neural mechanisms that underlie the function of central pattern generators (CPGs) presents a formidable challenge requiring sophisticated tools and well-chosen model systems. In this article, we describe recent work on vocalizations of the African clawed frog Xenopus laevis. These behaviors are driven by sexually differentiated CPGs and are exceptionally well suited to this objective. In particular, a simplified mechanism of vocal production (independent of respiratory musculature) allows straightforward interpretations of nerve activity with respect to behavior. Furthermore, the development of a fictively vocalizing isolated brain, together with the finding of rapid androgen-induced masculinization of female vocalizations, provides an invaluable tool for determining how new behaviors arise from existing circuits. PMID:18471902

  5. 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

  6. Attenuation of bone morphogenetic protein signaling during amphibian limb development results in the generation of stage-specific defects.

    PubMed

    Jones, Tamsin E M; Day, Robert C; Beck, Caroline W

    2013-11-01

    The vertebrate limb is one of the most intensively studied organs in the field of developmental biology. Limb development in tetrapod vertebrates is highly conserved and dependent on the interaction of several important molecular pathways. The bone morphogenetic protein (BMP) signaling cascade is one of these pathways and has been shown to be crucial for several aspects of limb development. Here, we have used a Xenopus laevis transgenic line, in which expression of the inhibitor Noggin is under the control of the heat-shock promoter hsp70 to examine the effects of attenuation of BMP signaling at different stages of limb development. Remarkably different phenotypes were produced at different stages, illustrating the varied roles of BMP in development of the limb. Very early limb buds appeared to be refractory to the effects of BMP attenuation, developing normally in most cases. Ectopic limbs were produced by overexpression of Noggin corresponding to a brief window of limb development at about stage 49/50, as recently described by Christen et al. (2012). Attenuation of BMP signaling in stage 51 or 52 tadpoles lead to a reduction in the number of digits formed, resulting in hypodactyly or ectrodactyly, as well as occasional defects in the more proximal tibia-fibula. Finally, inhibition at stage 54 (paddle stage) led to the formation of dramatically shortened digits resulting from loss of distal phalanges. Transcriptome analysis has revealed the possibility that more Noggin-sensitive members of the BMP family could be involved in limb development than previously suspected. Our analysis demonstrates the usefulness of heat-shock-driven gene expression as an effective method for inhibiting a developmental pathway at different times during limb development. PMID:23981117

  7. NMDAR-Dependent Control of Call Duration in Xenopus laevis

    PubMed Central

    Katzen, Abraham W.; Rhodes, Heather J.; Yamaguchi, Ayako

    2010-01-01

    Many rhythmic behaviors, such as locomotion and vocalization, involve temporally dynamic patterns. How does the brain generate temporal complexity? Here, we use the vocal central pattern generator (CPG) of Xenopus laevis to address this question. Isolated brains can elicit fictive vocalizations, allowing us to study the CPG in vitro. The X. laevis advertisement call is temporally modulated; calls consist of rhythmic click trills that alternate between fast (∼60 Hz) and slow (∼30 Hz) rates. We investigated the role of two CPG nuclei—the laryngeal motor nucleus (n.IX–X) and the dorsal tegmental area of the medulla (DTAM)—in setting rhythm frequency and call durations. We discovered a local field potential wave in DTAM that coincides with fictive fast trills and phasic activity that coincides with fictive clicks. After disrupting n.IX–X connections, the wave persists, whereas phasic activity disappears. Wave duration was temperature dependent and correlated with fictive fast trills. This correlation persisted when wave duration was modified by temperature manipulations. Selectively cooling DTAM, but not n.IX–X, lengthened fictive call and fast trill durations, whereas cooling either nucleus decelerated the fictive click rate. The N-methyl-d-aspartate receptor (NMDAR) antagonist dAPV blocked waves and fictive fast trills, suggesting that the wave controls fast trill activation and, consequently, call duration. We conclude that two functionally distinct CPG circuits exist: 1) a pattern generator in DTAM that determines call duration and 2) a rhythm generator (spanning DTAM and n.IX–X) that determines click rates. The newly identified DTAM pattern generator provides an excellent model for understanding NDMAR-dependent rhythmic circuits. PMID:20393064

  8. PHENOBARBITAL AFFECTS THYROID HISTOLOGY AND LARVAL DEVELOPMENT IN THE AFRICAN CLAWED FROG XENOPUS LAEVIS

    EPA Science Inventory

    The abstract highlights our recent study to explore endocrine disrupting effects of phenobarbital in the African clawed frog, Xenopus laevis. In mammals, this chemical is known to induce the biotransforming enzyme UDP-glucuronosyltransferase (UDPGT) resulting in increased thyroid...

  9. Pomphorhynchus laevis (Palaeacanthocephala) in the intestine of chub (Leuciscus cephalus) as an indicator of metal pollution.

    PubMed

    Sures, Bernd; Siddall, Roy

    2003-01-01

    Chub experimentally infected with the acanthocephalan parasite Pomphorhynchus laevis were exposed to 0.01 mg l(-1) Pb(2+) over 5 weeks. Lead was rapidly accumulated in the intestinal worms reaching a steady-state concentration after 4 weeks. This concentration was significantly greater than in the host muscle, liver and intestine and more than 9,000 times higher than the exposure concentration. Lead accumulation in P. laevis increased at a higher exposure concentration but was not correlated with either parasite intensity or with pooled or individual worm weight. The highest lead concentrations were recorded in those specimen of P. laevis that were attached in the posterior intestine. Interestingly, parasitised chub accumulated less lead in their own tissues than uninfected conspecifics. A mechanism of lead uptake by P. laevis and the application of acanthocephalans as accumulation indicators of metal pollution is discussed. PMID:12547347

  10. 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

  11. Stimulatory effects of distinct members of the bone morphogenetic protein family on ligament fibroblasts

    PubMed Central

    Bobacz, K; Ullrich, R; Amoyo, L; Erlacher, L; Smolen, J S; Graninger, W B

    2006-01-01

    Objective To investigate effects of cartilage derived morphogenetic protein‐1 and ‐2 (CDMP‐1, CDMP‐2), bone morphogenetic protein (BMP)‐7 and BMP‐6 on metabolism of ligament fibroblasts and their osteogenic or chondrogenic differentiation potential. Methods Ligament fibroblasts were obtained from 3 month old calves, plated as monolayers or micromass cultures, and incubated with or without CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6. Expression of the indicated growth factors was assessed by RT‐PCR and western immunoblotting. The presence of their respective type I and II receptors, and lineage related markers, was investigated in stimulated and unstimulated cells by RT‐PCR and northern blotting. Biosynthesis of matrix proteoglycans was assessed by [35S]sulphate incorporation in monolayers. Alcian blue and toluidine blue staining was done in micromass cultures. Results CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 were detected on mRNA and on the protein level. Type I and II receptors were endogenously expressed in unstimulated ligament fibroblasts. The growth factors significantly stimulated total proteoglycan synthesis as assessed by [35S]sulphate incorporation. Toluidine blue staining showed cartilage‐specific metachromasia in the growth factor treated micromass cultures. Transcription analysis of stimulated ligament fibroblasts demonstrated coexpression of chondrocyte markers but no up regulation of osteogenic markers. Conclusion CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 and their receptors were expressed in ligament tissue. These growth factors induced matrix synthesis in fibroblasts derived from bovine ligament. The preferential expression of cartilage markers in vitro suggests that CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 have the potential to induce differentiation towards a chondrogenic phenotype in ligament fibroblasts. Thus, fibroblasts from ligaments may serve as a source for chondrogenesis and tissue repair. PMID:15975973

  12. 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. PMID:24580675

  13. Copy number variation and genetic diversity of MHC Class IIb alleles in an alien population of Xenopus laevis.

    PubMed

    Mable, Barbara K; Kilbride, Elizabeth; Viney, Mark E; Tinsley, Richard C

    2015-10-01

    Xenopus laevis (the African clawed frog), which originated through hybridisation and whole genome duplication, has been used as a model for genetics and development for many years, but surprisingly little is known about immune gene variation in natural populations. The purpose of this study was to use an isolated population of X. laevis that was introduced to Wales, UK in the past 50 years to investigate how variation at the MHC compares to that at other loci, following a severe population bottleneck. Among 18 individuals, we found nine alleles based on exon 2 sequences of the Class IIb region (which includes the peptide binding region). Individuals carried from one to three of the loci identified from previous laboratory studies. Genetic variation was an order of magnitude higher at the MHC compared with three single-copy nuclear genes, but all loci showed high levels of heterozygosity and nucleotide diversity and there was not an excess of homozygosity or decrease in diversity over time that would suggest extensive inbreeding in the introduced population. Tajima's D was positive for all loci, which is consistent with a bottleneck. Moreover, comparison with published sequences identified the source of the introduced population as the Western Cape region of South Africa, where most commercial suppliers have obtained their stocks. These factors suggest that despite founding by potentially already inbred individuals, the alien population in Wales has maintained substantial genetic variation at both adaptively important and neutral genes. PMID:26329765

  14. 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.

  15. 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

  16. Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

    PubMed Central

    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 differentiation1-16. 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 effects of

  17. 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

  18. Bone morphogenetic proteins in tumour associated angiogenesis and implication in cancer therapies.

    PubMed

    Ye, Lin; Jiang, Wen G

    2016-10-01

    Bone morphogenetic protein (BMP) belongs to transforming growth factor-β superfamily. To date, more than 20 BMPs have been identified in humans. BMPs play a critical role in embryonic and postnatal development, and also in maintaining homeostasis in different organs and tissues by regulating cell differentiation, proliferation, survival and motility. They play important roles in the development and progression of certain malignancies, including prostate cancer, breast cancer, lung cancer, etc. Recently, more evidence shows that BMPs are also involved in tumour associated angiogenesis. For example BMP can either directly regulate the functions of vascular endothelial cells or indirectly influence the angiogenesis via regulation of angiogenic factors, such as vascular endothelial growth factor (VEGF). Such crosstalk can also be reflected in the interaction with other angiogenic factors, like hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF). All these factors are involved in the orchestration of the angiogenic process during tumour development and progression. Review of the relevant studies will provide a comprehensive prospective on current understanding and shed light on the corresponding therapeutic opportunity. PMID:26639195

  19. Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

    PubMed Central

    Filas, Benjamen A.; Varner, Victor D.; Voronov, Dmitry A.; Taber, Larry A.

    2011-01-01

    Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes. PMID:22025033

  20. Scaling of morphogenetic patterns in reaction-diffusion systems.

    PubMed

    Rasolonjanahary, Manan'Iarivo; Vasiev, Bakhtier

    2016-09-01

    Development of multicellular organisms is commonly associated with the response of individual cells to concentrations of chemical substances called morphogens. Concentration fields of morphogens form a basis for biological patterning and ensure its properties including ability to scale with the size of the organism. While mechanisms underlying the formation of morphogen gradients are reasonably well understood, little is known about processes responsible for their scaling. Here, we perform a formal analysis of scaling for chemical patterns forming in continuous systems. We introduce a quantity representing the sensitivity of systems to changes in their size and use it to analyse scaling properties of patterns forming in a few different systems. Particularly, we consider how scaling properties of morphogen gradients forming in diffusion-decay systems depend on boundary conditions and how the scaling can be improved by passive modulation of morphogens or active transport in the system. We also analyse scaling of morphogenetic signal caused by two opposing gradients and consider scaling properties of patterns forming in activator-inhibitor systems. We conclude with a few possible mechanisms which allow scaling of morphogenetic patterns. PMID:27255960

  1. Labeling strategy and signal broadening mechanism of Protein NMR spectroscopy in Xenopus laevis oocytes.

    PubMed

    Ye, Yansheng; Liu, Xiaoli; Chen, Yanhua; Xu, Guohua; Wu, Qiong; Zhang, Zeting; Yao, Chendie; Liu, Maili; Li, Conggang

    2015-06-01

    We used Xenopus laevis oocytes, a paradigm for a variety of biological studies, as a eukaryotic model system for in-cell protein NMR spectroscopy. The small globular protein GB1 was one of the first studied in Xenopus oocytes, but there have been few reports since then of high-resolution spectra in oocytes. The scarcity of data is at least partly due to the lack of good labeling strategies and the paucity of information on resonance broadening mechanisms. Here, we systematically evaluate isotope enrichment and labeling methods in oocytes injected with five different proteins with molecular masses of 6 to 54 kDa. (19) F labeling is more promising than (15) N, (13) C, and (2) H enrichment. We also used (19) F NMR spectroscopy to quantify the contribution of viscosity, weak interactions, and sample inhomogeneity to resonance broadening in cells. We found that the viscosity in oocytes is only about 1.2 times that of water, and that inhomogeneous broadening is a major factor in determining line width in these cells. PMID:25965532

  2. 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. PMID:25698756

  3. 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

  4. Color and intensity discrimination in Xenopus laevis tadpoles.

    PubMed

    Rothman, Gabriel R; Blackiston, Douglas J; Levin, Michael

    2016-09-01

    Investigations into the physiology of Xenopus laevis have the potential to greatly accelerate biomedical research, especially concerning neural plasticity and sensory systems, but are limited by the lack of available information on behavioral learning in this species. Here, we attempt to lay the foundations for a behavioral assay in Xenopus that can be used in conjunction with biological manipulations. We tested cohorts of Xenopus tadpoles across four light-mediated active-avoidance experiments, using either wavelength or intensity as the salient discriminative cue. In the wavelength task, we determine a baseline learning rate and characterize retention of learning, identifying active extinction effects as far more potent than the passage of time in the loss of behavior. In the intensity task, we examine the effects of varying differences between the discriminative stimuli on acquisition and extinction and identify a critical range of intensity differences where learning changes. The results of our experiments demonstrate that Xenopus is a tractable model organism for cognitive research and can learn a variety of associative tasks in the laboratory settings. These data reveal new aspects of the Xenopus larval visual processing system and facilitate future research between cognitive methods and biological/chemical manipulations to study mechanisms of brain structure and function. PMID:27146661

  5. Posttranslational modification and secretion of vitellogenin in Xenopus laevis

    SciTech Connect

    Gottlieb, T.A.

    1981-08-01

    The thesis represents an attempt to elucidate the intracellular pathway linking the synthesis and secretion of the yolk precursor protein vitellogenin. More specifically, how the hepatocyte attaches both phosphate and carbohydrate to the peptide backbone prior to secretion is considered. A model which summarizes the accumulated data depicting various aspects of these processes is also presented. Pulse-chase experiments demonstrate the subcellular sites of vitellogenin phosphorylation. The results of these experiments indicate that approximately 70% of the phsosphate residues are covalently attached to vitellogenin during its intracellular translocation through the smooth microsomes, while the rough microsomes can account for the remainder of the total incorporated phosphate. The presence of a divalent-cation requiring protein kinase (E.C. 2.7.1,37, ATP:protein phosphotransferase) is demonstrated in the microsomes derived from the liver of estrogenized female Xenopus laevis. We present data showing that this protein kinase is responsible for phosphorylating hepatic precursors to serum vitellogenin in vivo. Pulse-chase experiments measuring the rates of incorporation of radiolabeled glucosamine and galactose into intracellular vitellogenin show that glycosylation of this multicomponent protein occurs in a Golgi-enriched fraction isolated from homogenized liver slices. The results indicate that the oligosaccharide component of vitellogenin in Xenopus is a complex type of carbohydrate unit which is linked via an N-glycosidic bond between an asparagine residue and N-acetylglucosamine. With respect to subcellular localization of glycoprotein assembly in Xenopus liver, there is a significant departure from currently accepted models of glycoprotein synthesis.

  6. Thymidine kinase-mediated shut down of bone morphogenetic protein-4 expression allows regulated bone production.

    PubMed

    Lombardo, Barbara; Rocco, Teresa; Esposito, Maria T; Cantilena, Bruno; Gargiulo, Sara; Greco, Adelaide; Montanaro, Donatella; Brunetti, Arturo; Pastore, Lucio

    2013-06-01

    Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor. PMID:23317056

  7. [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

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

    PubMed

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

    2016-06-10

    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

  10. Astrocytes phagocytose focal dystrophies from shortening myelin segments in the optic nerve of Xenopus laevis at metamorphosis

    PubMed Central

    Mills, Elizabeth A.; Davis, Chung-ha O.; Bushong, Eric A.; Boassa, Daniela; Kim, Keun-Young; Ellisman, Mark H.; Marsh-Armstrong, Nicholas

    2015-01-01

    Oligodendrocytes can adapt to increases in axon diameter through the addition of membrane wraps to myelin segments. Here, we report that myelin segments can also decrease their length in response to optic nerve (ON) shortening during Xenopus laevis metamorphic remodeling. EM-based analyses revealed that myelin segment shortening is accomplished by focal myelin-axon detachments and protrusions from otherwise intact myelin segments. Astrocyte processes remove these focal myelin dystrophies using known phagocytic machinery, including the opsonin milk fat globule-EGF factor 8 (Mfge8) and the downstream effector ras-related C3 botulinum toxin substrate 1 (Rac1). By the end of metamorphic nerve shortening, one-quarter of all myelin in the ON is enwrapped or internalized by astrocytes. As opposed to the removal of degenerating myelin by macrophages, which is usually associated with axonal pathologies, astrocytes selectively remove large amounts of myelin without damaging axons during this developmental remodeling event. PMID:26240339

  11. Astrocytes phagocytose focal dystrophies from shortening myelin segments in the optic nerve of Xenopus laevis at metamorphosis.

    PubMed

    Mills, Elizabeth A; Davis, Chung-ha O; Bushong, Eric A; Boassa, Daniela; Kim, Keun-Young; Ellisman, Mark H; Marsh-Armstrong, Nicholas

    2015-08-18

    Oligodendrocytes can adapt to increases in axon diameter through the addition of membrane wraps to myelin segments. Here, we report that myelin segments can also decrease their length in response to optic nerve (ON) shortening during Xenopus laevis metamorphic remodeling. EM-based analyses revealed that myelin segment shortening is accomplished by focal myelin-axon detachments and protrusions from otherwise intact myelin segments. Astrocyte processes remove these focal myelin dystrophies using known phagocytic machinery, including the opsonin milk fat globule-EGF factor 8 (Mfge8) and the downstream effector ras-related C3 botulinum toxin substrate 1 (Rac1). By the end of metamorphic nerve shortening, one-quarter of all myelin in the ON is enwrapped or internalized by astrocytes. As opposed to the removal of degenerating myelin by macrophages, which is usually associated with axonal pathologies, astrocytes selectively remove large amounts of myelin without damaging axons during this developmental remodeling event. PMID:26240339

  12. 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

  13. Ultrastructural and neurochemical architecture of the pituitary neural lobe of Xenopus laevis.

    PubMed

    van Wijk, Diane C W A; Meijer, Kari H; Roubos, Eric W

    2010-09-01

    The melanotrope cell in the amphibian pituitary pars intermedia is a model to study fundamental aspects of neuroendocrine integration. They release alpha-melanophore-stimulating hormone (alphaMSH), under the control of a large number of neurochemical signals derived from various brain centers. In Xenopus laevis, most of these signals are produced in the hypothalamic magnocellular nucleus (Mg) and are probably released from neurohemal axon terminals in the pituitary neural lobe, to stimulate alphaMSH-release, causing skin darkening. The presence in the neural lobe of at least eight stimulatory factors implicated in melanotrope cell control has led us to investigate the ultrastructural architecture of this neurohemal organ, with particular attention to the diversity of neurohemal axon terminals and their neurochemical contents. Using regular electron microscopy, we here distinguish six types of neurohemal axon terminal, on the basis of the size, shape and electron-density of their secretory granule contents. Subsequently, we have identified the neurochemical contents of these terminal types by immuno-electron microscopy and antisera raised against not only the 'classical' neurohormones vasotocin and mesotocin but also brain-derived neurotrophic factor, cocaine- and amphetamine-regulated transcript peptide, corticotropin-releasing factor, metenkephalin, pituitary adenylyl cyclase-activating polypeptide, thyrotropin-releasing hormone and urocortin-1. This has revealed that each terminal type possesses a unique set of neurochemical messengers, containing at least four, but in some cases up to eight messengers. These results reveal the potential of the Mg/neural lobe system to release a wide variety of neurochemical messengers in a partly co-ordinated and partly differential way to control melanotrope cell activity as well as ion and water balance regulatory organs, in response to various, continuously changing, environmental stimuli. PMID:20067800

  14. 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

  15. Hemojuvelin and bone morphogenetic protein (BMP) signaling in iron homeostasis.

    PubMed

    Core, Amanda B; Canali, Susanna; Babitt, Jodie L

    2014-01-01

    Mutations in hemojuvelin (HJV) are the most common cause of the juvenile-onset form of the iron overload disorder hereditary hemochromatosis. The discovery that HJV functions as a co-receptor for the bone morphogenetic protein (BMP) family of signaling molecules helped to identify this signaling pathway as a central regulator of the key iron hormone hepcidin in the control of systemic iron homeostasis. This review highlights recent work uncovering the mechanism of action of HJV and the BMP-SMAD signaling pathway in regulating hepcidin expression in the liver, as well as additional studies investigating possible extra-hepatic functions of HJV. This review also explores the interaction between HJV, the BMP-SMAD signaling pathway and other regulators of hepcidin expression in systemic iron balance. PMID:24860505

  16. Recombinant human bone morphogenetic protein induces bone formation.

    PubMed Central

    Wang, E A; Rosen, V; D'Alessandro, J S; Bauduy, M; Cordes, P; Harada, T; Israel, D I; Hewick, R M; Kerns, K M; LaPan, P

    1990-01-01

    We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans. Images PMID:2315314

  17. Studies of Bone Morphogenetic Protein based Surgical Repair

    PubMed Central

    Lo, Kevin W.-H.; Ulery, Bret D.; Ashe, Keshia M.; Laurencin, Cato T.

    2012-01-01

    Over the past several decades, recombinant human bone morphogenetic proteins (rhBMPs) have been the most extensively studied and widely used osteoinductive agents for clinical bone repair. Since rhBMP-2 and rhBMP-7 were approved by the U.S. Food and Drug Administration for certain clinical uses, millions of patients worldwide have been treated with rhBMPs for various musculoskeletal disorders. Current clinical applications include treatment of long bone fracture non-unions, spinal surgeries, and oral maxillofacial surgeries. Considering the growing number of recent publications related to clincal research of rhBMPs, there exists enormous promise for these proteins to be used in bone regenerative medicine. The authors take this opportunity to review the rhBMP literature paying specific attention to the current applications of rhBMPs in bone repair and spine surgery. The prospective future of rhBMPs delivered in combination with tissue engineered scaffolds is also reviewed. PMID:22512928

  18. The role of human bone morphogenetic proteins in spinal fusion.

    PubMed

    Zlotolow, D A; Vaccaro, A R; Salamon, M L; Albert, T J

    2000-01-01

    The attainment of a stable arthrodesis is critical to the successful management of some types of spinal disorders. Autologous iliac-crest bone graft has been the most commonly utilized substance associated with predictable healing in spinal fusion applications. Although alternative graft substances exist, these have not been shown to be as uniformly effective in achieving spinal fusion. Because of the morbidity associated with bone autograft harvest, there is increasing interest in alternative graft substances and especially in the osteoinductive abilities of bone morphogenetic proteins (BMPs). Several animal models have demonstrated that BMP-containing allograft or synthetic carrier medium is as effective as or superior to autograft bone in promoting spinal fusion. Furthermore, the limited number of human trials utilizing BMPs to treat nonunions in the appendicular skeleton indicate that the results found in animal models will be reproducible in the clinical setting. PMID:10666648

  19. A morphogenetic regulatory role for ethyl alcohol in Candida albicans.

    PubMed

    Chauhan, Nitin M; Raut, Jayant S; Karuppayil, S Mohan

    2011-11-01

    Regulation of morphogenesis through the production of chemical signalling molecules such as isoamyl alcohol, 2-phenylethyl alcohol, 1-dodecanol, E-nerolidol and farnesol is reported in Candida albicans. The present study focuses on the effect of ethyl alcohol on C. albicans dimorphism and biofilm development. Ethyl alcohol inhibited germ tube formation induced by the four standard inducers in a concentration-dependent manner. The germ tube inhibitory concentration (4%) did not have any effect on the growth and viability of C. albicans cells. Ethyl alcohol also inhibited the elongation of germ tubes. Four percentage of ethyl alcohol significantly inhibited biofilm development on polystyrene and silicone surfaces. We suggest a potential morphogenetic regulatory role for ethyl alcohol, which may influence dissemination, virulence and establishment of infection. PMID:21605190

  20. A model for reading morphogenetic gradients: autocatalysis and competition at the gene level.

    PubMed Central

    Kerszberg, M; Changeux, J P

    1994-01-01

    How are morphogenetic gradients interpreted in terms of embryonic gene transcription patterns within a syncytium such as the Drosophila blastoderm? We propose a hypothetical model based on recent findings in the molecular biology of transcription factors. The model postulates a morphogen which is itself a spatially distributed transcription factor M or which generates a distribution of such a factor. We posit the existence of an additional, zygotically transcribed "vernier" factor V. M and V form all possible dimers: MM, MV, and VV. These are differentially translocated to the nuclei and bind with various affinities to responsive elements in the V promoter, thereby contributing to activation/inactivation of V transcription. We find four generic regimes. In order of complexity, they are as follows: (i) MM activates V; the M gradient gives rise to a sharp transcriptional boundary for V and to a secondary gradient in the concentration of protein V; (ii) MV activates V; a sharp boundary in transcription and distribution of V arises; (iii) MM and MV compete for binding; a stationary stripe of active V transcription is generated; (iv) MM and VV are in competition; a stripe of V transcription moves from one end of the embryo toward the other and may stop and/or dwindle at an intermediate position. Tentative interpretations in terms of Drosophila genes such as bicoid and hunchback are presented. Images PMID:8016073

  1. [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

  2. 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.

  3. Heparin Microparticle Effects on Presentation and Bioactivity of Bone Morphogenetic Protein-2

    PubMed Central

    Hettiaratchi, Marian H.; Miller, Tobias; Temenoff, Johnna S.; Guldberg, Robert E.; McDevitt, Todd C.

    2014-01-01

    Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration. PMID:24881028

  4. 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.

  5. An RNA molecule copurifies with RNase P activity from Xenopus laevis oocytes.

    PubMed Central

    Doria, M; Carrara, G; Calandra, P; Tocchini-Valentini, G P

    1991-01-01

    Utilizing a procedure for the purification of RNase P from Xenopus laevis germinal vesicle (GV) extracts, according to which the contamination by a large, cytoplasmic, cylindrical structure (1) is avoided, we demonstrate that the X.laevis enzyme, like the HeLa RNase P, is precipitated by anti-Th antibodies and an RNA molecule (XL RNA), 320 nucleotides long, copurifies with the activity. The sequence of XL RNA is 60% homologous to HeLa H1 RNA, therefore the two molecules seem related. Images PMID:1710353

  6. 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...

  7. The structure and development of Xenopus laevis cornea.

    PubMed

    Hu, Wanzhou; Haamedi, Nasrin; Lee, Jaehoon; Kinoshita, Tsutomu; Ohnuma, Shin-ichi

    2013-11-01

    The African clawed frog, Xenopus laevis, is a widely used model organism for tissue development. We have followed the process of corneal development closely in Xenopus and examined the corneal ultrastructure at each stage during its formation. Xenopus cornea development starts at stage 25 from a simple embryonic epidermis overlying the developing optic vesicle. After detachment of the lens placode which takes place around stage 30, cranial neural crest cells start to invade the space between the lens and the embryonic epidermis to construct the corneal endothelium. At stage 41, a second wave of migratory cells containing presumptive keratocytes invades the matrix leading to the formation of inner cornea and outer cornea. Three-dimensional electron microscopic examination shows that a unique cell mass, the stroma attracting center, connects the two layers like the center pole of a tent. After stage 48, many secondary stromal keratocytes individually migrate to the center and form the stroma layer. At stage 60, the stroma space is largely filled by collagen lamellae and keratocytes, and the stroma attracting center disappears. At early metamorphosis, the embryonic epithelium gradually changes to the adult corneal epithelium, which is covered by microvilli. Around stage 62 the embryonic epithelium thickens and a massive cell death is observed in the epithelium, coinciding with eyelid opening. After metamorphosis, the frog cornea has attained the adult structure of three cellular layers, epithelium, stroma, and endothelium, and two acellular layers between the cellular layers, namely the Bowman's layer and Descemet's membrane. After initial completion, Xenopus cornea, in particular the stroma, continues to thicken and enlarge throughout the lifetime of the animal. In the adult, a p63 positive limbus-like wavy structure is observed at the peripheral edge of the cornea. Proliferation analysis shows that the basal corneal epithelial cells actively divide and there are a

  8. 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

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

    PubMed

    De Busschere, Charlotte; 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

  10. 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

  11. 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 ...

  12. 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...

  13. 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

  14. 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. PMID:23884640

  15. Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development

    PubMed Central

    Holtzhausen, Alisha; Golzio, Christelle; How, Tam; Lee, Yong-Hun; Schiemann, William P.; Katsanis, Nicholas; Blobe, Gerard C.

    2014-01-01

    The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and cellular homeostasis, with aberrant BMP signaling resulting in a broad spectrum of human disease. We report that BMPs unexpectedly signal through the canonical transforming growth factor β (TGF-β)-responsive Smad2 and Smad3. BMP-induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP-binding TGF-β superfamily receptors, activin receptor-like kinase (ALK)3/6, and the Smad2/3 phosphorylating receptors ALK5/7. BMP signaling through Smad2 mediates, in part, dorsoventral axis patterning in zebrafish embryos, whereas BMP signaling through Smad3 facilitates cancer cell invasion. Consistent with increased BMP-mediated Smad2/3 signaling during cancer progression, Smad1/5 and Smad 2/3 signaling converge in human cancer specimens. Thus, the signaling mechanisms used by BMPs and TGF-β superfamily receptors are broader than previously appreciated.—Holtzhausen, A., Golzio, C., How, T., Lee, Y.-H., Schiemann, W. P., Katsanis, N., Blobe, G. C. Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development. PMID:24308972

  16. Dietary exposure to Aroclor 1254 alters gene expression in Xenopus laevis frogs.

    PubMed

    Jelaso, Anna M; DeLong, Cari; Means, Jay; Ide, Charles F

    2005-05-01

    Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that contribute to worldwide health problems. Despite data associating PCBs with adverse health effects, decisions to clean up contaminated sites remain controversial. Cleanup decisions are typically based on risk assessment methods that are not sensitive enough to detect subtle changes in health. We have recently shown that gene expression signatures can serve as sensitive molecular biomarkers of exposure and related health effects. Our initial studies were carried out with developing Xenopus laevis tadpoles that were exposed to the PCB mixture Aroclor 1254 (A1254) for 2 days. A1254 was dissolved in dimethyl sulfoxide and added to the aquarium water for rapid loading of PCBs into the tadpole tissue. These studies showed that increases in the expression of specific genes occurred independent of adverse health effects, and decreases in specific genes correlated with the appearance of observable health effects, including decreased survival and gross morphological and behavioral abnormalities. In this report, we extend our previous work to test the use of gene expression signatures as biomarkers in frogs exposed to PCBs through the diet from early tadpole stages through metamorphosis. This work showed that chronic low-dose exposure to A1254 (24 ppm) in food produced tissue levels of 17 ppm and increased gene expression of nerve growth factor and proopiomelanocortin independent of adverse health effects. Exposure to higher doses of A1254 (200 ppm) produced tissue levels of 80 ppm and increased expression of p450 1A1, also, independent of adverse health effects. This work provides further evidence for the use of gene expression changes as biomarkers of exposure to PCBs. PMID:15721885

  17. Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation.

    PubMed Central

    Bellier, S; Dubois, M F; Nishida, E; Almouzni, G; Bensaude, O

    1997-01-01

    Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34(cdc2)off kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells. PMID:9032270

  18. 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

  19. 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

  20. Bone Morphogenetic Proteins Regulate Enteric Gliogenesis by Modulating ErbB3 Signaling

    PubMed Central

    Chalazonitis, Alcmène; D’Autréaux, Fabien; Pham, Tuan D.; Kessler, John A.; Gershon, Michael D.

    2010-01-01

    The neural crest-derived cell population that colonizes the bowel (ENCDC) contains proliferating neural/glial progenitors. We tested the hypothesis that bone morphogenetic proteins (BMPs 2 and 4), which are known to promote enteric neuronal differentiation at the expense of proliferation, function similarly in gliogenesis. Enteric gliogenesis was analyzed in mice that overexpress the BMP antagonist, noggin, or BMP4 in the primordial ENS. Noggin-induced loss-of-function decreased, while BMP4-induced gain-of-function increased the glial density and glia/neuron ratio. When added to immunoisolated ENCDC, BMPs provoked nuclear translocation of phosphorylated SMAD proteins and enhanced both glial differentiation and expression of the neuregulin receptor ErbB3. ErbB3 transcripts were detected in E12 rat gut, before glial markers are expressed; moreover, expression of the ErbB3 ligand, glial growth factor 2 (GGF2) escalated rapidly after its first detection at E14. ErbB3-immunoreactive cells were located in the ENS of fetal and adult mice. GGF2 stimulated gliogenesis and proliferation and inhibited glial cell derived neurotrophic factor (GDNF)-promoted neurogenesis. Enhanced glial apoptosis occurred following GGF2 withdrawal; BMPs intensified this GGF2-dependence and reduced GGF2-stimulated proliferation. These observations support the hypotheses that BMPs are required for enteric gliogenesis and act by promoting responsiveness of ENCDC to ErbB3 ligands such as GGF2. PMID:21094638

  1. 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

  2. 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. PMID:26765958

  3. Recurrence Methods for the Identification of Morphogenetic Patterns

    PubMed Central

    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

  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. Heparan sulfate is required for bone morphogenetic protein-7 signaling.

    PubMed

    Irie, Atsushi; Habuchi, Hiroko; Kimata, Koji; Sanai, Yutaka

    2003-09-01

    Although genetic studies have suggested that heparan sulfate (HS) is involved in bone morphogenetic protein (BMP)-mediated embryonic morphogenesis, it is unclear whether HS is directly involved in BMP-mediated signaling. Here, we investigate the involvement of HS in BMP-7 signaling. We show that HS and heparin chains specifically bind to BMP-7. Digestion of cell-surface HS with heparitinase interferes with BMP-7-mediated Smad phosphorylation in ROS 17/2.8 osteoblastic cells. Inhibiting sulfation of cell-surface HS with chlorate also causes interruption of Smad phosphorylation. Addition of exogenous heparin to ROS 17/2.8 cells prevents BMP-7-mediated Smad phosphorylation rather than enhances the BMP-7 signal, suggesting that HS should be anchored on the plasma membrane for BMP signaling. Moreover, BMP-7 binding to ROS 17/2.8 cells is inhibited by chlorate treatment and exogenous application of heparin. These results demonstrate that BMP-7 specifically binds to cell-surface HS and the BMP-7-HS interaction is required for BMP-7 signaling. PMID:12927798

  6. Lineage and morphogenetic analysis of the cardiac valves.

    PubMed

    de Lange, Frederik J; Moorman, Antoon F M; Anderson, Robert H; Männer, Jörg; Soufan, Alexandre T; de Gier-de Vries, Corrie; Schneider, Michael D; Webb, Sandra; van den Hoff, Maurice J B; Christoffels, Vincent M

    2004-09-17

    We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (alphaMHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development. PMID:15297379

  7. 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

  8. Morphogenetic expression of Moraxella bovis fimbriae (pili) in Pseudomonas aeruginosa.

    PubMed Central

    Beard, M K; Mattick, J S; Moore, L J; Mott, M R; Marrs, C F; Egerton, J R

    1990-01-01

    Type 4 fimbriae (pili) are found in a wide variety of gram-negative bacteria and are composed of small structural subunits which share significant sequence homology among different species, especially at their amino-terminal ends. Previous studies demonstrating morphogenetic expression of Bacteroides nodosus fimbriae from cloned subunit genes in Pseudomonas aeruginosa suggested that there is a common mechanism for type 4 fimbriae assembly and that the structural subunits are interchangeable (J. S. Mattick et al., J. Bacteriol. 169:33-41, 1987). Here we have examined the expression of Moraxella bovis fimbrial subunits in P. aeruginosa. M. bovis subunits were assembled into extracellular fimbriae in this host, in some cases as a homopolymer but in others as a mosaic with the indigenous subunit, indicating structural equivalence. This result contrasts with other studies in which recombinant P. aeruginosa expressing different subunits produced fimbriae composed almost exclusively of one subunit or the other (T. C. Elleman and J. E. Peterson, Mol. Microbiol. 1:377-380, 1987). Both observations can be explained by reversibility of subunit-subunit interactions at the site of assembly, with the forward equilibrium favoring chain extension between compatible subunits. Images PMID:1970564

  9. The history and histology of bone morphogenetic protein.

    PubMed

    Murray, Samuel S; Brochmann Murray, Elsa J; Wang, Jeffrey C; Duarte, Maria Eugenia Leite

    2016-07-01

    Bone morphogenetic proteins are a group of structurally related proteins within the TGF-β superfamily of proteins with a diverse repertoire of functions in embryonic and adult organisms. As is apparent from the name, the members first characterized participate in bone growth, development, and remodeling. The "morphogenic" activity per se is defined as the induction of a recapitulation of endochondral bone formation by appropriate stem cells. The regenerative capacity of bone has been recognized since ancient times. The mechanism, applications, and conceptual basis of bone transplantation, bone implantation, ectopic bone formation, and exogenously induced bone formation have been studied by many investigators for more than a century. This review examines the efforts to characterize this activity in the European and American literature over approximately the last century. Because of the inherently complex nature of the process induced by these molecules (inflammation, stem cell proliferation, cartilage differentiation, replacement of cartilage with bone) it is important to evaluate previous investigations through a histological perspective. The cellular basis of the contemporary bioassay for BMP activity is illustrated and discussed from the histological point of view. PMID:26907674

  10. Development of the Upper Lip: Morphogenetic and Molecular Mechanisms

    PubMed Central

    Jiang, Rulang; Bush, Jeffrey O.; Lidral, Andrew C.

    2008-01-01

    The vertebrate upper lip forms from initially freely projecting maxillary, medial nasal and lateral nasal prominences at the rostral and lateral boundaries of the primitive oral cavity. These facial prominences arise during early embryogenesis from ventrally migrating neural crest cells in combination with the head ectoderm and mesoderm and undergo directed growth and expansion around the nasal pits to actively fuse with each other. Initial fusion is between lateral and medial nasal processes and is followed by fusion between maxillary and medial nasal processes. Fusion between these prominences involves active epithelial filopodial and adhering interactions as well as programmed cell death. Slight defects in growth and patterning of the facial mesenchyme or epithelial fusion result in cleft lip with or without cleft palate, the most common and disfiguring craniofacial birth defect. Recent studies of craniofacial development in animal models have identified components of several major signaling pathways, including Bmp, Fgf, Shh and Wnt signaling, that are critical for proper midfacial morphogenesis and/or lip fusion. There is also accumulating evidence that these signaling pathways cross-regulate genetically as well as crosstalk intracellularly to control cell proliferation and tissue patterning. This review will summarize the current understanding of the basic morphogenetic processes and molecular mechanisms underlying upper lip development and discuss the complex interactions of the various signaling pathways and challenges for understanding cleft lip pathogenesis. PMID:16292776

  11. Racemose inflorescences of monocots: structural and morphogenetic interaction at the flower/inflorescence level

    PubMed Central

    Remizowa, Margarita V.; Rudall, Paula J.; Choob, Vladimir V.; Sokoloff, Dmitry D.

    2013-01-01

    Background Understanding and modelling early events of floral meristem patterning and floral development requires consideration of positional information regarding the organs surrounding the floral meristem, such as the flower-subtending bracts (FSBs) and floral prophylls (bracteoles). In common with models of regulation of floral patterning, the simplest models of phyllotaxy consider only unbranched uniaxial systems. Racemose inflorescences and thyrses offer a useful model system for investigating morphogenetic interactions between organs belonging to different axes. Scope This review considers (1) racemose inflorescences of early-divergent and lilioid monocots and their possible relationship with other inflorescence types, (2) hypotheses on the morphogenetic significance of phyllomes surrounding developing flowers, (3) patterns of FSB reduction and (4) vascular patterns in the primary inflorescence axis and lateral pedicels. Conclusions Racemose (partial) inflorescences represent the plesiomorphic condition in monocots. The presence or absence of a terminal flower or flower-like structure is labile among early-divergent monocots. In some Alismatales, a few-flowered racemose inflorescence can be entirely transformed into a terminal ‘flower’. The presence or absence and position of additional phyllomes on the lateral pedicels represent important taxonomic markers and key features in regulation of flower patterning. Racemose inflorescences with a single floral prophyll are closely related to thyrses. Floral patterning is either unidirectional or simultaneous in species that lack a floral prophyll or possess a single adaxial floral prophyll and usually spiral in the outer perianth whorl in species with a transversely oriented floral prophyll. Inhibitory fields of surrounding phyllomes are relevant but insufficient to explain these patterns; other important factors are meristem space economy and/or the inhibitory activity of the primary inflorescence axis. Two

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

    PubMed

    Martinovic, Snjezana; Mazic, Sanja; Kisic, Veronika; Basic, Nikolina; Jakic-Razumovic, Jasminka; Borovecki, Fran; Batinic, Drago; Simic, Petra; Grgurevic, Lovorka; Labar, Boris; Vukicevic, Slobodan

    2004-09-01

    Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors. PMID:15314083

  18. Retinoic acid signaling regulates sonic hedgehog and bone morphogenetic protein signalings during genital tubercle development.

    PubMed

    Liu, Liqing; Suzuki, Kentaro; Nakagata, Naomi; Mihara, Kenichiro; Matsumaru, Daisuke; Ogino, Yukiko; Yashiro, Kenta; Hamada, Hiroshi; Liu, Zhonghua; Evans, Sylvia M; Mendelsohn, Cathy; Yamada, Gen

    2012-02-01

    Retinoic acid (RA) plays pivotal roles in organogenesis, and both excessive and reduced amounts of RA cause developmental abnormalities. Reproductive organs are susceptible to teratogen toxigenicity, and the genital tubercle (GT) is one such representative organ. The physiological function of endogenous RA signaling and the mechanisms of RA-induced teratogenicity are poorly understood during the GT development. The objective of this study is to understand the developmental and teratogenic roles of RA during GT development by analyzing genetically modified mouse models. We found dynamic patterns of gene expression for the RA-synthesizing enzyme, Raldh2, and for the RA-catabolizing enzyme, Cyp26b1, during GT development. Rarb, an indicator gene for RA signaling, starts its expression in the prospective corpus cavernosum penis and in the urethral plate epithelium (UE), which plays central roles during GT development. Excessive RA signaling in Cyp26b1(-/-) mutants leads to abnormal extents of cell proliferation and differentiation during GT development, and also upregulates expression of growth factor signalings. They include Sonic hedgehog (Shh) signaling and Bone morphogenetic protein (Bmp) signaling, which are expressed in the UE and its bilateral mesenchyme. RA signaling positively regulatesShh and Bmp4 expression during GT development as testified also by the experiment of RA administration and analyses of loss-of-function of RA signaling mutants. Thus, RA signaling is involved in the developmental cascade necessary for UE formation and GT development. PMID:22127979

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

    PubMed

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

    2016-05-15

    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

  20. Bone morphogenetic protein 4 inhibits liposaccharide-induced inflammation in the airway.

    PubMed

    Li, Zhengtu; Wang, Jian; Wang, Yan; Jiang, Hua; Xu, Xiaoming; Zhang, Chenting; Li, Defu; Xu, Chuyi; Zhang, Kedong; Qi, Yafei; Gong, Xuefang; Tang, Chun; Zhong, Nanshan; Lu, Wenju

    2014-11-01

    Bone morphogenetic protein 4 (BMP4) is a multifunctional growth factor that belongs to the TGF-β superfamily. The role of BMP4 in lung diseases is not fully understood. Here, we demonstrate that BMP4 was upregulated in lungs undergoing lipopolysaccharide (LPS)-induced inflammation, and in airway epithelial cells treated with LPS or TNF-α. BMP4 mutant (BMP4(+/-) ) mice presented with more severe lung inflammation in response to LPS or Pseudomonas aeruginosa, and lower bacterial load compared with that in BMP4(+/+) mice. Knockdown of BMP4 by siRNA increased LPS and TNF-α-induced IL-8 expression in 16HBE human airway epithelial cells and in primary human bronchial epithelial cells. Similarly, peritoneal macrophages from BMP4(+/-) mice produced greater levels of TNF-α and keratinocyte chemoattractant (KC) upon LPS treatment compared with cells from BMP4(+/+) mice. Administration of exogenous BMP4 attenuated the upregulation of TNF-α, IL-8, or KC induced by LPS and/or TNF-α in airway epithelial cells, and peritoneal macrophages. Finally, partial deficiency of BMP4 in BMP4(+/-) mice protected the animals from restrictive lung function reduction upon chronic LPS exposure. These results indicate that BMP4 plays an important anti-inflammatory role, controlling the strength and facilitating the resolution of acute lung inflammation; yet, BMP4 also contributes to lung function impairment during chronic lung inflammation. PMID:25142202

  1. Induction of a dopaminergic phenotype in cultured striatal neurons by bone morphogenetic proteins.

    PubMed

    Stull, N D; Jung, J W; Iacovitti, L

    2001-09-23

    In the present study, we examined whether the bone morphogenetic proteins (BMPs), which are important in the developmental specification of transmitter type in certain classes of neurons, might also play a role in signaling the differentiation of a dopaminergic (DA) phenotype. We found that BMP-2, -4 and -6 were each capable of inducing, in a dose and time dependent manner, moderate levels of the DA enzyme tyrosine hydroxylase (TH) in cultured neurons from the mouse embryonic striatum. In contradistinction to other TH-inducing agents, BMPs initiated de novo TH expression without the required synergy of exogenous growth factors or co-activating substances and in neurons presumably aged (E16) beyond the critical period for induction. However, the appearance of TH in induced cells was short-lived (24 h) and could not be prolonged by repeated supplementation with the BMPs. Inhibitors of the mitogen-activated protein kinase (MAPK/ERK) signaling pathway, PD98059 and apigenin, did not prevent TH induction by BMP-4, as they did other TH inducing agents, indicating that the MAPK/ERK pathway does not mediate BMPs effects on TH expression. We conclude that BMP-2, -4 and -6 can be added to the expanding inventory of agents capable of inducing TH, making them potentially important in the specification of a DA phenotype in stem/precursor cells for the treatment of Parkinson's disease. PMID:11557097

  2. [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

  3. 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.

  4. 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....

  5. The roles of Bcl-xL in modulating apoptosis during development of Xenopus laevis

    PubMed Central

    Johnston, Jillian; Chan, Robert; Calderon-Segura, Maria; McFarlane, Sarah; Browder, Leon W

    2005-01-01

    Background Apoptosis is a common and essential aspect of development. It is particularly prevalent in the central nervous system and during remodelling processes such as formation of the digits and in amphibian metamorphosis. Apoptosis, which is dependent upon a balance between pro- and anti-apoptotic factors, also enables the embryo to rid itself of cells damaged by gamma irradiation. In this study, the roles of the anti-apoptotic factor Bcl-xL in protecting cells from apoptosis were examined in Xenopus laevis embryos using transgenesis to overexpress the XR11 gene, which encodes Bcl-xL. The effects on developmental, thyroid hormone-induced and γ-radiation-induced apoptosis in embryos were examined in these transgenic animals. Results Apoptosis was abrogated in XR11 transgenic embryos. However, the transgene did not prevent the apoptotic response of tadpoles to thyroid hormone during metamorphosis. Post-metamorphic XR11 frogs were reared to sexual maturity, thus allowing us to produce second-generation embryos and enabling us to distinguish between the maternal and zygotic contributions of Bcl-xL to the γ-radiation apoptotic response. Wild-type embryos irradiated before the mid-blastula transition (MBT) underwent normal cell division until reaching the MBT, after which they underwent massive, catastrophic apoptosis. Over-expression of Bcl-xL derived from XR11 females, but not males, provided partial protection from apoptosis. Maternal expression of XR11 was also sufficient to abrogate apoptosis triggered by post-MBT γ-radiation. Tolerance to post-MBT γ-radiation from zygotically-derived XR11 was acquired gradually after the MBT in spite of abundant XR11 protein synthesis. Conclusion Our data suggest that Bcl-xL is an effective counterbalance to proapoptotic factors during embryonic development but has no apparent effect on the thyroid hormone-induced apoptosis that occurs during metamorphosis. Furthermore, post-MBT apoptosis triggered by irradiation before the

  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. Bone Morphogenetic Protein Signaling Regulates Gastric Epithelial Cell Development and Proliferation in Mice

    PubMed Central

    Shinohara, Masahiko; Mao, Maria; Keeley, Theresa M.; El–Zaatari, Mohamad; Lee, Hyuk–Joon; Eaton, Kathryn A.; Samuelson, Linda C.; Merchant, Juanita L.; Goldenring, James R.; Todisco, Andrea

    2010-01-01

    BACKGROUND & AIMS We investigated the role of bone morphogenetic protein (BMP) signaling in the regulation of gastric epithelial cell growth and differentiation by generating transgenic mice that express the BMP inhibitor noggin in the stomach. METHODS The promoter of the mouse H+/K+-ATPase β-subunit gene, which is specifically expressed in parietal cells, was used to regulate expression of noggin in the gastric epithelium of mice. The transgenic mice were analyzed for noggin expression, tissue morphology, cellular composition of the gastric mucosa, gastric acid content, and plasma levels of gastrin. Tissues were analyzed by immunohistochemical, quantitative real-time polymerase chain reaction, immunoblot, microtitration, and radioimmunoassay analyses. RESULTS In the stomachs of the transgenic mice, phosphorylation of Smad1, 5, and 8 decreased, indicating inhibition of BMP signaling. Mucosa were of increased height, with dilated glands, cystic structures, reduced numbers of parietal cells, and increased numbers of cells that coexpressed intrinsic factor, trefoil factor 2, and griffonia simplicifolialectin II, compared with wild-type mice. In the transgenic mice, levels of the H+/K+-ATPase α-subunit protein and messenger RNA were reduced, whereas those of intrinsic factor increased. The transgenic mice were hypochloridric and had an increased number of Ki67- and proliferating cell nuclear antigen-positive cells; increased levels of plasma gastrin; increased expression of transforming growth factor-α, amphiregulin, and gastrin; and activation of extracellular signal-regulated kinase 2. CONCLUSIONS Inhibiting BMP signaling in the stomachs of mice by expression of noggin causes loss of parietal cells, development of transitional cells that express markers of mucus neck and zymogenic lineages, and activation of proliferation. BMPs are therefore important regulators of gastric epithelial cell homeostasis. PMID:20826155

  8. 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

  9. 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

  10. Bone morphogenetic protein-7 accelerates fracture healing in osteoporotic rats

    PubMed Central

    Diwan, Ashish D; Leong, Anthony; Appleyard, Richard; Bhargav, Divya; Fang, Zhi Ming; Wei, Aiqun

    2013-01-01

    Background: Osteoporosis is characterized by low bone mass, bone fragility and increased susceptibility to fracture. Fracture healing in osteoporosis is delayed and rates of implant failure are high with few biological treatment options available. This study aimed to determine whether a single dose of bone morphogenetic protein-7 (BMP-7) in a collagen/carboxy-methyl cellulose (CMC) composite enhanced fracture healing in an osteoporotic rat model. Materials and Methods: An open femoral midshaft osteotomy was performed in female rats 3 months post-ovarectomy. Rats were randomized to receive either BMP-7 composite (n = 30) or composite alone (n = 30) at the fracture site during surgery. Thereafter calluses were collected on days 12, 20 and 31. Callus cross-sectional area, bone mineral density, biomechanical stiffness and maximum torque, radiographic bony union and histological callus maturity were evaluated at each time point. Results: There were statistically significant increases in bone mineral density and callus cross-section area at all time points in the BMP-7 group as compared to controls and biomechanical readings showed stronger bones at day 31 in the BMP-7 group. Histological and radiographic evaluation indicated significant acceleration of bony union in the BMP-7 group as compared to controls. Conclusion: This study demonstrated that BMP-7 accelerates fracture healing in an oestrogen-deficient environment in a rat femoral fracture healing model to scientific relevance level I. The use of BMP-7 composite could offer orthopedic surgeons an advantage over oestrogen therapy, enhancing osteoporotic fracture healing with a single, locally applied dose at the time of surgery, potentially overcoming delays in healing caused by the osteoporotic state. PMID:24379457

  11. A glass polyalkenoate cement carrier for bone morphogenetic proteins.

    PubMed

    Alhalawani, Adel M F; Rodriguez, Omar; Curran, Declan J; Co, Russell; Kieran, Sean; Arshad, Saad; Keenan, Timothy J; Wren, Anthony W; Crasto, Gazelle; Peel, Sean A F; Towler, Mark R

    2015-03-01

    This work considers a glass polyalkenoate cement (GPC)-based carrier for the effective delivery of bone morphogenetic proteins (BMPs) at an implantation site. A 0.12 CaO-0.04 SrO-0.36 ZnO-0.48 SiO2 based glass and poly(acrylic acid) (PAA, Mw 213,000) were employed for the fabrication of the GPC. The media used for the water source in the GPC reaction was altered to produce a series of GPCs. The GPC liquid media was either 100 % distilled water with additions of albumin at 0, 2, 5 and 8 wt% of the glass content, 100 % formulation buffer (IFB), and 100 % BMP (150 µg rhBMP-2/ml IFB). Rheological properties, compressive strength, ion release profiles and BMP release were evaluated. Working times (Tw) of the formulated GPCs significantly increased with the addition of 2 % albumin and remained constant with further increases in albumin content or IFB solutions. Setting time (Ts) experienced an increase with 2 and 5 % albumin content, but a decrease with 8 % albumin. Changing the liquid source to IFB containing 5 % albumin had no significant effect on Ts compared to the 8 % albumin-containing BT101. Replacing the albumin with IFB/BMP-2 did not significantly affect Tw. However, Ts increased for the BT101_BMP-2 containing GPCs, compared to all other samples. The compressive strength evaluated 1 day post cement mixing was not affected significantly by the incorporation of BMPs, but the ion release did increase from the cements, particularly for Zn and Sr. The GPCs released BMP after the first day, which decreased in content during the following 6 days. This study has proven that BMPs can be immobilized into GPCs and may result in novel materials for clinical applications. PMID:25773232

  12. 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

  13. 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

  14. 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

  15. 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

  16. Effect of recombinant human bone morphogenetic protein-2 on bisphosphonate-treated osteoblasts

    PubMed Central

    Kwon, Taek-Kyun; Song, Jae-Min; Kim, In-Ryoung; Park, Bong-Soo; Kim, Chul-Hoon; Cheong, In-Kyo

    2014-01-01

    Objectives Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a side effect of bisphophonate therapy that has been reported in recent years. Osteoclastic inactivity by bisphosphonate is the known cause of BRONJ. Bone morphogenetic protein-2 (BMP-2) plays an important role in the development of bone. Recombinant human BMP-2 (rhBMP-2) is potentially useful as an activation factor for bone repair. We hypothesized that rhBMP-2 would enhance the osteoclast-osteoblast interaction related to bone remodeling. Materials and Methods Human fetal osteoblast cells (hFOB 1.19) were treated with 100 µM alendronate, and 100 ng/mL rhBMP-2 was added. Cells were incubated for a further 48 hours, and cell viability was measured using an MTT assay. Expression of the three cytokines from osteoblasts, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF), were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results Cell viability was decreased to 82.75%±1.00% by alendronate and then increased to 110.43%±1.35% after treatment with rhBMP-2 (P<0.05, respectively). OPG, RANKL, and M-CSF expression were all decreased by alendronate treatment. RANKL and M-CSF expression were increased, but OPG was not significantly affected by rhBMP-2. Conclusion rhBMP2 does not affect OPG gene expression in hFOB, but it may increase RANKL and M-CSF gene expression. PMID:25551094

  17. Bone morphogenetic protein 7 induces cementogenic differentiation of human periodontal ligament-derived mesenchymal stem cells.

    PubMed

    Torii, D; Tsutsui, T W; Watanabe, N; Konishi, K

    2016-01-01

    Bone morphogenetic protein 7 (BMP-7) is a multifunctional differentiation factor that belongs to the transforming growth factor superfamily. BMP-7 induces gene expression of protein tyrosine phosphatase-like, member A/cementum attachment protein (PTPLA/CAP) and cementum protein 1 (CEMP1), both of which are markers of cementoblasts and cementocytes. In the previous study, we reported that BMP-7 treatment enhanced PTPLA/CAP and CEMP1 expression in both normal and immortal human periodontal ligament (PDL) cells. To elucidate the molecular mechanisms of the gene expression of these molecules, in this study, we identified a functional transcription activator binding region in the promoter region of PTPLA/CAP and CEMP1 that is responsive to BMP signals. Here, we report that some short motifs termed GC-rich Smad-binding elements (GC-SBEs) that are located in the human PTPLA/CAP promoter and CEMP1 promoter are BMP-7 responsive as analyzed with luciferase promoter assays. On the other hand, we found that transcription of Sp7/Osterix and PTPLA/CAP was up-regulated after 1 week of BMP-7 treatment on purified normal human PDL cells as a result of gene expression microarray analysis. Furthermore, transcription of Sp7/Osterix, runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP) was up-regulated after 2 weeks of BMP-7 treatment, whereas gene expression of osteo/odontogenic markers such as integrin-binding sialoprotein (IBSP), collagen, type I, alpha 1 (COL1A1), dentin matrix acidic phosphoprotein 1 (DMP1), and dentin sialophosphoprotein (DSPP) was not up-regulated in purified normal or immortal human PDL cells as a result of qRT-PCR. The results suggest that BMP-7 mediates cementogenesis via GC-SBEs in human PDL cells and that its molecular mechanism is different from that for osteo/odontogenesis. PMID:25464857

  18. Negative feedback in the bone morphogenetic protein 4 (BMP4) synexpression group governs its dynamic signaling range and canalizes development

    PubMed Central

    Paulsen, Malte; Legewie, Stefan; Eils, Roland; Karaulanov, Emil; Niehrs, Christof

    2011-01-01

    What makes embryogenesis a robust and canalized process is an important question in developmental biology. A bone morphogenetic protein (BMP) morphogen gradient plays a key role in embryonic development, and we are beginning to understand how the self-regulating properties of its signaling circuitry ensure robust embryonic patterning. An unexplored question is why the BMP signaling circuit is organized as a modular synexpression group, with a prevalence of feedback inhibitors. Here, we provide evidence from direct experimentation and mathematical modeling that the synexpressed feedback inhibitors BAMBI, SMAD6, and SMAD7 (i) expand the dynamic BMP signaling range essential for proper embryonic patterning and (ii) reduce interindividual phenotypic and molecular variability in Xenopus embryos. Thereby, negative feedback linearizes signaling responses and confers robust patterning, thus promoting canalized development. The presence of negative feedback inhibitors in other growth factor synexpression groups suggests that these properties may constitute a general principle. PMID:21633009

  19. Generation of the germ layers along the animal-vegetal axis in Xenopus laevis.

    PubMed

    Yasuo, H; Lemaire, P

    2001-01-01

    After completion of gastrulation, typical vertebrate embryos consist of three cell sheets, called germ layers. The outer layer, the ectoderm, which produces the cells of the epidermis and the nervous system; the inner layer, the endoderm, producing the lining of the digestive tube and its associated organs (pancreas, liver, lungs etc.) and the middle layer, the mesoderm, which gives rise to several organs (heart, kidney, gonads), connective tissues (bone, muscles, tendons, blood vessels), and blood cells. The formation of the germ layers is one of the earliest embryonic events to subdivide multicellular embryos into a few compartments. In Xenopus laevis, the spatial domains of three germ layers are largely separated along the animal-vegetal axis even before gastrulation; ectoderm in the animal pole region; mesoderm in the equatorial region and endoderm in the vegetal pole region. In this review, we summarise the recent advances in our understanding of the formation of the germ layers in Xenopus laevis. PMID:11291851

  20. 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

  1. 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

  2. Usage of the three termination codons in a single eukaryotic cell, the Xenopus laevis oocyte.

    PubMed Central

    Bienz, M; Kubli, E; Kohli, J; deHenau, S; Huez, G; Marbaix, G; Grosjean, H

    1981-01-01

    Oocytes from Xenopus laevis were injected with purified amber (UAG), ochre (UAA), and opal (UGA) suppressor tRNAs from yeasts. The radioactively labeled proteins translated from the endogenous mRNAs were then separated on two-dimensional gels. All three termination codons are used in a single cell, the Xenopus laevis oocyte. But a surprisingly low number of readthrough polypeptides were observed from the 600 mRNAs studied in comparison to uninjected oocytes. The experimental data are compared with the conclusions obtained from the compilation of all available termination sequences on eukaryotic and prokaryotic mRNAs. This comparison indicates that the apparent resistance of natural termination codons against readthrough, as observed by the microinjection experiments, cannot be explained by tandem or very close second stop codons. Instead it suggests that specific context sequences around the termination codons may play a role in the efficiency of translation termination. Images PMID:7024919

  3. 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

  4. 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.

  5. Sterility and Gene Expression in Hybrid Males of Xenopus laevis and X. muelleri

    PubMed Central

    Malone, John H.; Chrzanowski, Thomas H.; Michalak, Pawel

    2007-01-01

    Background Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus) and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri). Methodology/Principal Findings Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri) were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. Conclusions/Significance Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly contribute to growing

  6. 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

  7. 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.

  8. Effects of Silicon-Limitation on Growth and Morphology of Triparma laevis NIES-2565 (Parmales, Heterokontophyta)

    PubMed Central

    Yamada, Kazumasa; Yoshikawa, Shinya; Ichinomiya, Mutsuo; Kuwata, Akira; Kamiya, Mitsunobu; Ohki, Kaori

    2014-01-01

    The order Parmales (Heterokontophyta) is a group of small-sized unicellular marine phytoplankton, which is distributed widely from tropical to polar waters. The cells of Parmales are surrounded by a distinctive cell wall, which consists of several siliceous plates fitting edge to edge. Phylogenetic and morphological analyses suggest that Parmales is one of the key organisms for elucidating the evolutionary origin of Bacillariophyceae (diatoms), the most successful heterokontophyta. The effects of silicon-limitation on growth and morphogenesis of plates were studied using a strain of Triparma laevis NIES-2565, which was cultured for the first time in artificial sea water. The cells of T. laevis were surrounded by eight plates when grown with sufficient silicon. However, plate formation became incomplete when cells were cultured in a medium containing low silicate (ca. <10 µM). Cells finally lost almost all plates in a medium containing silicate concentrations lower than ca. 1 µM. However, silicon-limitation did not affect growth rate; cells continued to divide without changing their growth rate, even after all plates were lost. Loss of plates was reversible; when cells without plates were transferred to a medium containing sufficient silicate, regeneration of shield and ventral plates was followed by the formation of girdle and triradiate plates. The results indicate that the response to silicon-limitation of T. laevis is different from that of diatoms, where cell division becomes inhibited under such conditions. PMID:25054645

  9. 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.

  10. 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.

  11. Gene expression analysis of the ovary of hybrid females of Xenopus laevis and X. muelleri

    PubMed Central

    2008-01-01

    Background Interspecific hybrids of frogs of the genus Xenopus result in sterile hybrid males and fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern of misexpression in hybrid males compared to the two parental species with relatively few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis) and dramatically more genes misexpressed in hybrids compared to the paternal species (X. muelleri). In this work, we examine the gene expression pattern in hybrid females of X. laevis × X. muelleri to determine if this asymmetrical pattern of expression also occurs in hybrid females. Results We find a similar pattern of asymmetry in expression compared to males in that there were more genes differentially expressed between hybrids and X. muelleri compared to hybrids and X. laevis. We also found a dramatic increase in the number of misexpressed genes with hybrid females having about 20 times more genes misexpressed in ovaries compared to testes of hybrid males and therefore the match between phenotype and expression pattern is not supported. Conclusion We discuss these intriguing findings in the context of reproductive isolation and suggest that divergence in female expression may be involved in sterility of hybrid males due to the inherent sensitivity of spermatogenesis as defined by the faster male evolution hypothesis for Haldane's rule. PMID:18331635

  12. 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

  13. 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

  14. 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. PMID:26562177

  15. 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

  16. Tolerance to Isoflurane Does Not Occur in Developing Xenopus laevis Tadpoles

    PubMed Central

    Milutinovic, Pavle S.; Zhao, Jing

    2009-01-01

    Introduction Tolerance is observed for a variety of central nervous system depressants including ethanol, which is an anesthetic, but has not been convincingly demonstrated for a potent halogenated volatile anesthetic. Failure to demonstrate tolerance to these agents may be the result of inadequate exposure to anesthetic. In this study, we exposed Xenopus laevis tadpoles to surgical anesthetic concentrations of isoflurane for one week. Methods Xenopus laevis tadpoles were produced by in vitro fertilization, and exposed to isoflurane (0.59%, 0.98%, 1.52%) or oxygen for one week starting from the time of fertilization. Results Changes in anesthetic EC50 were small and not in a consistent direction. Control animals had an anesthetic EC50 of 0.594% ± 0.003% isoflurane. Tadpoles exposed to 1.52% isoflurane had a lower EC50 than controls (by 16%), while tadpoles raised under 0.59 and 0.98% isoflurane had higher EC50s than control (by 4.7% and 7.4%, respectively). Conclusion We provide the first description of week-long exposures of vertebrates to surgical anesthetic concentrations of isoflurane, and the first report of such exposures in developing vertebrates. Tolerance to isoflurane does not occur in developing Xenopus laevis tadpoles. Taken together with studies in other organisms, the development of tolerance to ethanol but not isoflurane suggests that mechanisms these drugs share probably do not account for the development of tolerance. PMID:19095846

  17. Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

    PubMed Central

    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. PMID:25058588

  18. Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectives.

    PubMed

    Marques, Cátia L; Fernández, Ignacio; Viegas, Michael N; Cox, Cymon J; Martel, Paulo; Rosa, Joana; Cancela, M Leonor; Laizé, Vincent

    2016-02-01

    BMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage- and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to down-regulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events. PMID:26341094

  19. Does bone morphogenetic protein 6 (BMP6) affect female fertility in the mouse?

    PubMed

    Sugiura, Koji; Su, You-Qiang; Eppig, John J

    2010-12-01

    Bone morphogenetic protein 6 (BMP6) is a transforming growth factor beta superfamily member produced by mammalian oocytes as well as other cell types. Despite well-characterized effects of recombinant BMP6 on granulosa cells in vitro, the function of BMP6 in vivo has been ill-defined. Therefore, the effects of genetic deletion of the Bmp6 gene on female mouse fertility were assessed. The mean litter size of Bmp6(-/-) females was reduced by 22% (P < 0.05) compared to Bmp6(+/+) controls. Not only did Bmp6(-/-) females naturally ovulate 24% fewer eggs, but competence of in vitro-matured oocytes to complete preimplantation development after fertilization in vitro was decreased by 50%. No apparent effect of Bmp6 deletion on either the morphology or the dynamics of follicular development was apparent. Nevertheless, levels of luteinizing hormone (LH)/human chorionic gonadotropin (hCG)-induced transcripts, which encode proteins required for cumulus expansion (HAS2, PTGS2, PTX3, and TNFAIP6), and of epidermal growth factor-like peptides (AREG, BTC, and EREG) were lower in Bmp6(-/-) mice than in controls after administration of a reduced dose of hCG (1 IU) in vivo. LH receptor (Lhcgr) transcript levels were not significantly lower in Bmp6(-/-) granulosa cells, suggesting that BMP6 is required for processes downstream of LH receptors. To assess whether another oocyte-derived BMP, BMP15, could have BMP6-redundant functions in vivo, the fertility of Bmp15/Bmp6 double mutants was assessed. Fertility was not significantly reduced in double-homozygous mutants compared with that in double-heterozygous controls. Therefore, BMP6 promotes normal fertility in female mice, at least in part, by enabling appropriate responses to LH and normal oocyte quality. Thus, Bmp6 probably is part of the complex genetic network that determines female fertility. PMID:20702851

  20. 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

  1. 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. PMID:19535030

  2. Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2.

    PubMed

    Zhao, Yong-Gang; Meng, Fan-Xing; Li, Bing-Wei; Sheng, You-Ming; Liu, Ming-Ming; Wang, Bing; Li, Hong-Wei; Xiu, Rui-Juan

    2016-02-01

    Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Beta-glycerophosphate (β-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle α-actin (SM-α-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by β-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification. PMID:26797522

  3. 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

  4. Elafin Reverses Pulmonary Hypertension via Caveolin-1–Dependent Bone Morphogenetic Protein Signaling

    PubMed Central

    Nickel, Nils P.; Spiekerkoetter, Edda; Gu, Mingxia; Li, Caiyun G.; Li, Hai; Kaschwich, Mark; Diebold, Isabel; Hennigs, Jan K.; Kim, Ki-Yoon; Miyagawa, Kazuya; Wang, Lingli; Cao, Aiqin; Sa, Silin; Jiang, Xinguo; Stockstill, Raymond W.; Nicolls, Mark R.; Zamanian, Roham T.; Bland, Richard D.

    2015-01-01

    Rationale: Pulmonary arterial hypertension is characterized by endothelial dysfunction, impaired bone morphogenetic protein receptor 2 (BMPR2) signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates hypoxic pulmonary hypertension in mice, but its potential to improve endothelial function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown. Objectives: To assess elafin-mediated regression of pulmonary vascular pathology in rats and in lung explants from patients with pulmonary hypertension. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells and to elucidate the underlying mechanism. Methods: Rats with pulmonary hypertension induced by vascular endothelial growth factor receptor blockade and hypoxia (Sugen/hypoxia) as well as lung organ cultures from patients with pulmonary hypertension were used to assess elafin-mediated reversibility of pulmonary vascular disease. Pulmonary arterial endothelial cells from patients and control subjects were used to determine the efficacy and mechanism of elafin-mediated BMPR2 signaling. Measurements and Main Results: In Sugen/hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a BMPR2 target. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and decreased neointimal lesions in lung organ culture. In normal and patient pulmonary artery endothelial cells, elafin promoted angiogenesis by increasing pSMAD-dependent and -independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via

  5. Hyaluronan regulates bone morphogenetic protein-7-dependent prevention and reversal of myofibroblast phenotype.

    PubMed

    Midgley, Adam C; Duggal, Lucy; Jenkins, Robert; Hascall, Vincent; Steadman, Robert; Phillips, Aled O; Meran, Soma

    2015-05-01

    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

  6. Bone morphogenetic protein 2 and decorin expression in old fracture fragments and surrounding tissues.

    PubMed

    Han, X G; Wang, D K; Gao, F; Liu, R H; Bi, Z G

    2015-01-01

    Bone morphogenetic protein 2 (BMP-2) can promote fracture healing. Although the complex role BMP-2 in bone formation is increasingly understood, the role of endogenous BMP-2 in nonunion remains unclear. Decorin (DCN) can promote the formation of bone matrix and calcium deposition to control bone morphogenesis. In this study, tissue composition and expression of BMP-2 and DCN were detected in different parts of old fracture zones to explore inherent anti-fibrotic ability and osteogenesis. Twenty-three patients were selected, including eight cases of delayed union and 15 cases of nonunion. Average duration of delayed union or nonunion was 15 months. Fracture fragments and surrounding tissues, including bone grafts, marrow cavity contents, and sticking scars, were categorically sampled during surgery. Through observation and histological testing, component comparisons were made between fracture fragments and surrounding tissue. The expression levels of DCN and BMP-2 in different tissues were detected by immunohistochemical staining and real-time polymerase chain reaction. The expression of DCN and BMP- 2 in different parts of the nonunion area showed that, compared with bone graft and marrow cavity contents, sticking scars had the highest expression of BMP-2. Compared with the marrow cavity contents and sticking scars, bone grafts had the highest expression of DCN. The low antifibrotic and osteogenic activity of the nonunion area was associated with non-co-expression of BMP-2 and DCN. Therefore, the co-injection of osteogenic factor BMP and DCN into the nonunion area can improve the induction of bone formation and enhance the conversion of the old scar, thereby achieving better nonunion treatment. PMID:26400336

  7. High Doses of Bone Morphogenetic Protein 2 Induce Structurally Abnormal Bone and Inflammation In Vivo

    PubMed Central

    Zara, Janette N.; Siu, Ronald K.; Zhang, Xinli; Shen, Jia; Ngo, Richard; Lee, Min; Li, Weiming; Chiang, Michael; Chung, Jonguk; Kwak, Jinny; Wu, Benjamin M.; Ting, Kang

    2011-01-01

    The major Food and Drug Association–approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL. PMID:21247344

  8. 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

  9. Glucocorticoid-induced differentiation of fetal rat calvarial osteoblasts is mediated by bone morphogenetic protein-6.

    PubMed

    Boden, S D; Hair, G; Titus, L; Racine, M; McCuaig, K; Wozney, J M; Nanes, M S

    1997-07-01

    Glucocorticoids (GCs) at physiological concentrations promote osteoblast differentiation from fetal calvarial cells, calvarial organ cultures, and bone marrow stromal cells; however, the cellular pathways involved are not known. Bone morphogenetic proteins (BMPs) are recognized as important mediators of osteoblast differentiation. Specific roles for individual BMPs during postembryonic membranous bone formation have yet to be determined. We recently reported that GC potentiated the osteoblast differentiation effects of BMP-2 and BMP-4, but not of BMP-6, which, by itself, was the most potent of the three. In the present study, we used fetal rat secondary calvarial cultures to study the role of BMP-6 during early osteoblast differentiation. Treatment with the GC triamcinolone (10(-9) M) resulted in a 5- to 8-fold increase in BMP-6 steady-state messenger RNA levels, peaking at 12 h. In contrast, BMPs -2, -4, -5, -7, and transforming growth factor (TGF)-beta1 messenger RNA levels increased by less than 2-fold, after GC treatment, compared with untreated control cultures at 24 h. BMP-6 protein secretion increased 6- to 7-fold by 12 h and 12-fold (from 7.5 to 90 ng/ml) by 24 h, as measured by quantitative Western analysis. Treatment of cells with oligodeoxynucleotides antisense to BMP-6 diminished secretion of BMP-6 protein and significantly inhibited the GC-induced differentiation, as determined by a 10-fold decrease in the number of mineralized bone nodules, compared with controls that were treated with sense oligonucleotides or no oligonucleotides (ANOVA, P < 0.05). The antisense oligonucleotide inhibition of differentiation was rescued by treatment with exogenous recombinant human BMP-6. We conclude that GC-induced differentiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-6 has an important and unique role during early osteoblast differentiation. PMID:9202223

  10. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.

    PubMed

    Zara, Janette N; Siu, Ronald K; Zhang, Xinli; Shen, Jia; Ngo, Richard; Lee, Min; Li, Weiming; Chiang, Michael; Chung, Jonguk; Kwak, Jinny; Wu, Benjamin M; Ting, Kang; Soo, Chia

    2011-05-01

    The major Food and Drug Association-approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL. PMID:21247344

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Transgenic overexpression of bone morphogenetic protein 11 propeptide in skeleton enhances bone formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bone morphogenetic protein 11 (BMP11) is a key regulatory protein in skeletal development. BMP11 propeptide has been shown to antagonize GDF11 activity in vitro. To explore the role of BMP11 propeptide in skeletal formation in vivo, we generated transgenic mice with skeleton-specific overexpression...

  16. 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

  17. Waterborne exposure to triadimefon causes thyroid endocrine disruption and developmental delay in Xenopus laevis tadpoles.

    PubMed

    Li, Meng; Li, Shuying; Yao, Tingting; Zhao, Renjie; Wang, Qiangwei; Zhu, Guonian

    2016-08-01

    Triadimefon (TDF) is a triazole-derivative fungicide that is detectable in the environment and target agricultural products, prompting concern over its risk to wildlife and human health. In our study, Nieuwkoop & Faber stage 51 Xenopus laevis tadpoles were exposed to different nominal concentrations TDF (0, 0.112, and 1.12mg/L) for 21 days while the tadpoles were undergoing pre-morphological development. Developmental condition, bioaccumulation and thyroid hormone levels, and mRNA expression of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis were examined. Exposure to TDF caused a reduction in developmental rates on pre-metamorphosis of X. laevis. TDF exposure significantly decreased thyroid hormone (T4 and T3) concentrations, indicating thyroid endocrine disruption. The downregulation of thyroglobulin and upregulation of genes related to thyroid hormone metabolism (ugt1ab) might be responsible for the decreased thyroid hormone concentrations. Treatment with TDF also significantly increased mRNA expression of genes involved in thyroid-stimulating hormone as a compensatory mechanism response to decreased thyroid hormone concentrations. Gene expression and in silico ligand docking studies were combined to study the interaction between TDF and thyroid hormone receptor. Results showed that TDF could consequently affect the HPT axis signaling pathway. In addition, bioconcentration of TDF was observed in tadpoles, indicating the bioactivity of this compound. Taken together, the results suggest that TDF alters the HPT axis-related genes and changes thyroid hormone contents in X. laevis tadpoles, thus causing thyroid endocrine disruption and consequently delaying thyroid hormones-dependent metamorphic development. PMID:27289584

  18. 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

  19. Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

    PubMed Central

    Dzementsei, Aliaksandr; Schneider, David; Janshoff, Andreas; Pieler, Tomas

    2013-01-01

    Summary The directional migration of primordial germ cells (PGCs) to the site of gonad formation is an advantageous model system to study cell motility. The embryonic development of PGCs has been investigated in different animal species, including mice, zebrafish, Xenopus and Drosophila. In this study we focus on the physical properties of Xenopus laevis PGCs during their transition from the passive to the active migratory state. Pre-migratory PGCs from Xenopus laevis embryos at developmental stages 17–19 to be compared with migratory PGCs from stages 28–30 were isolated and characterized in respect to motility and adhesive properties. Using single-cell force spectroscopy, we observed a decline in adhesiveness of PGCs upon reaching the migratory state, as defined by decreased attachment to extracellular matrix components like fibronectin, and a reduced adhesion to somatic endodermal cells. Data obtained from qPCR analysis with isolated PGCs reveal that down-regulation of E-cadherin might contribute to this weakening of cell-cell adhesion. Interestingly, however, using an in vitro migration assay, we found that movement of X. laevis PGCs can also occur independently of specific interactions with their neighboring cells. The reduction of cellular adhesion during PGC development is accompanied by enhanced cellular motility, as reflected in increased formation of bleb-like protrusions and inferred from electric cell-substrate impedance sensing (ECIS) as well as time-lapse image analysis. Temporal alterations in cell shape, including contraction and expansion of the cellular body, reveal a higher degree of cellular dynamics for the migratory PGCs in vitro. PMID:24285703

  20. Effects of 17α-trenbolone and melengestrol acetate on Xenopus laevis growth, development, and survival.

    PubMed

    Finch, Bryson E; Blackwell, Brett R; Faust, Derek R; Wooten, Kimberly J; Maul, Jonathan D; Cox, Stephen B; Smith, Philip N

    2013-02-01

    The synthetic growth-promoting hormones trenbolone and melengestrol acetate have been detected in the environment near beef cattle feedlots and are reportedly transported via wind-borne particulate matter. Therefore, movement of synthetic hormones from beef cattle feedlots to water bodies via particulate matter is possible. Our objective was to evaluate potential effects of 17α-trenbolone (17α-TB), melengestrol acetate (MGA), and combinations of both on growth, development, and survival of Xenopus laevis larvae. On post-hatch day 2 (stage 33/34), X. laevis larvae were exposed to three nominal concentrations of 17α-TB (10, 100, and 500 ng/L), MGA (1, 10, and 100 ng/L), a combination of both (1/10, 10/100, and 100/500 ng/L MGA/17α-TB), frog embryo teratogenesis assay-Xenopus medium, or a solvent control. Significant increases in all X. laevis growth metrics were observed among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB and 10 ng/L MGA + 100 ng/L 17α-TB treatments. Stage of development was increased among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB treatment group and significantly decreased among those in the 500 ng/L 17α-TB treatment. Total body mass and snout-vent length of X. laevis larvae were significantly reduced in the 100 ng/L MGA and 100 ng/L MGA + 500 ng/L 17α-TB treatment groups. Larvae exposed to 500 ng/L 17α-TB had decreased total body mass, snout-vent length, and total length. In general, growth measurements decreased with increasing concentration of MGA, 17α-TB, or a combination of both. Survival among all treatments was not significantly different from controls. Amphibians exposed to MGA and 17α-TB in the environment may experience alterations in growth and development. PMID:22890510

  1. Trichodina xenopodus, a ciliated protozoan, in a laboratory-maintained Xenopus laevis.

    PubMed

    Collymore, Chereen; White, Julie R; Lieggi, Christine

    2013-08-01

    A postmortem evaluation of a domestically bred, adult, female Xenopus laevis revealed the presence of a urinary bladder protozoan consistent with Trichodina xenopodus. T. xenopodus is considered an incidental finding, as its presence in the urinary bladder in frogs has not been correlated with disease or with urinary bladder epithelial lesions. Trichodina spp. are ciliated protozoa known to colonize many species of amphibians and fish. These protozoa frequently inhabit the skin and gills, but may also be present in the urinary bladder of infected animals. Their presence on the skin and gills in low numbers is not related to disease; however, large numbers may indicate poor water quality and overcrowding. PMID:24209965

  2. 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.

  3. The developmental expression of two Xenopus laevis steel homologues, Xsl-1 and Xsl-2.

    PubMed

    Martin, Benjamin L; Harland, Richard M

    2004-12-01

    We have investigated the expression patterns of two Xenopus laevis genes with similarity to mouse steel (sl) using whole-mount in situ hybridization. Areas of overlapping expression include the epidermis, stomodeum, proctodeum, and otic placodes. Xsl-1 is specifically expressed in the pronephros, neural tube, gil arches, and a superficial region of the somite, while Xsl-2 is in the ganglion and facial nerve projecting to visceral arch 3, the mandibular arch, and the clefts of anterior somites. Thus, both transcripts exhibit partially overlapping expression patterns, but their tissue distribution differs significantly from that of sl expression in the mouse and chicken. PMID:15567720

  4. 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

  5. Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro

    PubMed Central

    Hanocq, F.; Kirsch-Volders, M.; Hanocq-Quertier, J.; Baltus, E.; Steinert, G.

    1972-01-01

    High molecular weight DNA was isolated from the yolk platelets of Xenopus laevis oocytes ovulated in vitro. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded. Yolk DNA behaves like nuclear DNA, and not like mitochondrial DNA, upon renaturation after denaturation. The molecules are always linear. Cytochemical and biochemical controls preclude the possibility that the yolk DNA might be contaminated by nuclear or mitochondrial DNA. We conclude that the yolk DNA is an endogenous component of the yolk platelets. Images PMID:4113867

  6. Root-to-Shoot transfer and distribution of endosulfan in the wetland macrophyte Bidens laevis L.

    PubMed

    Pérez, Débora J; Menone, Mirta L; Doucette, William J

    2013-11-01

    Endosulfan is genotoxic in somatic cells of Bidens laevis, and reproduction could be affected if translocated from roots to flower buds. Hydroponic experiments were conducted to quantify this transfer. While the root uptake of [(14) C] endosulfan and its transfer to aboveground tissues was relatively low, the resulting average flower bud concentration (1.01 ± 0.76 ng/g) after 30 d of exposure to an aqueous concentration of 5 μg/L could still represent a genotoxic risk for germ cells. PMID:23929711

  7. 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

  8. 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

  9. 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

  10. Prediction of Functionally Important Phospho-Regulatory Events in Xenopus laevis Oocytes

    PubMed Central

    Johnson, Jeffrey R.; Santos, Silvia D.; Johnson, Tasha; Pieper, Ursula; Strumillo, Marta; Wagih, Omar; Sali, Andrej; Krogan, Nevan J.; Beltrao, Pedro

    2015-01-01

    The African clawed frog Xenopus laevis is an important model organism for studies in developmental and cell biology, including cell-signaling. However, our knowledge of X. laevis protein post-translational modifications remains scarce. Here, we used a mass spectrometry-based approach to survey the phosphoproteome of this species, compiling a list of 2636 phosphosites. We used structural information and phosphoproteomic data for 13 other species in order to predict functionally important phospho-regulatory events. We found that the degree of conservation of phosphosites across species is predictive of sites with known molecular function. In addition, we predicted kinase-protein interactions for a set of cell-cycle kinases across all species. The degree of conservation of kinase-protein interactions was found to be predictive of functionally relevant regulatory interactions. Finally, using comparative protein structure models, we find that phosphosites within structured domains tend to be located at positions with high conformational flexibility. Our analysis suggests that a small class of phosphosites occurs in positions that have the potential to regulate protein conformation. PMID:26312481

  11. Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles.

    PubMed

    Jelaso, Anna M; Lehigh-Shirey, Elisabeth; Means, Jay; Ide, Charles F

    2003-01-01

    Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that pose global ecological and human health problems. Although it is well established that PCBs are associated with a variety of adverse health effects in wildlife and in humans, it is often difficult to determine direct cause-and-effect relationships between exposure and specific health outcomes. In this study, gene expression signatures were used to relate exposure to PCBs with altered physiological responses and/or specific health effects. Real-time PCR was used to measure gene expression levels for 10 genes in Xenopus laevis tadpoles (18 days postfertilization, PF) after acute exposure (2 days) to the PCB mixture Aroclor 1254. Specific gene expression signatures correlated with exposure and were predictive of adverse health effects. Exposure to low levels of Aroclor 1254 (5-50 ppb) significantly increased expression of six genes, independent of any health effects; exposure to midlevel concentrations (300-400 ppb) significantly decreased expression levels of two genes, NGF and beta-actin, prior to the onset of observable health effects; exposure to higher doses (500-700 ppb) significantly decreased NGF and beta-actin expression concomitant with the appearance of gross morphological abnormalities, behavioral deficits, and a statistically significant decrease in survival. This study expands upon our previous work that demonstrated an age-dependent susceptibility to Aroclor 1254 in Xenopus laevis tadpoles and that defined specific gene expression signatures as useful bioindicators of exposure and as predictors of overt or impending health effects. PMID:12874807

  12. 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

  13. Melatonin deacetylation: retinal vertebrate class distribution and Xenopus laevis tissue distribution.

    PubMed

    Grace, M S; Cahill, G M; Besharse, J C

    1991-09-13

    Deacetylation is a rapid clearance mechanism for ocular melatonin. We have studied the distribution of retinal melatonin deacetylase activity among vertebrate classes. Exogenous radiolabeled melatonin is metabolized by ocular tissue prepared from the amphibian Xenopus laevis, the reptile Anolis carolinensis, the teleost fish Carassius auratus, and the bird Gallus domesticus. In contrast, we were unable to detect ocular melatonin breakdown in rat or pig. In each species exhibiting ocular melatonin breakdown, melatonin is first deacetylated to 5-methoxytryptamine, which is deaminated, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. Deacetylation of melatonin is inhibited by eserine (physostigmine), causing a reduction in the levels of all 3 metabolites. Deamination of 5-methoxytryptamine is inhibited by the monoamine oxidase inhibitor pargyline, such that 5-methoxyindoleacetic acid and 5-methoxytryptophol levels are decreased while levels of 5-methoxytryptamine are increased. Incubation with the deacetylase inhibitor eserine increases endogenous melatonin levels in Xenopus and Carassius eyecups, indicating that endogenous melatonin is metabolized via the deacetylase. We also studied the tissue distribution of the deacetylase in Xenopus laevis. Melatonin deacetylation occurs in retina, retinal pigment epithelium, and skin, all of which are sites of melatonin action. These results indicate that among non-mammalian vertebrates, deacetylation is a common clearance mechanism for ocular melatonin, and may degrade melatonin at other sites of action as well. Melatonin deacetylation may help regulate local melatonin concentration, and generates other biologically active methoxyindoles. PMID:1782560

  14. 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

  15. Sulcia symbiont of the leafhopper Macrosteles laevis (Ribaut, 1927) (Insecta, Hemiptera, Cicadellidae: Deltocephalinae) harbors Arsenophonus bacteria.

    PubMed

    Kobiałka, Michał; Michalik, Anna; Walczak, Marcin; Junkiert, Łukasz; Szklarzewicz, Teresa

    2016-05-01

    The leafhopper Macrosteles laevis, like other plant sap-feeding hemipterans, lives in obligate symbiotic association with microorganisms. The symbionts are harbored in the cytoplasm of large cells termed bacteriocytes, which are integrated into huge organs termed bacteriomes. Morphological and molecular investigations have revealed that in the bacteriomes of M. laevis, two types of bacteriocytes are present which are as follows: bacteriocytes with bacterium Sulcia and bacteriocytes with Nasuia symbiont. We observed that in bacteriocytes with Sulcia, some cells of this bacterium contain numerous cells of the bacterium Arsenophonus. All types of symbionts are transmitted transovarially between generations. In the mature female, the bacteria Nasuia, bacteria Sulcia, and Sulcia with Arsenophonus inside are released from the bacteriocytes and start to assemble around the terminal oocytes. Next, the bacteria enter the cytoplasm of follicular cells surrounding the posterior pole of the oocyte. After passing through the follicular cells, the symbionts enter the space between the oocyte and follicular epithelium, forming a characteristic "symbiont ball." PMID:26188921

  16. Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.

    PubMed Central

    Swick, A G; Janicot, M; Cheneval-Kastelic, T; McLenithan, J C; Lane, M D

    1992-01-01

    Heterologous proteins can be expressed in Xenopus laevis oocytes by cytoplasmic microinjection of mRNA. To circumvent limitations inherent in this approach we investigate direct nuclear injection of strong viral expression vectors to drive transcription and subsequent translation of cDNAs encoding cytoplasmic, secreted, and plasma membrane proteins. After several viral promoters had been tested, the pMT2 vector was found to be a superior expression vector for X. laevis oocytes capable of directing expression of high levels of functional heterologous proteins. Typically the amount of protein derived from transcription-translation of the microinjected cDNA accounts for approximately 1% of total non-yolk protein. Moreover, the inefficiency usually associated with nuclear injections was overcome by coinjection of pMT2 driving expression of a secreted alkaline phosphatase as an internal control to select positive-expressing oocytes. Using this method, we have successfully expressed high levels of chloramphenicol acetyltransferase, the adipocyte-specific cytosolic 422(aP2) protein, and the membrane-associated glucose transporter GLUT1. The system described should be applicable to a wide variety of proteins for which cDNAs are available. Hence, the cumbersome and often inefficient in vitro synthesis of mRNA for studying ion channels, receptors, and transporters as well as for expression cloning in Xenopus oocytes should no longer be necessary. Images PMID:1542676

  17. 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

  18. 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

  19. 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

  20. A Ca2+-activated channel from Xenopus laevis oocyte membranes reconstituted into planar bilayers.

    PubMed Central

    Young, G P; Young, J D; Deshpande, A K; Goldstein, M; Koide, S S; Cohn, Z A

    1984-01-01

    Plasma membrane fractions from Xenopus laevis oocytes were incorporated into planar lipid bilayers. We show the existence of numerous Ca2+-activated nonspecific channels that are more permeable to anions. These channels are activated by Ca2+ at micromolar concentration but not by Mg2+, Zn2+, or Mn2+, even at millimolar concentrations. Decreasing Ca2+ concentration to less than 1 microM decreases the time of channel opening until channels close completely in the absence of Ca2+ and in the presence of EGTA. I- and Br- are more permeable through this channel than Cl-. The time during which the channels remain open is also voltage-dependent, with the channels switching off at higher voltages in both polarities. Single-channel activity shows a conductance of 380 pS in 1 M NaCl and 1 mM CaCl2, with an average open lifetime of 1.5 s at 40 mV. Similar channels are found in different stages of oocyte maturation. These observations support the hypothesis that an increase in oocyte-free Ca2+ activates directly these channels, and the resultant Cl- efflux forms the ionic basis for the fertilization potential in X. laevis. PMID:6089180

  1. 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

  2. Subcellular Metabolite and Lipid Analysis of Xenopus laevis Eggs by LAESI Mass Spectrometry

    PubMed Central

    Reschke, Brent R.; Henderson, Holly D.; Powell, Matthew J.; Moody, Sally A.; Vertes, Akos

    2014-01-01

    Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis. PMID:25506922

  3. 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.

  4. Systems Biology of the Self-regulating Morphogenetic Gradient of the Xenopus Gastrula

    PubMed Central

    Plouhinec, Jean-Louis; De Robertis, E. M.

    2009-01-01

    The morphogenetic field concept was proposed by experimental embryologists to account for the self-regulative behavior of embryos. Such fields have remained an abstract concept until the recent identification of their molecular components using a combination of genetics, biochemistry, and theoretical modeling. One of the best studied models of a morphogenetic field is the Dorsal-Ventral (D-V) patterning of the early frog embryo. This patterning system is regulated by the bone morphogenetic protein (BMP) signaling pathway and an intricate network of secreted protein antagonists. This biochemical pathway of interacting proteins functions in the extracellular space to generate a D-V gradient of BMP signaling, which is maintained during extensive morphogenetic movements of cell layers during gastrulation. The D-V field is divided into a dorsal and a ventral center, in regions of low and high BMP signaling respectively, under opposite transcriptional control by BMPs. The robustness of the patterning is assured at two different levels. First, in the extracellular space by secreted BMP antagonists that generate a directional flow of BMP ligands to the ventral side. The flow is driven by the regulated proteolysis of the Chordin inhibitor and by the presence of a molecular sink on the ventral side that concentrates BMP signals. The tolloid metalloproteinases and the Chordin-binding protein Crossveinless-2 (CV2) are key components of this ventral sink. Second, by transcriptional feedback at the cellular level: The dorsal and ventral signaling centers adjust their size and level of BMP signaling by transcriptional feedback. This allows cells on one side of a gastrula containing about 10,000 cells to communicate with cells in the opposite pole of the embryo. PMID:20066084

  5. 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

  6. 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…

  7. Blocking c-Met signaling enhances bone morphogenetic protein-2-induced osteoblast differentiation

    PubMed Central

    Shibasaki, Seiji; Kitano, Sachie; Karasaki, Miki; Tsunemi, Sachi; Sano, Hajime; Iwasaki, Tsuyoshi

    2015-01-01

    We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c-Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c-Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3-E1 murine pre-osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)-2 or osteoblast-inducer reagent in the presence or absence of either HGF antagonist (NK4) or c-Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme-linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription-PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP-2-stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast-inducer reagent-stimulated MC3T3-E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c-Met signaling pathway. However, both mitogen-activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP-2-stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP-2-stimulated C2C12 cells. These results indicate that although the c-Met-MEK-ERK-Smad and c-Met-PI3K-AKT-Smad signaling pathways positively regulate osteoblast differentiation, c-Met signaling negatively regulates osteoblast differentiation, independent of the MEK-ERK-Smad and PI3

  8. Blocking c-Met signaling enhances bone morphogenetic protein-2-induced osteoblast differentiation.

    PubMed

    Shibasaki, Seiji; Kitano, Sachie; Karasaki, Miki; Tsunemi, Sachi; Sano, Hajime; Iwasaki, Tsuyoshi

    2015-01-01

    We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c-Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c-Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3-E1 murine pre-osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)-2 or osteoblast-inducer reagent in the presence or absence of either HGF antagonist (NK4) or c-Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme-linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription-PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP-2-stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast-inducer reagent-stimulated MC3T3-E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c-Met signaling pathway. However, both mitogen-activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP-2-stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP-2-stimulated C2C12 cells. These results indicate that although the c-Met-MEK-ERK-Smad and c-Met-PI3K-AKT-Smad signaling pathways positively regulate osteoblast differentiation, c-Met signaling negatively regulates osteoblast differentiation, independent of the MEK-ERK-Smad and PI3

  9. A fucoidan from Nemacystus decipiens disrupts angiogenesis through targeting bone morphogenetic protein 4.

    PubMed

    Wang, Wucheng; Chen, Huanjun; Zhang, Lei; Qin, Yi; Cong, Qifei; Wang, Peipei; Ding, Kan

    2016-06-25

    A sulfated and acetylated fucoidan, named NDH01, was extracted from seaweed Nemacystus decipiens. NDH01 was composed of mannose, glucuronic acid, fucose, sulfate group and acetyl group in the molar ratio of 3.0: 14.4: 82.6: 34.3: 13.9. The backbone of NDH01 was fucose-free core, composed of α-d-1,2-Manp and β-d-1,4-GlcpA disaccharide repeat unit. The branches were attached at the C3, C4 and C6 of α-d-1,2-Manp. The sidechain was composed of α-l-1,3,4-Fucp, α-l-1,4-Fucp, α-l-1,3-Fucp and α-l-1,4-GlcpA. The sulfate group was linked to C4 of α-l-1,3,4-Fucp, whereas, acetyl group was branched on C2 of α-l-1,2,3-Fucp. NDH01 could disrupt tube formation and inhibit the migration as well as cell growth of human microvascular endothelial cells. Besides, phosphorylation of Smad/1/5/8, Erk and FAK was significantly inhibited by NDH01. Further studies uncovered that NDH01 blocked Smad1/5/8 signaling via interacting with bone morphogenetic protein 4 and downregulating bone morphogenetic protein 4 expression. The results suggested that NDH01 might be an angiogenesis inhibitor through targeting bone morphogenetic protein 4. PMID:27083822

  10. The role of voltage-gated calcium channels in neurotransmitter phenotype specification: Coexpression and functional analysis in Xenopus laevis

    PubMed Central

    Lewis, Brittany B; Miller, Lauren E; Herbst, Wendy A; Saha, Margaret S

    2014-01-01

    Calcium activity has been implicated in many neurodevelopmental events, including the specification of neurotransmitter phenotypes. Higher levels of calcium activity lead to an increased number of inhibitory neural phenotypes, whereas lower levels of calcium activity lead to excitatory neural phenotypes. Voltage-gated calcium channels (VGCCs) allow for rapid calcium entry and are expressed during early neural stages, making them likely regulators of activity-dependent neurotransmitter phenotype specification. To test this hypothesis, multiplex fluorescent in situ hybridization was used to characterize the coexpression of eight VGCC α1 subunits with the excitatory and inhibitory neural markers xVGlut1 and xVIAAT in Xenopus laevis embryos. VGCC coexpression was higher with xVGlut1 than xVIAAT, especially in the hindbrain, spinal cord, and cranial nerves. Calcium activity was also analyzed on a single-cell level, and spike frequency was correlated with the expression of VGCC α1 subunits in cell culture. Cells expressing Cav2.1 and Cav2.2 displayed increased calcium spiking compared with cells not expressing this marker. The VGCC antagonist diltiazem and agonist (−)BayK 8644 were used to manipulate calcium activity. Diltiazem exposure increased the number of glutamatergic cells and decreased the number of γ-aminobutyric acid (GABA)ergic cells, whereas (−)BayK 8644 exposure decreased the number of glutamatergic cells without having an effect on the number of GABAergic cells. Given that the expression and functional manipulation of VGCCs are correlated with neurotransmitter phenotype in some, but not all, experiments, VGCCs likely act in combination with a variety of other signaling factors to determine neuronal phenotype specification. J. Comp. Neurol. 522:2518–2531, 2014. PMID:24477801

  11. Probing the molecular dimensions of general anaesthetic target sites in tadpoles (Xenopus laevis) and model systems using cycloalcohols.

    PubMed Central

    Curry, S.; Moss, G. W.; Dickinson, R.; Lieb, W. R.; Franks, N. P.

    1991-01-01

    1. The series of cycloalcohols C6, C7, C8 and C10 have been used to probe the molecular dimensions of a variety of general anaesthetic target sites. 2. The general anaesthetic EC50 concentrations of the cycloalcohols were determined for tadpoles (Xenopus laevis). All of the cycloalcohols tested were found to be potent general anaesthetics (on average EC50/Csat = 0.03). 3. The effects of the cycloalcohols on highly purified luciferase enzymes from fireflies (Photinus pyralis) and bacteria (Vibrio harveyi) were also investigated. Both enzymes were inhibited competitively, with the cycloalcohols competing with firefly luciferin for binding to the firefly enzyme and with n-decanal for binding to the bacterial enzyme. 4. The binding site on the firefly enzyme could accommodate two molecules of cycloalcohols C6 and C7 but only a single molecule of the larger cycloalcohols (C8 and C10), implying a volume of the binding site of about 250 cm3 mol-1. In contrast, the binding site on the bacterial luciferase could bind only a single cycloalcohol molecule between C6 and C10. 5. While all of the cycloalcohols were potent inhibitors of the firefly luciferase enzyme (on average EC50/Csat = 0.015), they were very weak inhibitors of the bacterial luciferase enzyme (on average EC50/Csat = 0.12). Since both enzymes bind long-chain aliphatic n-alcohols tightly, the differing affinities of the cycloalcohols for the two enzymes is probably a consequence of geometrical factors. 6. The cycloalcohols produced very small effects on lipid bilayers. At EC50 concentrations which produce general anaesthesia, lipid bilayer phase transitions were shifted, on average, by only 0.43 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2043920

  12. Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos.

    PubMed

    Iraha, Fumie; Saito, Yoshinari; Yoshida, Keiko; Kawakami, Masatoki; Izutsu, Yumi; Daar, Ira Owen; Maéno, Mitsugu

    2002-10-01

    In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie-2, a Xenopus homolog of the tie-2 receptor tyrosine kinase, was examined. Whole-mount in situ hybridization analysis revealed that Xtie-2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie-2-positive cells are predominantly present just outside the boundary of alpha-globin-positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie-2 and alpha-globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a "dorsal determinant" from the vegetal cytoplasm at the 1-cell stage. Second, expression of both Xtie-2 and alpha-globin were markedly induced in ectodermal explants (animal caps) from embryos co-injected with activin and bone morphogenetic protein (BMP)-4 RNA. Furthermore, both Xtie-2 and alpha-globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP-4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP-4-dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages. PMID:12392573

  13. Hermes is a localized factor regulating cleavage of vegetal blastomeres in Xenopus laevis.

    PubMed

    Zearfoss, N R; Chan, A P; Wu, C F; Kloc, M; Etkin, L D

    2004-03-01

    We have identified the RNA-binding protein Hermes in a screen for vegetally localized RNAs in Xenopus oocytes. The RNA localizes to the vegetal cortex through both the message transport organizer (METRO) and late pathways. Hermes mRNA and protein are both detected at the vegetal cortex of the oocyte; however, the protein is degraded within a several hour period during oocyte maturation. Injection of antisense morpholino oligonucleotides (HE-MO) against Hermes caused a precocious reduction in Hermes protein present during maturation and resulted in a phenotype characterized by cleavage defects in vegetal blastomeres. The phenotype can be partially rescued by injecting Hermes mRNA. These results demonstrate that the localized RNA-binding protein Hermes functions during oocyte maturation to regulate the cleavage of specific vegetally derived cell lineages. Hermes most likely performs its function by regulating the translation or processing of one or more target RNAs. This is an important mechanism by which the embryo can generate unique cell lineages. The regulation of region-specific cell division is a novel function for a localized mRNA. PMID:14975717

  14. 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

  15. 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

  16. 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

  17. Dehydration mediated microRNA response in the African clawed frog Xenopus laevis.

    PubMed

    Wu, Cheng-Wei; Biggar, Kyle K; Storey, Kenneth B

    2013-10-25

    Exposure to various environmental stresses induces metabolic rate depression in many animal species, an adaptation that conserves energy until the environment is again conducive to normal life. The African clawed frog, Xenopus laevis, is periodically subjected to arid summers in South Africa, and utilizes entry into the hypometabolic state of estivation as a mechanism of long term survival. During estivation, frogs must typically deal with substantial dehydration as their ponds dry out and X. laevis can endure >30% loss of its body water. We hypothesize that microRNAs play a vital role in establishing a reversible hypometabolic state and responding to dehydration stress that is associated with amphibian estivation. The present study analyzes the effects of whole body dehydration on microRNA expression in three tissues of X. laevis. Compared to controls, levels of miR-1, miR-125b, and miR-16-1 decreased to 37±6, 64±8, and 80±4% of control levels during dehydration in liver. By contrast, miR-210, miR-34a and miR-21 were significantly elevated by 3.05±0.45, 2.11±0.08, and 1.36±0.05-fold, respectively, in the liver. In kidney tissue, miR-29b, miR-21, and miR-203 were elevated by 1.40±0.09, 1.31±0.05, and 2.17±0.31-fold, respectively, in response to dehydration whereas miR-203 and miR-34a were elevated in ventral skin by 1.35±0.05 and 1.74±0.12-fold, respectively. Bioinformatic analysis of the differentially expressed microRNAs suggests that these are mainly involved in two processes: (1) expression of solute carrier proteins, and (2) regulation of mitogen-activated protein kinase signaling. This study is the first report that shows a tissue specific mode of microRNA expression during amphibian dehydration, providing evidence for microRNAs as crucial regulators of metabolic depression. PMID:23958654

  18. 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.

  19. 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

  20. Thyroid hormone regulates stromelysin expression, protease secretion and the morphogenetic potential of normal polarized mammary epithelial cells.

    PubMed Central

    López-Barahona, M; Fialka, I; González-Sancho, J M; Asunción, M; González, M; Iglesias, T; Bernal, J; Beug, H; Muñoz, A

    1995-01-01

    Stromelysins are a group of proteases which degrade the extracellular matrix and activate other secreted proteases. Stromelysin (ST)-1 and ST-2 genes are induced by tumor promoters, oncogenes and growth factors, and have been involved in acquisition of the malignant phenotype. We show here that the thyroid hormone (T3) increases ST-1 and ST-2 expression in a non-transformed mouse mammary epithelial cell line (EpH4) in a way that is dependent on the level of thyroid receptor/c-erbA (TR alpha-1) expression. In agreement with this, T3 increases the secreted stromelysin activity and enhances the gelatinolytic activity of type IV collagenase. We have also demonstrated that T3 affects the epithelial polarity of EpH4 cells, diminishing the transepithelial electrical resistance of monolayers cultured on permeable filters, causing an abnormal distribution of polarization markers and the disruption of the organized 3-D structures formed by these cells in type I collagen gels. These results indicate that the ligand-activated TR alpha-1 plays an important role in regulating the morphogenetic and invasive capacities of mammary epithelial cells. Because the c-erbA locus is altered in several types of carcinoma, an altered or deregulated TR alpha-1 expression may also be important for breast cancer development and metastasis. Images PMID:7720705

  1. 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

  2. Protein serine/threonine phosphatase PPM1A dephosphorylates Smad1 in the bone morphogenetic protein signaling pathway.

    PubMed

    Duan, Xueyan; Liang, Yao-Yun; Feng, Xin-Hua; Lin, Xia

    2006-12-01

    Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-beta (TGF-beta) superfamily that activates a broad range of biological responses in the metazoan organism. The BMP-initiated signaling pathway is under tight control by processes including regulation of the ligands, the receptors, and the key downstream intracellular effector Smads. A critical point of control in BMP signaling is the phosphorylation of Smad1, Smad5, and Smad8 in their C-terminal SXS motif. Although such phosphorylation, which is mediated by the type I BMP receptor kinases in response to BMP stimulation, is well characterized, biochemical mechanisms underlying Smad dephosphorylation remain to be elucidated. In this study, we have found that PPM1A, a metal ion-dependent protein serine/threonine phosphatase, physically interacts with and dephosphorylates Smad1 both in vitro and in vivo. Functionally, overexpression of PPM1A abolishes BMP-induced transcriptional responses, whereas RNA interference-mediated knockdown of PPM1A enhances BMP signaling. Collectively, our study suggests that PPM1A plays an important role in controlling BMP signaling through catalyzing Smad dephosphorylation. PMID:16931515

  3. The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular Remodeling by Activating NFAT and the Bone Morphogenetic Protein Signaling Pathway ▿

    PubMed Central

    Chan, Mun Chun; Weisman, Alexandra S.; Kang, Hara; Nguyen, Peter H.; Hickman, Tyler; Mecker, Samantha V.; Hill, Nicholas S.; Lagna, Giorgio; Hata, Akiko

    2011-01-01

    Pulmonary artery hypertension (PAH) is characterized by elevated pulmonary artery resistance and increased medial thickness due to deregulation of vascular remodeling. Inactivating mutations of the BMPRII gene, which encodes a receptor for bone morphogenetic proteins (BMPs), are identified in ∼60% of familial PAH (FPAH) and ∼30% of idiopathic PAH (IPAH) patients. It has been hypothesized that constitutive reduction in BMP signal by BMPRII mutations may cause abnormal vascular remodeling by promoting dedifferentiation of vascular smooth muscle cells (vSMCs). Here, we demonstrate that infusion of the amiloride analog phenamil during chronic-hypoxia treatment in rat attenuates development of PAH and vascular remodeling. Phenamil induces Tribbles homolog 3 (Trb3), a positive modulator of the BMP pathway that acts by stabilizing the Smad family signal transducers. Through induction of Trb3, phenamil promotes the differentiated, contractile vSMC phenotype characterized by elevated expression of contractile genes and reduced cell growth and migration. Phenamil activates the Trb3 gene transcription via activation of the calcium-calcineurin-nuclear factor of activated T cell (NFAT) pathway. These results indicate that constitutive elevation of Trb3 by phenamil is a potential therapy for IPAH and FPAH. PMID:21135135

  4. Sex-specific response to hypoxia in a reduced brainstem preparation from Xenopus laevis.

    PubMed

    Rousseau, Jean-Philippe; Fournier, Stéphanie; Kinkead, Richard

    2016-04-01

    Respiratory reflexes and tolerance to hypoxia show significant sexual dimorphism. However, the data supporting this notion originates exclusively from mammals. To determine whether this concept is limited to this group of vertebrates, we examined the sex-specific response to acute hypoxia in an adult reduced brainstem preparation from Xenopus laevis. Within the first 5min of exposure to hypoxic aCSF (98% N2/2% CO2), recordings of respiratory-related activity show a stronger increase in fictive breathing frequency in males than females. This initial response was followed by a decrease in respiratory-related activity; this depression occurred 6min sooner in males than females. These results represent new evidences of sexual dimorphism in respiratory control in amphibians and provide potential insight in understanding the homology with other groups of vertebrates, including mammals. PMID:26528898

  5. Optical coherence tomography for detection of compound action potential in Xenopus Laevis sciatic nerve

    NASA Astrophysics Data System (ADS)

    Troiani, Francesca; Nikolic, Konstantin; Constandinou, Timothy G.

    2016-03-01

    Due to optical coherence tomography (OCT) high spatial and temporal resolution, this technique could be used to observe the quick changes in the refractive index that accompany action potential. In this study we explore the use of time domain Optical Coherence Tomography (TD-OCT) for real time action potential detection in ex vivo Xenopus Laevis sciatic nerve. TD-OCT is the easiest and less expensive OCT technique and, if successful in detecting real time action potential, it could be used for low cost monitoring devices. A theoretical investigation into the order of magnitude of the signals detected by a TD-OCT setup is provided by this work. A linear dependence between the refractive index and the intensity changes is observed and the minimum SNR for which the setup could work is found to be SNR = 2 x 104.

  6. 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.

  7. 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.

  8. Transmembrane Signal Transduction in Oocyte Maturation and Fertilization: Focusing on Xenopus laevis as a Model Animal

    PubMed Central

    Sato, Ken-ichi

    2014-01-01

    Fertilization is a cell biological phenomenon of crucial importance for the birth of new life in a variety of multicellular and sexual reproduction species such as algae, animal and plants. Fertilization involves a sequence of events, in which the female gamete “egg” and the male gamete “spermatozoon (sperm)” develop, acquire their functions, meet and fuse with each other, to initiate embryonic and zygotic development. Here, it will be briefly reviewed how oocyte cytoplasmic components are orchestrated to undergo hormone-induced oocyte maturation and sperm-induced activation of development. I then review how sperm-egg membrane interaction/fusion and activation of development in the fertilized egg are accomplished and regulated through egg coat- or egg plasma membrane-associated components, highlighting recent findings and future directions in the studies using Xenopus laevis as a model experimental animal. PMID:25546390

  9. In vivo pump-probe optical coherence tomography imaging in Xenopus laevis.

    PubMed

    Carrasco-Zevallos, Oscar; Shelton, Ryan L; Kim, Wihan; Pearson, Jeremy; Applegate, Brian E

    2015-01-01

    Currently, optical coherence tomography (OCT), is not capable of obtaining molecular information often crucial for identification of disease. To enable molecular imaging with OCT, we have further developed a technique that harnesses transient changes in light absorption in the sample to garner molecular information. A Fourier-domain Pump-Probe OCT (PPOCT) system utilizing a 532 nm pump and 830 nm probe has been developed for imaging hemoglobin. Methylene blue, a biological dye with well-know photophysics, was used to characterize the system before investigating the origin of the hemoglobin PPOCT signal. The first in vivo PPOCT images were recorded of the vasculature in Xenopus laevis. The technique was shown to work equally well in flowing and nonflowing vessels. Furthermore, PPOCT was compared with other OCT extensions which require flow, such as Doppler OCT and phase-variance OCT. PPOCT was shown to better delineate tortuous vessels, where nodes often restrict Doppler and phase-variance reconstruction. PMID:24282110

  10. Pre-meiotic transformation of germplasm-related structures during male gamete differentiation in Xenopus laevis.

    PubMed

    Reunov, Arkadiy A; Reunova, Yulia A

    2016-02-01

    To highlight the ultrastructural features of transformation occurring with germplasm-related structures (GPRS), the spermatogenic cells of Xenopus laevis were studied by transmission electron microscopy and quantitative analysis. It was determined that in spermatogonia and spermatocytes, the compact germinal granules underwent fragmentation into particles comparable with inter-mitochondrial cement (IMC). Fragments of IMC agglutinated some cell mitochondria and resulted in the creation of mitochondrial clusters. Clustered mitochondria responded with loss of their membranes that occurred by the twisting of membranous protrusions around themselves until multi-layered membranes were formed. The mitochondrial affinity of multi-layered membranes was proven by an immunopositive test for mitochondrial dihydrolipoamide acetyltransferase. As a consequence of mitochondrial membrane twisting, the naked mitochondrial cores appeared and presumably underwent dispersion, which is the terminal stage of GPRS transformation. As no GPRS were observed in spermatids and sperm, it was assumed that these structures are functionally assigned to early stages of meiotic differentiation. PMID:25511532

  11. 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

  12. Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage.

    PubMed

    Slaby, Sylvain; Lemière, Sébastien; Hanotel, Julie; Lescuyer, Arlette; Demuynck, Sylvain; Bodart, Jean-François; Leprêtre, Alain; Marin, Matthieu

    2016-08-01

    Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration. PMID:27218424

  13. Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

    PubMed Central

    Kaeser, Gwendolyn E.; Rabe, Brian A.; Saha, Margaret S.

    2011-01-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABAA and metabotropic GABAB receptors. During the development of the nervous system GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABAA and GABAB receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABAA α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. PMID:21384470

  14. 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

  15. 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

  16. Remyelination by Resident Oligodendrocyte Precursor Cells in a Xenopus laevis Inducible Model of Demyelination.

    PubMed

    Sekizar, Sowmya; Mannioui, Abdelkrim; Azoyan, Loris; Colin, Catherine; Thomas, Jean-Léon; Du Pasquier, David; Mallat, Michel; Zalc, Bernard

    2015-01-01

    We have generated a Xenopus laevis transgenic line, MBP-GFP-NTR, allowing conditional ablation of myelin-forming oligodendrocytes. In this transgenic line the transgene is driven by the proximal portion of the myelin basic protein regulatory sequence, specific to mature oligodendrocytes. The transgene protein is formed by the green fluorescent protein reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. The NTR enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. Ablation of oligodendrocytes by MTZ treatment of the tadpole induced demyelination, and here we show that myelin debris are subsequently eliminated by microglial cells. After cessation of MTZ treatment, remyelination proceeded spontaneously. We questioned the origin of remyelinating cells. Our data suggest that Sox10+ oligodendrocyte precursor cells (OPCs), which are already present in the optic nerve prior to the experimentally induced demyelination, are responsible for remyelination, and this required only minimal (if any) cell division of OPCs. © 2015 S. Karger AG, Basel. PMID:25896276

  17. 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

  18. 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

  19. Regulation of adenovirus transcription by an Ela gene in microinjected Xenopus laevis oocytes

    SciTech Connect

    Jones, N.C.; Richter, J.D.; Weeks, D.L.; Smith, L.D.

    1983-12-01

    The regulation of adenovirus type 5 gene expression by the E1a gene product was examined in microinjected Xenopus laevis oocytes. Chimeric genes were constructed which included the promoter region of early adenovirus type 5 gene 3 and the structural sequence which codes for the bacterial enzyme chloramphenicol-3-O-acetyltransferase (CAT). A plasmid containing this chimeric gene as well as plasmids containing the E1a gene were coinjected into oocyte nuclei. The presence of the E1a gene was shown to increase CAT activity by up to 8.5-fold over basal levels. Synthesis of the functional product from the E1a gene requires the removal of intron sequences by RNA splicing. The E1a gene and a derivative that precisely lacks the intron were equally effective in increasing CAT activity, suggesting that splicing of the primary E1a transcript is efficiently accomplished in the oocyte nucleus. This was confirmed by directly examining the E1a mRNAs by the S1 mapping procedure. A protein extract from adenovirus type 5-infected HeLa cells enriched for the E1a protein may supplant the E1a plasmid in enhancing CAT activity. Synthesis of the CAT enzyme after gene injection is invariant in oocytes from the same frog, but oocytes from different frogs show a high degree of variability in their ability to synthesize the CAT enzyme. Microinjected X. laevis oocytes appear to be an extremely useful system to study the effects of protein elements on transcription.

  20. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

    PubMed

    Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

    2014-11-01

    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. PMID:25277091

  1. Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy.

    PubMed

    Sadaghiani, B; Thiébaud, C H

    1987-11-01

    The Xenopus borealis quinacrine marker and scanning electron microscopy have been used to study the appearance, migration, and homing of neural crest cells in the embryo of Xenopus. The analysis shows that the primordium of the neural crest develops from the nervous layer of the ectoderm and consists of three segments at early neurula stages. This primordium is located in the lateral halves of the neural folds behind the prospective eye vesicles. The histological and experimental evidence shows that the neural crest cells also originate from the medial portion of the neural folds. The neural crest segments in the cephalic region start to migrate just before the closure of the neural tube. Isotopic and isochronic unilateral grafts of X. borealis neural crest into X. laevis embryos were performed in order to map the fate of the cranial crest segments and the vagal-truncal neural crest. The analysis of the X. laevis host embryos shows that the mandibular crest segment contributes to the lower jaw (Meckel's cartilage), quadrate, and ethmoid-trabecular cartilages, as well as to the ganglionic and Schwann cells of the trigeminus nerve, the connective tissues, the mesenchymal and choroid layers of the eye, and the cornea. The hyoid crest segment is located in the ceratohyal cartilage and in ganglia VII and VIII. The branchial crest segment migrates from the caudal part of the otic vesicle and divides into two portions which contribute to the cartilages of the gills. The vagal-truncal neural crest starts to migrate later at stage 25. It migrates by means of the vagus complex in a ventral direction and penetrates into the splanchnic layer of the digestive tract. The trunk neural crest cells disperse into three different pathways which differ from those of the avian embryo at this level. PMID:3666314

  2. 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

  3. Inhibitory effects of bark extracts from Ulmus laevis on endometrial carcinoma: an in-vitro study.

    PubMed

    Paschke, Daniel; Abarzua, Sibylle; Schlichting, André; Richter, Dagmar-Ulrike; Leinweber, Peter; Briese, Volker

    2009-04-01

    Anti-inflammatory effects of elm tree have been shown in several studies. Besides this, protective effects of components of elm bark on damaged tissue have also been described. This study was carried out to investigate the antitumour potential of an ethanolic extract isolated from Ulmus laevis in the hormone-dependent endometrial carcinoma cell line RL95-2. A range of 2.5-500 microg/ml of elm bark extract was used as standard concentrations. The molecular-chemical composition of the bark extract was analysed by pyrolysis-field ionization mass spectrometry. The influence of the bark extracts was determined on cell vitality [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test], cell proliferation (5-bromo-2-deoxyuridine test) and cytotoxicity (lactate dehydrogenase test) in the human endometrial carcinoma cell line RL 95-2. By pyrolysis-field ionization mass spectrometry, the main substance classes of the extract as a composition of sterols/triterpenes, free fatty acids and a group of phenols, lignin monomers and flavonoids was identified. Our study showed a significant inhibition of cell vitality and proliferation measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test up to 5 microg/ml extract and up to 100 microg/ml according to the 5-bromo-2-deoxyuridine test. Concentrations of 500 microg/ml induced a significant inhibition of cell vitality up to 80% and cell proliferation up to 81.5%. A significant cytotoxity was not observed. The results lead to the assumption that the bark extract from Ulmus laevis has antiproliferation and anticancer potential in hormone-dependent endometrial carcinoma cells. PMID:19337064

  4. Evaluation of Presurgical Skin Preparation Agents in African Clawed Frogs (Xenopus laevis).

    PubMed

    Philips, Blythe H; Crim, Marcus J; Hankenson, F Claire; Steffen, Earl K; Klein, Peter S; Brice, Angela K; Carty, Anthony J

    2015-11-01

    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

  5. 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

  6. 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

  7. 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. PMID:26971167

  8. Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells.

    PubMed

    Muñoz, Rosana; Edwards-Faret, Gabriela; Moreno, Mauricio; Zuñiga, Nikole; Cline, Hollis; Larraín, Juan

    2015-12-15

    Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2(/)3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2(/)3(+) cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2(/)3(+) neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism

  9. Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

    SciTech Connect

    Bahouth, S.W.; Malbon, C.C.

    1987-05-01

    Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in the control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.

  10. 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

  11. Mapping the morphogenetic potential of antler fields through deleting and transplanting subregions of antlerogenic periosteum in sika deer (Cervus nippon).

    PubMed

    Gao, Zhiguang; Yang, Fuhe; McMahon, Chris; Li, Chunyi

    2012-02-01

    Morphogenetic fields are a localised and regionally regulated group of cells capable of responding to signals leading to the development of organs. In this study, we sought to determine if antlers develop from such a field. We divided antler fields into four subregions: anterior, posterior, medial and lateral. The antlerogenic periosteum (AP) in each subregion (half of the AP) was deleted and then transplanted into an ectopic site. Antlers form from the cells exclusively residing in the AP, which is located in an antler field. The morphogenetic potential of each subregion was assessed by the antler growth from both the defective field and the transplantation site. The results showed that when the AP anterior half was absent, the fields formed antlers missing the first tine, whereas when the anterior half was present, the ectopic sites regenerated antlers containing the first tine. When the medial half was deleted, the fields could only grow spike antlers, and when the medial half was present, the ectopic sites developed branched antlers. In contrast, the antler fields were able to compensate the defects caused by ablation of the posterior or the lateral half to form relatively normal antlers; and the ectopic sites containing these grafted halves only formed spike antlers. Therefore, antler morphogenetic information was primarily held in the AP anterior-medial halves. This study substantiates the presence of morphogenetic fields in regulating the distinct pattern of antler growth, and demonstrates that antler development is a useful model for the study of morphogenetic fields. PMID:22122063

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Use of large-scale expression cloning screens in the Xenopus laevis tadpole to identify gene function.

    PubMed

    Grammer, T C; Liu, K J; Mariani, F V; Harland, R M

    2000-12-15

    We have conducted an expression cloning screen of approximately 50, 000 cDNAs from a tadpole stage Xenopus laevis cDNA library to functionally identify genes affecting a wide range of cellular and developmental processes. Fifty-seven cDNAs were isolated for their ability to alter gross tadpole morphology or the expression patterns of tissue-specific markers. Thirty-seven of the cDNAs have not been previously described for Xenopus, and 15 of these show little or no similarity to sequences in the NCBI database. The screen and the identified genes are presented in this paper to demonstrate the power, ease, speed, and flexibility of expression cloning in the X. laevis embryo. Future screens such as this one can be done on a larger scale and will complement the sequence-based screens and genome-sequencing projects which are producing a large body of novel genes without ascribed functions. PMID:11112324

  18. DNA sequence of the Xenopus laevis mitochondrial heavy and light strand replication origins and flanking tRNA genes.

    PubMed Central

    Wong, J F; Ma, D P; Wilson, R K; Roe, B A

    1983-01-01

    We have determined the primary structure of the two regions of the Xenopus laevis mitochondrial genome which encompass the origins of heavy (H) and light (L) strand replication. The first segment, which consists of 2398 nucleotides, contains the displacement loop (D-loop), the tRNA genes for threonine, proline and phenylalanine, the origin of H-strand replication, and the promoters of H- and L-strand transcription. The second segment, which consists of 447 nucleotides, contains the L-strand replication origin flanked by the tRNA genes for tryptophan, alanine, asparagine, cysteine, and tyrosine. A comparison of the sequences of the Xenopus laevis mitochondrial L-strand replication origin region and the eight tRNA genes with their counterparts from the mammalian mitochondrial genomes reveals that these regions are quite homologous, while its D-loop region shows only slight homology with those of the mammalian mitochondrial genomes. PMID:6308566

  19. Molecular cloning and sequencing of mRNAs coding for minor adult globin polypeptides of Xenopus laevis.

    PubMed Central

    Knöchel, W; Meyerhof, W; Hummel, S; Grundmann, U

    1983-01-01

    Globin mRNA was isolated from immature red blood cells of an adult Xenopus laevis female. mRNA/cDNA hybrids were integrated in the Pst I cleavage site of pBR 322 by G/C tailing, and cloned in Escherichia coli strain HB 101. By restriction site analysis as well as hybridization behaviour we identified two clones coding for minor adult alpha and beta globin chains. Nucleotide sequence analysis and derived amino acid sequences are presented. PMID:6298748

  20. Patterns of bone morphogenetic protein-2 expression in smooth muscle tumors of the uterine corpus and other uterine tissues.

    PubMed

    Fadare, Oluwole; Renshaw, Idris L; Liang, Sharon X

    2011-07-01

    Bone morphogenetic proteins (BMPs) are extracellular, multifunctional growth factors that constitute the largest subset of the transforming growth factor β superfamily. BMP2 is involved in cardiovascular embryogenesis, in addition to a variety of other postnatal functions, such as neovascularization, osteoinduction, tumor signaling, and in the uterus, stromal decidualization at the implantation site. Estrogen receptor signaling is common in smooth muscle tumors of the uterus, and preclinical models suggest significant interactions between BMP2 and estrogen receptor-mediated signaling. The purpose of this study is to define the patterns of BMP2 expression, as assessed by immunohistochemistry, in smooth muscle tumors and other tissues of the uterine corpus, and to establish whether BMP2 expression has any prognostic significance in uterine leiomyosarcomas. BMP2 was positive (cytoplasmic pattern, typically focal) in 24% of leiomyosarcomas and 20.7% of leiomyomata, but was either infrequently expressed or not expressed in all other tissues evaluated, including normal myometrium and endometrium, endometrial stromal tumors, typical adenomyoma, adenomyosis, and serosal endometriosis. The endothelial cells of small, thin-walled vessels were frequently, but not invariably immunoreactive for BMP2. There was no significant difference between BMP2⁺ and BMP⁻ leiomyosarcomas regarding average tumor size, average patient age, microvessel density, and proportions with high tumor grade, advanced stage and frequency of death from disease on follow-up. Two (29%) of 7 BMP2⁺ leiomyosarcomas were estrogen receptor+, compared with 5 (50%) of 10 BMP2⁻ leiomyosarcomas, a statistically insignificant difference (P=0.62). It is concluded that BMP2 is only expressed in a minority of smooth muscle tumors of the uterine corpus, and lacks prognostic significance in leiomyosarcomas. BMP2 is rarely expressed in the other nonendothelial tissues of the human uterine corpus that were

  1. Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2.

    PubMed

    Xu, Xian; Jha, Amit K; Duncan, Randall L; Jia, Xinqiao

    2011-08-01

    We are interested in developing hydrophilic particulate systems that are capable of sequestering growth factors, regulating their release and potentiating their biological functions. To this end heparin (HP)-decorated, hyaluronic acid (HA)-based hydrogel particles (HGPs) were synthesized using an inverse emulsion polymerization technique employing divinyl sulfone as the crosslinker. By varying the feed composition of the aqueous phase the amount of HP integrated in the particles can be systematically tuned. The resulting microscopic particles are spherical in shape and contain nanosized pores suitable for growth factor encapsulation. The covalently immobilized HP retained its ability to bind bone morphogenetic protein-2 (BMP-2) specifically, and its release kinetics can be adjusted by tuning the particle composition. Compared with pure HA particles the hybrid HA/HP HGPs show a higher BMP-2 loading capacity. While BMP-2 was released from HA HGPs with a significant initial burst, a near zero order release kinetics was observed from HA/HP hybrid particles with an optimized heparin content of 0.55 μg per mg HGPs. The ability of HA/HP hybrid particles to present BMP-2 in a controlled manner, combined with the innate bioactivity of HA, induced robust and consistent chondrogenic differentiation of murine mesenchymal stem cells, as shown by up-regulation of the mRNA levels of chondrogenic markers and the production of cartilage-specific extracellular matrix components. The simplicity of the particle synthesis, combined with the defined biological activities of the constituent building blocks, renders the HP-decorated, HA-based hydrogel particle system an attractive candidate for the sustained release of BMP-2, possibly for cartilage repair and regeneration. PMID:21550426

  2. 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. PMID:23782946

  3. Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2

    PubMed Central

    Xu, Xian; Jha, Amit K.; Duncan, Randall L.; Jia, Xinqiao

    2011-01-01

    We are interested in developing hydrophilic particulate systems that are capable of sequestering growth factors, regulating their release and potentiating their biological functions. Towards this end, heparin (HP)-decorated, hyaluronic acid (HA)-based, hydrogel particles (HGPs) were synthesized using an inverse emulsion polymerization technique employing divinyl sulfone as the crosslinker. By varying the feed composition of the aqueous phase, the amount of heparin integrated in the particles can be systematically tuned. The resulting microscopic particles are spherical in shape and contain nanosized pores suitable for growth factor encapsulation. The covalently immobilized heparin retained its ability to bind bone morphogenetic protein 2 (BMP-2) specifically, and its release kinetics can be adjusted by tuning the particle composition. Compared to the pure HA particles, the hybrid HA/HP HGPs show a higher BMP-2 loading capacity. While BMP-2 was released from HA HGPs with a significant initial burst, a near zero-order release kinetics was observed from HA/HP hybrid particles with an optimized heparin content of 0.55 μg per milligram HGPs. The ability of HA/HP hybrid particles to present BMP-2 in a controlled manner, combined with the innate bioactivity of HA, induced robust and consistent chondrogenic differentiation of murine mesenchymal stem cells, as evidenced by the up-regulation of mRNA levels of chondrogenic markers and the production of cartilage-specific extracellular matrix components. The simplicity of the particle synthesis, combined with the defined biological activities of the constituent building blocks, renders the HP-decorated, HA-based hydrogel particle system an attractive candidate for the sustained release of BMP-2, possibly for cartilage repair and regeneration. PMID:21550426

  4. 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

  5. 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

  6. 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. PMID:25742953

  7. 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

  8. 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

  9. [Ectopic osteogenesis in vivo using bone morphogenetic protein-2 derived peptide loaded biodegradable hydrogel].

    PubMed

    Zhao, Jingjing; Fang, Zhenhua; Huang, Ruokun; Xiao, Kai; Li, Jing; Xie, Ming; Kan, Wusheng

    2014-08-01

    We investigated the development of an injectable, biodegradable hydrogel composite of poly(trimethylene carbonate)-F127-poly(trimethylene carbonate)(PTMC11-F127-PTMC11 )loaded with bone morphogenetic protein-2 (BMP-2) derived peptide P24 for ectopic bone formation in vivo and evaluated its release kinetics in vitro. Then we evaluated P24 peptide release kinetics from different concentration of PTMC11-F127-PTMC11 hydrogel in vitro using bicinchoninic acid (BCA)assay. P24/ PTMC11-F127-PTMC11 hydrogel was implanted into each rat's erector muscle of spine and ectopic bone formation of the implanted gel in vivo was detected by hematoxylin and eosin stain (HE). PTMC11-F127-PTMC11 hydrogel with concentration more than 20 percent showed sustained slow release for one month after the initial burst release. Bone trabeculae surround the P24/ PTMC11-F127-PTMC11 hydrogel was shown at the end of six weeks by hematoxylin and eosin stain. These results indicated that encapsulated bone morphogenetic protein (BMP-2) derived peptide P24 remained viable in vivo, thus suggesting the potential of PTMC11-F127-PT- MC11 composite hydrogels as part of a novel strategy for localized delivery of bioactive molecules. PMID:25508424

  10. [Ectopic osteogenesis in vivo using bone morphogenetic protein-2 derived peptide loaded biodegradable hydrogel].

    PubMed

    Zhao, Jingjing; Fang, Zhenhua; Huang, Ruokun; Xiao, Kai; Li, Jing; Xie, Ming; Kan, Wusheng

    2014-08-01

    We investigated the development of an injectable, biodegradable hydrogel composite of poly(trimethylene carbonate)-F127-poly(trimethylene carbonate)(PTMC11-F127-PTMC11 )loaded with bone morphogenetic protein-2 (BMP-2) derived peptide P24 for ectopic bone formation in vivo and evaluated its release kinetics in vitro. Then we evaluated P24 peptide release kinetics from different concentration of PTMC11-F127-PTMC11 hydrogel in vitro using bicinchoninic acid (BCA)assay. P24/ PTMC11-F127-PTMC11 hydrogel was implanted into each rat's erector muscle of spine and ectopic bone formation of the implanted gel in vivo was detected by hematoxylin and eosin stain (HE). PTMC11-F127-PTMC11 hydrogel with concentration more than 20 percent showed sustained slow release for one month after the initial burst release. Bone trabeculae surround the P24/ PTMC11-F127-PTMC11 hydrogel was shown at the end of six weeks by hematoxylin and eosin stain. These results indicated that encapsulated bone morphogenetic protein (BMP-2) derived peptide P24 remained viable in vivo, thus suggesting the potential of PTMC11-F127-PT- MC11 composite hydrogels as part of a novel strategy for localized delivery of bioactive molecules. PMID:25464793

  11. 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

  12. 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

  13. 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

  14. The role of the bone morphogenetic proteins in leukaemic stem cell persistence.

    PubMed

    Toofan, Parto; Irvine, David; Hopcroft, Lisa; Copland, Mhairi; Wheadon, Helen

    2014-08-01

    CML (chronic myeloid leukaemia) is characterized by the presence of the oncogenic tyrosine kinase fusion protein BCR (breakpoint cluster region)-Abl, responsible for driving the disease. Current TKI (tyrosine kinase inhibitor) therapies effectively inhibit BCR-Abl to control CML in the majority of patients, but do not eliminate the LSC (leukaemic stem cell) population, which becomes quiescent following treatment. Patients require long-term treatment to sustain remission; alternative strategies are therefore required, either alone or in combination with TKIs to eliminate the LSCs and provide a cure. The embryonic morphogenetic pathways play a key role in haemopoiesis with recent evidence suggesting LSCs are more dependent on these signals following chemotherapy than normal HSCs (haemopoietic stem cells). Recent evidence in the literature and from our group has revealed that the BMP (bone morphogenetic protein) pathway is differentially expressed in CML patients compared with normal donors. In the present review, we explore the role that BMP signalling plays in oesteoblast differentiation, HSC maintenance and the implication of altered BMP signalling on LSC persistence in the BM (bone marrow) niche. Overall, we highlight the BMP pathway as a potential target for developing LSC-directed therapies in CML in the future. PMID:25109962

  15. Peripheral myelin of Xenopus laevis: role of electrostatic and hydrophobic interactions in membrane compaction.

    PubMed

    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

    2008-04-01

    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 X. 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 role and the

  16. 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

  17. Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling

    PubMed Central

    Vogt, Janis; Dingwell, Kevin S.; Herhaus, Lina; Gourlay, Robert; Macartney, Thomas; Campbell, David; Smith, James C.; Sapkota, Gopal P.

    2014-01-01

    Bone morphogenetic proteins (BMPs) control multiple cellular processes in embryos and adult tissues. BMPs signal through the activation of type I BMP receptor kinases, which then phosphorylate SMADs 1/5/8. In the canonical pathway, this triggers the association of these SMADs with SMAD4 and their translocation to the nucleus, where they regulate gene expression. BMPs can also signal independently of SMAD4, but this pathway is poorly understood. Here, we report the discovery and characterization of PAWS1/FAM83G as a novel SMAD1 interactor. PAWS1 forms a complex with SMAD1 in a SMAD4-independent manner, and BMP signalling induces the phosphorylation of PAWS1 through BMPR1A. The phosphorylation of PAWS1 in response to BMP is essential for activation of the SMAD4-independent BMP target genes NEDD9 and ASNS. Our findings identify PAWS1 as the first non-SMAD substrate for type I BMP receptor kinases and as a novel player in the BMP pathway. We also demonstrate that PAWS1 regulates the expression of several non-BMP target genes, suggesting roles for PAWS1 beyond the BMP pathway. PMID:24554596

  18. Knockdown of Pex11β reveals its pivotal role in regulating peroxisomal genes, numbers, and ROS levels in Xenopus laevis A6 cells.

    PubMed

    Fox, Mark A; Nieuwesteeg, Michelle A; Willson, Jessica A; Cepeda, Mario; Damjanovski, Sashko

    2014-04-01

    Peroxisomes are organelles that are ubiquitously found in all eukaryotic cells. Enzymes within their lumen are responsible for a variety of processes including the metabolism of fatty acids and eradication (neutralization) of free radicals. Peroxisomes are dynamic organelles, able to alter their numbers in response to a variety of different metabolic and cell-specific cues. Changes in peroxisome numbers can occur through division of preexisting peroxisomes or through de novo biogenesis from the ER. Proteins such as the Pex11 family of peroxins have been implicated as regulatory factors involved in peroxisome division. Division of peroxisomes involves elongation and membrane constriction followed by fission, which requires Pex11β. The regulation of peroxisome numbers in different cell types and tissues is variable and poorly understood. Here, we examine how knockdown of Pex11β affects peroxisomal genes, proteins, and peroxisome numbers in A6 kidney epithelial cells derived from Xenopus laevis. Pex11β morpholino use subsequently decreased mRNA levels of Pex1, PMP70, and PPARγ. Moreover, the Pex11β morpholino decreased PMP70 protein levels and PMP70-positive structures. Furthermore, the marker GFP-SKL revealed fewer peroxisome-like structures. These decreases resulted in increased levels of H2O2 and cellular and mitochondrial reactive oxygen species as measured by Amplex Red, DCFDA, and MitoTracker assays, respectively. PMID:24234511

  19. Modulating Hydrogel Crosslink Density and Degradation to Control Bone Morphogenetic Protein Delivery and In Vivo Bone Formation

    PubMed Central

    Holloway, Julianne L.; Ma, Henry; Rai, Reena; Burdick, Jason A.

    2014-01-01

    Bone morphogenetic proteins (BMPs) show promise in therapies for improving bone formation after injury; however, the high supraphysiological concentrations required for desired osteoinductive effects, off-target concerns, costs, and patient variability have limited the use of BMP-based therapeutics. To better understand the role of biomaterial design in BMP delivery, a matrix metalloprotease (MMP)-sensitive hyaluronic acid (HA)-based hydrogel was used for BMP-2 delivery to evaluate the influence of hydrogel degradation rate on bone repair in vivo. Specifically, maleimide-modified HA (MaHA) macromers were crosslinked with difunctional MMP-sensitive peptides to permit protease-mediated hydrogel degradation and growth factor release. The compressive, rheological, and degradation properties of MaHA hydrogels were characterized as a function of crosslink density, which was varied through either MaHA concentration (1–5 wt%) or maleimide functionalization (10–40 %f). Generally, the compressive moduli increased, the time to gelation decreased, and the degradation rate decreased with increasing crosslink density. Furthermore, BMP-2 release increased with either a decrease in the initial crosslink density or an increase in collagenase concentration (non-specific MMP degradation). Lastly, two hydrogel formulations with distinct BMP-2 release profiles were evaluated in a critical-sized calvarial defect model in rats. After six weeks, minimal evidence of bone repair was observed within defects left empty or filled with hydrogels alone. For hydrogels that contained BMP-2, similar volumes of new bone tissue were formed; however, the faster degrading hydrogel exhibited improved cellular invasion, bone volume to total volume ratio, and overall defect filling. These results illustrate the importance of coordinating hydrogel degradation with the rate of new tissue formation. PMID:24905414

  20. Positive Selection in Bone Morphogenetic Protein 15 Targets a Natural Mutation Associated with Primary Ovarian Insufficiency in Human

    PubMed Central

    Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  1. Hollow hydroxyapatite microspheres: a novel bioactive and osteoconductive carrier for controlled release of bone morphogenetic protein-2 in bone regeneration

    PubMed Central

    Xiao, Wei; Fu, Hailuo; Rahaman, Mohamed N.; Liu, Yonxing; Bal, B. Sonny

    2013-01-01

    The regeneration of large bone defects is a common and significant clinical problem. Limitations associated with existing treatments such as autologous bone grafts and allografts have increased the need for synthetic bone graft substitutes. The objective of this study was to evaluate the capacity of novel hollow hydroxyapatite (HA) microspheres to serve as a carrier for controlled release of bone morphogenetic-2 (BMP2) in bone regeneration. Hollow HA microspheres (106–150 μm) with a high surface area (>100 m2/g) and a mesoporous shell wall (pore size 10–20 nm) were created using a glass conversion technique. The release of BMP2 from the microspheres into a medium composed of diluted fetal bovine serum in vitro was slow, but it occurred continuously for over 2 weeks. When implanted in rat calvarial defects for 3 or 6 weeks, the microspheres loaded with BMP2 (1 μg/defect) showed a significantly better capacity to regenerate bone than those without BMP2. The amount of new bone in the defects implanted with the BMP2-loaded microspheres was 40% and 43%, respectively, at 3 and 6 weeks, compared to 13% and 17%, respectively, for the microspheres without BMP2. Coating the BMP2-loaded microspheres with a biodegradable polymer, poly(lactic-co-glycolic acid), reduced the amount of BMP2 released in vitro and, above a certain coating thickness, significantly reduced bone regeneration in vivo. The results indicate that these hollow HA microspheres could provide a bioactive and osteoconductive carrier for growth factors in bone regeneration. PMID:23747325

  2. Bone Morphogenetic Protein Receptor Type II Deficiency and Increased Inflammatory Cytokine Production. A Gateway to Pulmonary Arterial Hypertension

    PubMed Central

    Soon, Elaine; Crosby, Alexi; Southwood, Mark; Yang, Peiran; Tajsic, Tamara; Toshner, Mark; Appleby, Sarah; Shanahan, Catherine M.; Bloch, Kenneth D.; Pepke-Zaba, Joanna; Upton, Paul

    2015-01-01

    Rationale: Mutations in bone morphogenetic protein receptor type II (BMPR-II) underlie most cases of heritable pulmonary arterial hypertension (PAH). However, disease penetrance is only 20–30%, suggesting a requirement for additional triggers. Inflammation is emerging as a key disease-related factor in PAH, but to date there is no clear mechanism linking BMPR-II deficiency and inflammation. Objectives: To establish a direct link between BMPR-II deficiency, a consequentially heightened inflammatory response, and development of PAH. Methods: We used pulmonary artery smooth muscle cells from Bmpr2+/− mice and patients with BMPR2 mutations and compared them with wild-type controls. For the in vivo model, we used mice heterozygous for a null allele in Bmpr2 (Bmpr2+/−) and wild-type littermates. Measurements and Main Results: Acute exposure to LPS increased lung and circulating IL-6 and KC (IL-8 analog) levels in Bmpr2+/− mice to a greater extent than in wild-type controls. Similarly, pulmonary artery smooth muscle cells from Bmpr2+/− mice and patients with BMPR2 mutations produced higher levels of IL-6 and KC/IL-8 after lipopolysaccharide stimulation compared with controls. BMPR-II deficiency in mouse and human pulmonary artery smooth muscle cells was associated with increased phospho-STAT3 and loss of extracellular superoxide dismutase. Chronic lipopolysaccharide administration caused pulmonary hypertension in Bmpr2+/− mice but not in wild-type littermates. Coadministration of tempol, a superoxide dismutase mimetic, ameliorated the exaggerated inflammatory response and prevented development of PAH. Conclusions: This study demonstrates that BMPR-II deficiency promotes an exaggerated inflammatory response in vitro and in vivo, which can instigate development of pulmonary hypertension. PMID:26073741

  3. Positive selection in bone morphogenetic protein 15 targets a natural mutation associated with primary ovarian insufficiency in human.

    PubMed

    Auclair, Sylvain; Rossetti, Raffaella; Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  4. Transient brown adipocyte-like cells derive from peripheral nerve progenitors in response to bone morphogenetic protein 2.

    PubMed

    Salisbury, Elizabeth A; Lazard, Zawaunyka W; Ubogu, Eroboghene E; Davis, Alan R; Olmsted-Davis, Elizabeth A

    2012-12-01

    Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 into wild-type mice, there was replication of beta-3 adrenergic receptor(+) (ADRB3(+)) cells within the sciatic nerve perineurium. Fluorescence-activated cell sorting and analysis of cells isolated from these nerves confirmed ADRB3(+) cell expansion and their expression of the neural migration marker HNK1. Similar analysis performed 4 days after BMP2 delivery revealed a significant decrease in ADRB3(+) cells from isolated sciatic nerves, with their concurrent appearance within the adjacent soft tissue, suggesting migration away from the nerve. These soft tissue-derived cells also expressed the brown adipose marker uncoupling protein 1 (UCP1). Quantification of ADRB3-specific RNA in total hind limb tissue revealed a 3-fold increase 2 days after delivery of BMP2, followed by a 70-fold increase in UCP1-specific RNA after 3 days. Expression levels then rapidly returned to baseline by 4 days. Interestingly, these ADRB3(+) UCP1(+) cells also expressed the neural guidance factor reelin. Reelin(+) cells demonstrated distinct patterns within the injected muscle, concentrated toward the area of BMP2 release. Blocking mast cell degranulation-induced nerve remodeling resulted in the complete abrogation of UCP1-specific RNA and protein expression within the hind limbs following BMP2 injection. The data collectively suggest that local BMP2 administration initiates a cascade of events leading to the expansion, migration, and differentiation of progenitors from the peripheral nerve perineurium to brown adipose-like cells in the mouse, a necessary prerequisite for associated nerve remodeling. PMID:23283549

  5. Sequence analysis of bone morphogenetic protein receptor type II mRNA from ascitic and nonascitic commercial broilers.

    PubMed

    Cisar, C R; Balog, J M; Anthony, N B; Donoghue, A M

    2003-10-01

    Ascites syndrome, also known as pulmonary hypertension syndrome (PHS), is a common metabolic disorder in rapidly growing meat-type chickens. Environmental factors, such as cold, altitude, and diet, play significant roles in development of the disease, but there is also an important genetic component to PHS susceptibility. The human disease familial primary pulmonary hypertension (FPPH) is similar to PHS in broilers both genetically and physiologically. Several recent studies have shown that mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are a cause of FPPH in humans. To determine whether mutations in the chicken BMPR2 gene play a similar role in PHS susceptibility, BMPR-II mRNA from ascitic and nonascitic commercial broilers were sequenced and compared with the published Leghorn chicken BMPR-II mRNA sequence. Fourteen single nucleotide polymorphisms (SNP) were identified in the commercial broiler BMPR-II mRNA. No mutations unique to ascites-susceptible broilers were present in the coding, 5' untranslated or 3' untranslated regions of BMPR-II mRNA. The twelve SNP present within the coding region of BMPR-II mRNA were synonymous substitutions and did not alter the BMPR-II protein sequence. In addition, analysis of BMPR2 gene expression by reverse transcriptase-PCR indicated that there were no differences in BMPR-II mRNA levels in ascitic and nonascitic birds. Therefore, it appears unlikely that mutations in the BMPR2 gene were responsible for susceptibility to PHS in these commercial broilers. PMID:14601724

  6. Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

    PubMed

    Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing

    2016-03-01

    SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish. PMID:26699903

  7. 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.

  8. Evidence for multiple, distinct ADAR-containing complexes in Xenopus laevis

    PubMed Central

    Schweidenback, Caterina T.H.; Emerman, Amy B.; Jambhekar, Ashwini

    2015-01-01

    ADAR (adenosine deaminase acting on RNA) is an RNA-editing enzyme present in most metazoans that converts adenosines in double-stranded RNA targets into inosines. Although the RNA targets of ADAR-mediated editing have been extensively cataloged, our understanding of the cellular function of such editing remains incomplete. We report that long, double-stranded RNA added to Xenopus laevis egg extract is incorporated into an ADAR-containing complex whose protein components resemble those of stress granules. This complex localizes to microtubules, as assayed by accumulation on meiotic spindles. We observe that the length of a double-stranded RNA influences its incorporation into the microtubule-localized complex. ADAR forms a similar complex with endogenous RNA, but the endogenous complex fails to localize to microtubules. In addition, we characterize the endogenous, ADAR-associated RNAs and discover that they are enriched for transcripts encoding transcriptional regulators, zinc-finger proteins, and components of the secretory pathway. Interestingly, association with ADAR correlates with previously reported translational repression in early embryonic development. This work demonstrates that ADAR is a component of two, distinct ribonucleoprotein complexes that contain different types of RNAs and exhibit diverse cellular localization patterns. Our findings offer new insight into the potential cellular functions of ADAR. PMID:25519486

  9. Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system.

    PubMed

    Sinzelle, L; Vallin, J; Coen, L; Chesneau, A; Du Pasquier, D; Pollet, N; Demeneix, B; Mazabraud, A

    2006-12-01

    Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure. PMID:16957880

  10. Features of vestibuloocular reflex modulations induced by altered gravitational forces in tadpoles ( Xenopus laevis)

    NASA Astrophysics Data System (ADS)

    Sebastian, C.; Horn, E.

    2001-01-01

    In Xenopus laevis tadpoles, we studied the static vestibuloocular reflex (rVOR) in relation to modifications of the gravitational environment to find basic mechanisms of how altered gravitational forces (AGF) affect this reflex. Animals were exposed to microgravity during space flight or hypergravity (3g) for 4 to 12 days. Basic observations were that (1) the development of the rVOR is significantly affected by altered gravitational conditions, (2) the duration of 1g-readaptation depends on the strength of the test stimulus, (3) μg induces malformations of the body which are related to the rVOR depression. Future studies are based on the hypotheses (1) that the vestibular nuclei play a key roll in the adaptation to AGF conditions, (2) that the stimulus transducing systems in the sense organ are affected by AGF conditions, and (3) that fertilized eggs will be converted to normal adults guided by physiological and morphological set points representing the genetic programs. Developmental retardation or acceleration, or otherwise occurring deviations from standard development during embryonic and postembryonic life will activate genes that direct the developmental processes towards normality.

  11. 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.

  12. Asymmetric dispersal structures a riverine metapopulation of the freshwater pearl mussel Margaritifera laevis

    PubMed Central

    Terui, Akira; Miyazaki, Yusuke; Yoshioka, Akira; Kaifu, Kenzo; Matsuzaki, Shin-ichiro S; Washitani, Izumi

    2014-01-01

    Unidirectional water flow results in the downstream-biased, asymmetric dispersal of many riverine organisms. However, little is known of how asymmetric dispersal influences riverine population structure and dynamics, limiting our ability to properly manage riverine organisms. A metapopulation of the freshwater pearl mussel Margaritifera laevis may be sensitive to river currents because mussels are repeatedly exposed to downstream drift during floods—a parasitic life stage is the only, limited period (∼40 days) during which larvae (glochidia) can move upstream with the aid of host fish. We hypothesized that water-mediated dispersal would overwhelm upstream dispersal via host fish, and therefore, that upstream subpopulations play a critical role as immigrant sources. To test this hypothesis, we examined the effects of both up- and downstream immigrant sources on the size of target subpopulations in the Shubuto River system, Hokkaido, Japan. We found that target subpopulation size was dependent on the upstream distribution range of reproductive subpopulations and the number of upstream tributaries, which are proxies for the number of potential immigrants moving downstream. In contrast, little influence was observed of downstream immigrant sources (proximity to downstream reproductive subpopulations). These results were consistent even after accounting for local environments and stream size. Our finding suggests that upstream subpopulations can be disproportionately important as immigrant sources when dispersal is strongly asymmetric. PMID:25247058

  13. Involvement of sperm proteases in the binding of sperm to the vitelline envelope in Xenopus laevis.

    PubMed

    Kubo, Hideo; Kotani, Masaharu; Yamamoto, Yukio; Hazato, Tadahiko

    2008-01-01

    Sperm binding to the vitelline envelope in dejellied Xenopus laevis eggs was effectively inhibited by inhibitors for trypsin (soybean trypsin inhibitor and p-toluenesulfonyl-L-lysine chloroethyl ketone) and aminopeptidase B (o-phenanthroline, bestatin, and arphamenine B). Likewise, synthetic 4-methylcoumaryl-7-amide (MCA) substrates (t-butoxycarbonyl-GlyArgArg-MCA, benzyloxycarbonyl-ArgArg-MCA, and Arg-MCA) inhibited binding. Consistently, when jellied eggs were inseminated in the presence of these substrates or inhibitors for proteases, fertilization was effectively blocked. The medium in which live sperm or the sperm membrane fraction were suspended exhibited hydrolyzing activities against the synthetic substrates mentioned above, and these activities were effectively inhibited by the protease inhibitors. Ultracentrifugal fractionation of the sperm suspension following induction of the acrosome reaction by a calcium ionophore, A23187, indicated that a considerable amount of the total tryptic and aminopeptidase B activity was released into the medium. On this occasion, part of the tryptic and aminopeptidase B activity was definitely estimated to be discharged in association with a vesiculated membrane, supporting the notion that the proteases involved in binding to the vitelline envelope are present on the sperm plasma membrane. PMID:18275249

  14. A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

    PubMed Central

    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. Cell specialization in the epithelium of the small intestine of feeding Xenopus laevis tadpoles.

    PubMed Central

    Marshall, J A; Dixon, K E

    1978-01-01

    The intestinal epithelium of feeding Xenopus laevis tadpoles was studied using light microscope, electron microscope and autoradiographic techniques. The wall of the small intestine differs from that of most other vertebrates studied in that it lacks villous-like folds. A single prominent longitudinal fold, the typhlosole, forms about stage 49, and a series of shallow longitudinal epithelial pleats is also present in some animals. The morphology of the epithelial cells indicates that there are no differences between the cells in their degree of specialization. Three epithelial cell types were recognized: principal cells, gland cells and endocrine cells, making up about 65%, 15% and 1%, respectively, of all cells present, while approximately 20% of the cells in the epithelium are lymphocytes, 1% appear to be discharged gland cells, and 2% degenerating cells. No Paneth, caveolated or undifferentiated cells were identified. The findings are discussed in relation to other studies on cell proliferation and on nuclear transplantation. Images Figs. 3-4 Figs. 5-6 Figs. 7-8 Fig. 9 Fig. 10 Fig. 11 PMID:649494

  16. Temperature-independent energy expenditure in early development of the African clawed frog Xenopus laevis.

    PubMed

    Nagano, Yatsuhisa; Ode, Koji L

    2014-08-01

    The thermal dissipation of activated eggs and embryos undergoing development from cleavage to the tailbud stage of the African clawed frog Xenopus laevis was measured as a function of incubation time at temperatures ranging from T = 288.2 K to 295.2 K, using a high-precision isothermal calorimeter. A23187-mediated activation of mature eggs induced stable periodic thermal oscillations lasting for 8-34 h. The frequency agreed well with the cell cycle frequency of initial cleavages at the identical temperature. In the developing embryo, energy metabolism switches from embryonic to adult features during gastrulation. The thermal dissipation after gastrulation fit well with a single modified Avrami equation, which has been used for modeling crystal-growth. Both the oscillation frequency of the activated egg and the growth rate of the embryo strongly depend on temperature with the same apparent activation energy of approximately 87 kJ mole(-1). This result suggests that early development proceeds as a single biological time, attributable to a single metabolic rate. A temperature-independent growth curve was derived by scaling the thermogram to the biological time, indicating that the amount of energy expenditure during each developmental stage is constant over the optimal temperature range. PMID:25078857

  17. Recovery capabilities of Xenopus laevis after exposure to Cadmium and Zinc.

    PubMed

    Mouchet, F; Teaniniuraitemoana, V; Baudrimont, M; Daffe, G; Gauthier, L; Gonzalez, P

    2015-11-01

    The present investigation evaluates the recovery capabilities of Xenopus laevis following 12days of exposure to 30μg CdL(-1) and 1000μg ZnL(-1) alone or mixed, followed by a depuration phase in laboratory conditions. Focused endpoints, which were investigated at different times of depuration, are bioaccumulation of Cd and Zn, micronucleus induction, quantification of metallothioneins (MTs), and expression of genes involved in metal toxicity mechanisms. The results show that at the end of the contamination phase, there was higher metal bioaccumulation capability and MT synthesis in remaining tissues than in the liver. An increased expression of genes involved in detoxification and oxidative stress mechanisms was observed, suggesting an additive effect of both metals and a higher Zn regulation in the liver. During the depuration phase, the results show the recovery capability of Xenopus from 7days of depuration related to metamorphosis processes, which were observed at the end of the experiment. The results confirm the relevance of the amphibian model and the complementarities between a marker of genotoxicity, MT production, bioaccumulation and transcriptional analysis in the evaluation of the ecotoxicological impact. The results also highlight the reversible effects of Cd and Zn toxicity. PMID:26073702

  18. Sensory physiology, anatomy and immunohistochemistry of Rohon-Beard neurones in embryos of Xenopus laevis.

    PubMed Central

    Clarke, J D; Hayes, B P; Hunt, S P; Roberts, A

    1984-01-01

    Rohon-Beard neurones show substance P-like immunoactivity in their somas and in their centrally projecting axons. Peripherally, the morphology of their free nerve endings within the trunk skin has been shown using horseradish peroxidase staining. The excitation of Rohon-Beard neurones by natural and electrical stimulation of the skin has been examined using intracellular micro-electrodes in the late embryo of Xenopus laevis. Rohon-Beard cells are sensitive to transient, local indentation of the trunk skin, responding with one or a few impulses. They adapt rapidly to repeated stimulation. They can also be excited by a brief current pulse to the skin. They are not sensitive to slow indentation of the skin, nor are they excited by epithelial action potentials. The responses to skin stimulation are not abolished by a Ringer solution containing 12 mM-Mg2+ and only 0.5 mM-Ca2+. Intracellularly evoked action potentials in single Rohon-Beard cells are sometimes sufficient to evoke sustained episodes of fictive swimming. The results indicate that Rohon-Beard cells are responsible for detecting light touch stimuli to the embryo's body and for initiating swimming in response to this stimulus. Images PLATE 2 PLATE 1 PMID:6201612

  19. 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.

  20. Temperature-independent energy expenditure in early development of the African clawed frog Xenopus laevis

    NASA Astrophysics Data System (ADS)

    Nagano, Yatsuhisa; Ode, Koji L.

    2014-08-01

    The thermal dissipation of activated eggs and embryos undergoing development from cleavage to the tailbud stage of the African clawed frog Xenopus laevis was measured as a function of incubation time at temperatures ranging from T = 288.2 K to 295.2 K, using a high-precision isothermal calorimeter. A23187-mediated activation of mature eggs induced stable periodic thermal oscillations lasting for 8-34 h. The frequency agreed well with the cell cycle frequency of initial cleavages at the identical temperature. In the developing embryo, energy metabolism switches from embryonic to adult features during gastrulation. The thermal dissipation after gastrulation fit well with a single modified Avrami equation, which has been used for modeling crystal-growth. Both the oscillation frequency of the activated egg and the growth rate of the embryo strongly depend on temperature with the same apparent activation energy of approximately 87 kJ mole-1. This result suggests that early development proceeds as a single biological time, attributable to a single metabolic rate. A temperature-independent growth curve was derived by scaling the thermogram to the biological time, indicating that the amount of energy expenditure during each developmental stage is constant over the optimal temperature range.

  1. 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 the enzymatic properties and phosphorylation state of purified HK from the muscle of control and dehydrated (30 % total body water lost) frogs. Bioinformatic tools were also applied to analyze the structural implication of HK phosphorylation in silico. HK from the muscle of dehydrated frogs showed a significantly higher Vmax (3.4-fold) and Km for glucose (2.4-fold) compared with control HK but the Km for ATP was unaltered. HK from dehydrated frogs also showed greater phosphoserine content (20 % increase) and lower phosphothreonine (22 % decrease) content compared to control HK. Control HK had a higher melting temperature (Tm = 61.9 °C) than from dehydrated (Tm = 54.2 °C) frogs when thermostability was tested using differential scanning fluorimetry. In silico phosphorylation of a Xenopus HK caused alterations in active site binding, corroborating phosphorylation as the probable mechanism for kinetic regulation. Physiological consequences of dehydration-induced HK phosphorylation appear to facilitate glycolytic metabolism in hypoxic situations. Augmented HK function increases the ability of Xenopus to overcome compromised oxidative phosphorylation associated with ischemia during dehydration. PMID:26797504

  2. STRUCTURE AND FUNCTION OF THE MIDDLE EAR APPARATUS OF THE AQUATIC FROG, XENOPUS LAEVIS.

    PubMed

    Mason, Mj; Wang, M; Narins, Pm

    2009-01-01

    We report the results of anatomical and vibrometric studies of the middle ear of the African clawed frog, Xenopus laevis. The cartilaginous tympanic disk of Xenopus shows pronounced sexual dimorphism, that of male frogs being much larger than that of females, relative to body size. The stapes footplate, however, is not enlarged in males. The cucullaris muscle was found to insert on the stapes in frogs of both sexes. Using laser interferometry to examine the response of middle ear structures to airborne sound, the stapes footplate was found to vibrate close to 180° out-of-phase with the tympanic disk across a range of frequencies, this resembling the relationship between tympanic membrane and footplate movement previously described in ranid frogs. By contrast, whereas there is a pronounced difference in vibration velocity between tympanic membrane and footplate in ranids, the footplate vibration velocity in Xenopus was found to be similar to that of the tympanic disk. This may be interpreted as an adaptation to improve the detection of sound underwater. PMID:20953303

  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. PMID:26434712

  4. Nucleolin from Xenopus laevis: cDNA cloning and expression during development.

    PubMed

    Caizergues-Ferrer, M; Mariottini, P; Curie, C; Lapeyre, B; Gas, N; Amalric, F; Amaldi, F

    1989-03-01

    Nucleolin is a key nucleolar protein in higher eukaryotic cells and is involved directly in ribosome biogenesis. Using an antiserum raised against hamster nucleolin, the homologous protein was detected in nucleoli of Xenopus laevis hepatocytes as well as in the amplified nucleoli of oocytes. A cDNA encoding Xenopus nucleolin has been isolated and sequenced. The deduced protein sequence reveals similar domains in Xenopus and in mammals, but they have undergone separate evolutions. In particular, each of the four RNA-binding domains has evolved differently--the carboxy-proximal domain is twice as conserved (87%) as the amino-proximal domain (42%). These data shed some light on the possible roles of each domain. The expression of nucleolin has been followed throughout oogenesis and embryogenesis. The appearance of nucleolin during early development precedes the transcription of rDNA and the synthesis of ribosomal proteins. The maximal accumulation of nucleolin at gastrulation coincides with nucleolar reformation. Furthermore, when ribosomal synthesis is activated during oogenesis and embryogenesis, peptides immunorelated to nucleolin appear and accumulate. The results suggest that nucleolin plays a role not only in ribosome assembly but also in nucleologenesis. PMID:2656405

  5. 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.

  6. Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones

    PubMed Central

    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 dynamics1,2. 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 polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. 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 types6-8. PMID:25225829

  7. 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

  8. 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

  9. 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

  10. Mortality and morbidity in African clawed frogs (Xenopus laevis) associated with construction noise and vibrations.

    PubMed

    Felt, Stephen A; Cowan, Andrea M; Luong, Richard; Green, Sherril L

    2012-03-01

    In Spring 2008, 175 adult female Xenopus laevis were exposed to construction-related vibrations that caused overt water rippling in the frog tanks. The 3 affected tanks were custom-built static, 300-gal 'pond-style' tanks that sat on the floor of the housing room. The water in the tank developed visible ripples as a result of the vibrations transmitted through the floor during jack-hammering in an adjacent room that was approximately 10 ftaway. All frogs in the tanks displayed buoyancy problems, excessive air gulping, and skin sloughing; ultimately 7 frogs died. In addition, these 7 animals were bloated, and 5 of these 7 had regurgitated and everted their stomach and distal esophagus into the oral cavity, resulting in airway obstruction and death. Gross pathologic findings included regurgitation and eversion of the stomach of the distal portion of the esophagus into the oral cavity, obstruction of the airway, and lung overinflation. No significant histologic lesions were observed. Construction vibrations transmitted through the water appeared to have disrupted the mechanoreceptive function of the lateral line system, resulting in overstimulation of the noxious feeding response, regurgitation, and eversion of the stomach and distal esophagus into the oral cavity and subsequent suffocation due to airway obstruction. After immediate cessation of the jack-hammering and relocation of the remaining frogs, no additional morbidities or mortalities occurred. PMID:22776127

  11. 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

  12. 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

  13. sox4 And sox11 Function during Xenopus laevis Eye Development

    PubMed Central

    Metzig, Marlen; Tao, Si; Hollemann, Thomas; Kühl, Michael; Kühl, Susanne J.

    2013-01-01

    SoxC genes are involved in many developmental processes such as cardiac, lymphoid, and bone development. The SoxC gene family is represented by Sox4, Sox11, and Sox12. Loss of either Sox4 or Sox11 function is lethal during mouse embryogenesis. Here, we demonstrate that sox4 and sox11 are strongly expressed in the developing eye, heart as well as brain in Xenopus laevis. Morpholino oligonucleotide mediated knock-down approaches in anterior neural tissue revealed that interference with either Sox4 or Sox11 function affects eye development. A detailed analysis demonstrated strong effects on eye size and retinal lamination. Neural induction was unaffected upon Sox4 or Sox11 MO injection and early eye field differentiation and cell proliferation were only mildly affected. Depletion of both genes, however, led independently to a significant increase in cell apoptosis in the eye. In summary, Sox4 and Sox11 are required for Xenopus visual system development. PMID:23874955

  14. Perturbation of Organogenesis by the Herbicide Atrazine in the Amphibian Xenopus laevis

    PubMed Central

    Lenkowski, Jenny R.; Reed, J. Michael; Deininger, Lisa; McLaughlin, Kelly A.

    2008-01-01

    Background Exposure to anthropogenic chemicals during development can disrupt the morphogenesis of organ systems. Use of the herbicide atrazine has been debated in recent years because of its implicated, but poorly characterized, effects on vertebrates. Previous studies primarily examined the effects of atrazine exposure during metamorphosis or early developmental stages of amphibians. Objectives We sought to identify and characterize the susceptibility during the often-overlooked developmental stage of organ morphogenesis. Methods We used a static renewal experimental treatment to investigate the effects of 10, 25, and 35 mg/L atrazine from early organ morphogenesis through the onset of tadpole feeding in the aquatic amphibian model system, Xenopus laevis. We quantified malformations of the body axis, heart, and intestine, as well as apoptosis in the midbrain and pronephric kidney. Results We found a significant dose-dependent increase in the percentage of atrazine-exposed tadpoles with malformations of multiple tissues including the main body axis, circulatory system, kidney, and digestive system. Incidence of apoptotic cells also increased in the both midbrain and kidney of atrazine-exposed tadpoles. Conclusions Our results demonstrate that acute atrazine exposure (10–35 mg/L for ≤ 48 hr) during early organ morphogenesis disrupts proper organ development in an amphibian model system. The concurrent atrazine-induced apoptosis in the pronephric kidney and midbrain begins to elucidate a mechanism by which atrazine may disrupt developmental processes in nontarget organisms. PMID:18288322

  15. 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

  16. 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

  17. 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

  18. 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

  19. Regulation of Respiratory and Vocal Motor Pools in the Isolated Brain of Xenopus laevis

    PubMed Central

    Zornik, Erik; Kelley, Darcy B.

    2010-01-01

    The aquatic frog Xenopus laevis uses a complex vocal repertoire during mating and male–male interactions. Calls are produced without breathing, allowing the frogs to vocalize for long periods underwater. The Xenopus vocal organ, the larynx, is innervated by neurons in cranial motor nucleus (n.) IX–X, which contains both vocal (laryngeal) and respiratory (glottal) motor neurons. The primary descending input to n.IX–X comes from the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM), located in the rostral hindbrain. We wanted to characterize premotor inputs to respiratory and vocal motor neurons and to determine what mechanisms might be involved in regulating two temporally distinct rhythmic behaviors: breathing and calling. Using isolated brain and larynx preparations, we recorded extracellular activity from the laryngeal nerve and muscles and intracellular activity in laryngeal and glottal motor neurons. Spontaneous nerve activities mimicking respiratory and vocal patterns were observed. DTAM projection neurons (DTAMIX–X neurons) provide direct input to glottal and laryngeal motor neurons. Electrical stimulation produced short-latency coordinated activity in the laryngeal nerve. DTAMIX–X neurons provide excitatory monosynaptic inputs to laryngeal motor neurons and mixed excitatory and inhibitory inputs to glottal motor neurons. DTAM stimulation also produced a delayed burst of glottal motor neuron activity. Together, our data suggest that neurons in DTAM produce vocal motor output by directly activating laryngeal motor neurons and that DTAM may coordinate vocal and respiratory motor activity. PMID:18199762

  20. 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.

  1. Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis.

    PubMed

    Pastori, R L; Moskaitis, J E; Buzek, S W; Schoenberg, D R

    1991-04-01

    Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver. PMID:1922078

  2. 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

  3. 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

  4. Examination of an amphibian-based assay using the larvae of Xenopus laevis and Ambystoma mexicanum.

    PubMed

    Saka, Masahiro

    2003-05-01

    Semistatic acute toxicity tests of amphibian larvae (Xenopus laevis and Ambystoma mexicanum) were conducted at different developmental stages and by different methods to establish a simple amphibian-based assay. Test substance was pentachlorophenol sodium salt (PCP-Na). The endpoint was mortality and the 24-, 48-, 72-, and 96-h LC50 values were calculated by probit analysis. Interspecific differences in larval responses were not clear. Larval sensitivity tended to increase with larval age. Newly hatched larvae were most resistant to PCP-Na. During the tests of well-developed larvae, concentrations of dissolved oxygen and PCP-Na in the test solutions greatly dropped owing to uptake by the larvae. Therefore, middle-developed (2-week-old) larvae were most suitable for the test. Toxicity tests for volatile substances would be also possible using 2-week-old larvae in closed vessels. Test individuals should be kept individually to avoid the effects of poisonous skin secretions released from dead larvae. PMID:12706392

  5. Effects of dietary exposure of polycyclic musk HHCB on the metamorphosis of Xenopus laevis.

    PubMed

    Pablos, María Victoria; Jiménez, María Ángeles; San Segundo, Laura; Martini, Federica; Beltrán, Eulalia; Fernández, Carlos

    2016-06-01

    The compound 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-[γ]-2-benzopyrane (HHCB; galaxolide, Chemical Abstracts Service number 1222-05-5) is a synthetic musk used extensively as a fragrance in many consumer products and classified as an emerging pollutant. The ecotoxicological information available for HHCB addresses exposure via water, but this compound is frequently adsorbed into particulate matter. The goal of the present study was to assess the effects of dietary exposure to several environmentally relevant HHCB concentrations adsorbed in food during Xenopus laevis metamorphosis. The authors sought to determine if such exposure to this synthetic musk resulted in histological changes in the thyroid gland in conjunction with changes in development (staging, timing to metamorphosis), body weight, and length. Developmental acceleration on day 14, together with hypertrophy of the thyroid follicular epithelium in tadpoles, suggested a possible agonistic effect of HHCB, which would have been compensated after metamorphosis by regulatory mechanisms to maintain homeostasis. Further research into the potential thyroid-related mechanisms of action of HHCB should be conducted. Environ Toxicol Chem 2016;35:1428-1435. © 2015 SETAC. PMID:26472276

  6. 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. PMID:26294036

  7. The embryonic development of Xenopus laevis under a low frequency electric field.

    PubMed

    Boga, Ayper; Binokay, Secil; Emre, Mustafa; Sertdemir, Yasar

    2012-06-01

    The aim of this study was to determine the effects of a low frequency electric field on the early embryonic development of frogs. The embryos of African clawed toads, Xenopus laevis, were exposed to a 20-μA electric current during the cleavage stages. The developmental processes of embryos during and after electric field exposure were monitored for teratogenic effects. All the embryos continuously exposed to the electric field died without undergoing any developmental processes. However, when the embryos were exposed to the electric field for 20-min periods (four times/over 2 d), the embryos developed into both normal tadpoles (70 %) and malformed tadpoles with light edema, reduced pigmentation, or axial anomalies, such as crooked tails. After exposure, the control embryos were at development stage 35.5 (2 d 2 h), while the normal embryos of the assay group were at developmental stage 41(3 d 4 h). There was a 1 d 2 h difference between the two developmental stages, revealing the importance of that time period for embryogenesis. In conclusion, the effects of electric current on Xenopus embryos are dependent on the initial developmental stage and the duration of exposure. PMID:22723004

  8. The Emergence of Cambodian Civil Society within Global Educational Governance: A Morphogenetic Approach to Agency and Structure

    ERIC Educational Resources Information Center

    Edwards, D. Brent, Jr.; Brehm, William C.

    2015-01-01

    This paper uses Margaret Archer's morphogenetic approach to analyze the emergence of civil society within global educational governance. The purpose is to understand the intersection of historical structures with global actors and spaces that have accompanied the globalization of education. Based on findings from a study on the impact in…

  9. Availability of chemosensory receptors is down-regulated by habituation of larvae to a morphogenetic signal.

    PubMed Central

    Trapido-Rosenthal, H G; Morse, D E

    1986-01-01

    Larvae of the gastropod mollusc Haliotis rufescens are induced to settle from the plankton and metamorphose in response to exogenous gamma-aminobutyric acid (GABA) and a number of GABA-mimetic compounds, including a GABA-mimetic inducer uniquely associated with the surfaces of the naturally recruiting algae. Previous evidence has shown that recognition of these inducers is mediated by specialized chemosensory receptors on the larval epithelium and that transduction of the morphogenetic signal then is mediated by cAMP and excitatory depolarization. We demonstrate here the specific and saturable labeling of a population of larval receptors with the GABA analog beta-(p-chlorophenyl)-[3H]GABA ([3H]baclofen); identification of these labeled receptors with those controlling metamorphosis is suggested by four independent criteria: the effectiveness of GABA and its close structural analogs to induce metamorphosis is closely correlated with the effectiveness of these compounds to compete for binding to this receptor; the natural inducer purified from the recruiting algae competes for binding to this receptor; (-)-[3H]baclofen specifically bound to the receptors is shed from the larvae after approximately 20 hr, at the time corresponding to the metamorphic abscission and shedding of sensory cilia and other structures from the larvae; and the availability of the receptors for labeling and the ability of the larvae to respond to GABA and GABA analogs can be down-regulated in parallel by habituation of the larvae early in their development. These down-regulated larvae are fully capable of settlement and metamorphosis in response to agents that elevate intracellular cAMP or depolarize the chemosensory membrane, confirming that down-regulation is confined to the receptors, with no effect on the postreceptor pathway. The results reported here thus suggest that the sensitivity of marine invertebrate larvae to morphogenetic stimuli from the environment can be down-regulated by

  10. The Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on the Mortality and Growth of Two Amphibian Species (Xenopus laevis and Pseudacris triseriata)

    PubMed Central

    Collier, Alex; Orr, Lowell; Morris, Julie; Blank, James

    2008-01-01

    We observed a slight drop in the growth of Xenopus laevis and Pseudacris triseriata larvae following acute exposure (24–48 h) during egg development to three concentrations of TCDD (0.3, 3.0, 30.0 μg/l). Our exposure protocol was modeled on a previous investigation that was designed to mimic the effects of maternal deposition of TCDD. The doses selected were consistent with known rates of maternal transfer between mother and egg using actual adult body burdens from contaminated habitats. Egg and embryonic mortality immediately following exposure increased only among 48 h X. laevis treatments. Control P. triseriata and X. laevis completed metamorphosis more quickly than TCDD-treated animals. The snout-vent length of recently transformed P. triseriata did not differ between treatments although controls were heavier than high-dosed animals. Likewise, the snout-vent length and weight of transformed X. laevis did not differ between control and TCDD treatments. These findings provide additional evidence that amphibians, including P. triseriata and X. laevis are relatively insensitive to acute exposure to TCDD during egg and embryonic development. Although the concentrations selected for this study were relatively high, they were not inconsistent with our current understanding of bioaccumulation via maternal transfer. PMID:19151431

  11. Enhanced release of bone morphogenetic proteins from demineralized bone matrix by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Sung, Nak-Yun; Choi, Jong-il

    2015-06-01

    Gamma irradiation is a useful method for sterilizing demineralized bone matrix (DBM), but its effect on the osteoinductivity of DBM is still controversial. In this study, the osteoinductive activity of gamma-irradiated DBM was examined using a mouse myoblastic cell line (C2C12). DBM was extracted from adult bovine bone and was irradiated at a dose of 25 kGy using a 60cobalt gamma-irradiator. Cell proliferation with DBM was not affected by gamma-irradiation, but alkaline phosphatase and osteocalcin productions were significantly increased in C2C12 cell groups treated with gamma-irradiated DBM. It was reasoned that bone morphogenetic proteins were more efficiently released from gamma-irradiated DBM than from the non-irradiated control. This result suggests the effectiveness of radiation sterilization of bone implants

  12. Polyphosphate: A Morphogenetically Active Implant Material Serving as Metabolic Fuel for Bone Regeneration.

    PubMed

    Müller, Werner E G; Tolba, Emad; Schröder, Heinz C; Wang, Xiaohong

    2015-09-01

    The initial mineralization centers during human bone formation onto osteoblasts are composed of CaCO3 . Those bioseeds are enzymatically formed via carbonic anhydrase(s) in close association with the cell surface of the osteoblasts. Subsequently, the bicarbonate/carbonate anions are exchanged non-enzymatically by inorganic phosphate [Pi ]. One source for the supply of Pi is polyphosphate [polyP] which is a physiological polymer, formed in the osteoblasts as well as in the platelets. The energy-rich acid anhydride bonds within the polyP chain are cleaved by phosphatase(s); during this reaction free-energy might be released that could be re-used, as metabolic fuel, for the maintenance of the steady-state concentrations of the substrates/products during mineralization. Finally it is outlined that polyP, as a morphogenetically active scaffold, is even suitable for 3D cell printing. PMID:25982003

  13. ATP-Driven Self-Assembly of a Morphogenetic Protein in Bacillus subtilis

    PubMed Central

    Ramamurthi, Kumaran S.; Losick, Richard

    2008-01-01

    SUMMARY A hallmark of morphogenesis is the orchestrated assembly of complex, supramolecular structures. One such structure is the proteineous coat that surrounds spores of the bacterium Bacillus subtilis. The coat is a multilayered shell that is composed of over fifty proteins. These proteins assemble around a basement layer composed of the morphogenetic protein SpoIVA. We show that SpoIVA harbors a Walker A box that is required for the proper deployment of the protein to the surface of the developing spore and proper assembly of the entire coat. We further show that purified SpoIVA both binds and hydrolyzes ATP and that the protein self-assembles into cable-like structures in a manner that depends on ATP hydrolysis. Self-assembly driven by ATP is an unusual mechanism for the construction of a large cellular structure. PMID:18691972

  14. Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma.

    PubMed

    Takaoka, K; Yoshikawa, H; Masuhara, K; Sugamoto, K; Tsuda, T; Aoki, Y; Ono, K; Sakamoto, Y

    1989-07-01

    A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions. PMID:2545399

  15. Morphogenetic circuitry regulating growth and development in the dimorphic pathogen Penicillium marneffei.

    PubMed

    Boyce, Kylie J; Andrianopoulos, Alex

    2013-02-01

    Penicillium marneffei is an emerging human-pathogenic fungus endemic to Southeast Asia. Like a number of other fungal pathogens, P. marneffei exhibits temperature-dependent dimorphic growth and grows in two distinct cellular morphologies, hyphae at 25°C and yeast cells at 37°C. Hyphae can differentiate to produce the infectious agents, asexual spores (conidia), which are inhaled into the host lung, where they are phagocytosed by pulmonary alveolar macrophages. Within macrophages, conidia germinate into unicellular yeast cells, which divide by fission. This minireview focuses on the current understanding of the genes required for the morphogenetic control of conidial germination, hyphal growth, asexual development, and yeast morphogenesis in P. marneffei. PMID:23204189

  16. Limits on the morphogenetic role of rain splash transport in hillslope evolution

    NASA Astrophysics Data System (ADS)

    Dunne, Thomas; Malmon, Daniel V.; Dunne, Kieran B. J.

    2016-03-01

    An experimentally parameterized equation for rain splash transport was generalized in order to compute annual sediment transport rates for a range of climates. The resulting equation was then used to examine the conditions under which rain splash transport can play a significant morphogenetic role in water-eroded landscapes. Rain splash can generate convex hillslopes on short, steep hillslopes such as the margins of gullies in unconsolidated sediments. However, comparison of predicted profiles with long, convex hillslopes in postorogenic landscapes indicates that rain splash cannot transport sufficient quantities of sediment to produce the observed convex hillslope profiles. Rates of biogenic transport estimated from the literature also appear to be insufficient. Sheetwash transport appears to be the only process capable of molding these extensive convex hillslope profiles despite the common assumption that it generates exclusively concave forms.

  17. The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

    PubMed Central

    Raja, Erna; Edlund, Karolina; Kahata, Kaoru; Zieba, Agata; Morén, Anita; Watanabe, Yukihide; Voytyuk, Iryna; Botling, Johan; Söderberg, Ola; Micke, Patrick; Pyrowolakis, George; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease. PMID:26701726

  18. The biological function of type I receptors of bone morphogenetic protein in bone

    PubMed Central

    Lin, Shuxian; Svoboda, Kathy K H; Feng, Jian Q; Jiang, Xinquan

    2016-01-01

    Bone morphogenetic proteins (BMPs) have multiple roles in skeletal development, homeostasis and regeneration. BMPs signal via type I and type II serine/threonine kinase receptors (BMPRI and BMPRII). In recent decades, genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone, cartilage, and muscles. In this review, we focus on all three types of BMPRI, which consist of activin-like kinase 2 (ALK2, also called type IA activin receptor), activin-like kinase 3 (ALK3, also called BMPRIA), and activin-like kinase 6 (ALK6, also called BMPRIB). The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis, and osteogenesis. Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future. PMID:27088043

  19. USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling

    PubMed Central

    Herhaus, Lina; Al-Salihi, Mazin A.; Dingwell, Kevin S.; Cummins, Timothy D.; Wasmus, Lize; Vogt, Janis; Ewan, Richard; Bruce, David; Macartney, Thomas; Weidlich, Simone; Smith, James C.; Sapkota, Gopal P.

    2014-01-01

    Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis. PMID:24850914

  20. The controversy surrounding bone morphogenetic proteins in the spine: a review of current research.

    PubMed

    Hustedt, Joshua W; Blizzard, Daniel J

    2014-12-01

    Bone morphogenetic proteins have been in use in spinal surgery since 2002. These proteins are members of the TGF-beta superfamily and guide mesenchymal stem cells to differentiate into osteoblasts to form bone in targeted tissues. Since the first commercial BMP became available in 2002, a host of research has supported BMPs and they have been rapidly incorporated in spinal surgeries in the United States. However, recent controversy has arisen surrounding the ethical conduct of the research supporting the use of BMPs. Yale University Open Data Access (YODA) recently teamed up with Medtronic to offer a meta-analysis of the effectiveness of BMPs in spinal surgery. This review focuses on the history of BMPs and examines the YODA research to guide spine surgeons in their use of BMP in spinal surgery. PMID:25506287

  1. A Survey of Strategies to Modulate the Bone Morphogenetic Protein Signaling Pathway: Current and Future Perspectives

    PubMed Central

    2016-01-01

    Bone morphogenetic proteins (BMPs) constitute the largest subdivision of the TGF-β family of ligands and are unequivocally involved in regulating stem cell behavior. Appropriate regulation of canonical BMP signaling is critical for the development and homeostasis of numerous human organ systems, as aberrations in the BMP pathway or its regulation are increasingly associated with diverse human pathologies. In this review, we provide a wide-perspective on strategies that increase or decrease BMP signaling. We briefly outline the current FDA-approved approaches, highlight emerging next-generation technologies, and postulate prospective avenues for future investigation. We also detail how activating other pathways may indirectly modulate BMP signaling, with a particular emphasis on the relationship between the BMP and Activin/TGF-β pathways. PMID:27433166

  2. ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis.

    PubMed

    Ramamurthi, Kumaran S; Losick, Richard

    2008-08-01

    A hallmark of morphogenesis is the orchestrated assembly of complex, supramolecular structures. One such structure is the proteineous coat that surrounds spores of the bacterium Bacillus subtilis. The coat is a multilayered shell that is composed of more than 50 proteins. These proteins assemble around a basement layer composed of the morphogenetic protein SpoIVA. We show that SpoIVA harbors a Walker A box that is required for the proper deployment of the protein to the surface of the developing spore and proper assembly of the entire coat. We further show that purified SpoIVA both binds and hydrolyzes ATP and that the protein self-assembles into cable-like structures in a manner that depends on ATP hydrolysis. Self-assembly driven by ATP is an unusual mechanism for the construction of a large cellular structure. PMID:18691972

  3. Enhanced healing of rat calvarial defects with sulfated chitosan-coated calcium-deficient hydroxyapatite/bone morphogenetic protein 2 scaffolds.

    PubMed

    Zhao, Jun; Shen, Gang; Liu, Changsheng; Wang, Shaoyi; Zhang, Wenjie; Zhang, Xiaochen; Zhang, Xiuli; Ye, Dongxia; Wei, Jie; Zhang, Zhiyuan; Jiang, Xinquan

    2012-01-01

    Calcium phosphate cements (CPCs), which are widely used in bone regeneration, possess good biocompatibility and osteoconductivity and have been demonstrated to be candidate carriers for bone growth factors. However, limited release of growth factors from CPCs and slow degradation of the materials are not desirable for certain clinical applications. Previous studies have shown that calcium-deficient hydroxyapatite (CDHA) from CPCs presents more rapid degradation rate than CPCs. In this study, a hybrid growth factor delivery system was prepared by using bone morphogenetic protein 2 (BMP-2) loaded CDHA porous scaffold with sulfated chitosan (SCS) coating for improved release profile. We tested the BMP-2 release characteristic of CDHA/BMP-2/SCS composite in vitro and its ability to repair rat calvarial bone defects. A higher percentage of BMP-2 was released when sulfated chitosan coating was present compared with CDHA/BMP-2 group. Eight weeks postoperation, the repaired crania were evaluated by microcomputed tomography, sequential fluorescent labeling, histological analysis, and immunohistochemistry. CDHA/BMP-2/SCS group promoted the most extensive new bone formation than CDHA/BMP-2 and CDHA groups. Our observations suggest that sulfated chitosan coating could enhance the release profile of CDHA/BMP-2 composite in vitro and promote new bone formation in vivo. The hybrid CDHA/BMP-2/SCS system is a promising growth factor delivery strategy for bone regeneration. PMID:21830854

  4. Morphogenetically active scaffold for osteochondral repair (polyphosphate/alginate/N,O-carboxymethyl chitosan).

    PubMed

    Müller, W E; Neufurth, M; Wang, S; Tolba, E; Schröder, H C; Wang, X

    2016-01-01

    Here we describe a novel bioinspired hydrogel material that can be hardened with calcium ions to yield a scaffold material with viscoelastic properties matching those of cartilage. This material consists of a negatively charged biopolymer triplet, composed of morphogenetically active natural inorganic polyphosphate (polyP), along with the likewise biocompatible natural polymers N,O-carboxymethyl chitosan (N,O-CMC) and alginate. The porosity of the hardened scaffold material obtained after calcium exposure can be adjusted by varying the pre-processing conditions. Various compression tests were applied to determine the local (nanoindentation) and bulk mechanical properties (tensile/compression test system for force measurements) of the N,O-CMC-polyP-alginate material. Determinations of the Young's modulus revealed that the stiffness of this comparably water rich (and mouldable) material increases during successive compression cycles to values measured for native cartilage. The material not only comprises viscoelastic properties suitable for a cartilage substitute material, but also displays morphogenetic activity. It upregulates the expression of genes encoding for collagen type II and aggrecan, the major proteoglycan within the articular cartilage, in human chondrocytes, and the expression of alkaline phosphatase in human bone-like SaOS-2 cells, as revealed in RT qPCR experiments. Further, we demonstrate that the new polyP-based material can be applied for manufacturing 3D solid models of cartilage bone such as of the tibial epiphyseal plate and the superior articular cartilage surface. Since the material is resorbable and enhances the activity of cells involved in regeneration of cartilage tissue, this material has the potential to be used for artificial articular cartilage implants. PMID:26898843

  5. The Deep-Sea Natural Products, Biogenic Polyphosphate (Bio-PolyP) and Biogenic Silica (Bio-Silica), as Biomimetic Scaffolds for Bone Tissue Engineering: Fabrication of a Morphogenetically-Active Polymer

    PubMed Central

    Wang, Xiaohong; Schröder, Heinz C.; Feng, Qingling; Draenert, Florian; Müller, Werner E. G.

    2013-01-01

    Bone defects in human, caused by fractures/nonunions or trauma, gain increasing impact and have become a medical challenge in the present-day aging population. Frequently, those fractures require surgical intervention which ideally relies on autografts or suboptimally on allografts. Therefore, it is pressing and likewise challenging to develop bone substitution materials to heal bone defects. During the differentiation of osteoblasts from their mesenchymal progenitor/stem cells and of osteoclasts from their hemopoietic precursor cells, a lineage-specific release of growth factors and a trans-lineage homeostatic cross-talk via signaling molecules take place. Hence, the major hurdle is to fabricate a template that is functioning in a way mimicking the morphogenetic, inductive role(s) of the native extracellular matrix. In the last few years, two naturally occurring polymers that are produced by deep-sea sponges, the biogenic polyphosphate (bio-polyP) and biogenic silica (bio-silica) have also been identified as promoting morphogenetic on both osteoblasts and osteoclasts. These polymers elicit cytokines that affect bone mineralization (hydroxyapatite formation). In this manner, bio-silica and bio-polyP cause an increased release of BMP-2, the key mediator activating the anabolic arm of the hydroxyapatite forming cells, and of RANKL. In addition, bio-polyP inhibits the progression of the pre-osteoclasts to functionally active osteoclasts. Based on these findings, new bioinspired strategies for the fabrication of bone biomimetic templates have been developed applying 3D-printing techniques. Finally, a strategy is outlined by which these two morphogenetically active polymers might be used to develop a novel functionally active polymer. PMID:23528950

  6. Comparison between lead accumulation of Pomphorhynchus laevis (Palaeacanthocephala) in the intestine of chub (Leuciscus cephalus) and in the body cavity of goldfish (Carassius auratus auratus).

    PubMed

    Sures, B; Siddall, R

    2001-05-15

    This experimental study assessed the role of the microhabitat in the uptake of metals by adult acanthocephalans. We examined the accumulation of lead by adult Pomphorhynchus laevis in the intestine of chub (Leuciscus cephalus) and compared it with that in goldfish, Carassius auratus auratus, in which the parasites penetrate the intestinal wall and enter the body cavity. Chub and goldfish experimentally infected with adult Pomphorhynchus laevis were exposed to 0.01 mg l(-1) Pb(2+) over 3 weeks. Lead was rapidly accumulated in the intestinal acanthocephalans reaching a mean concentration of 7.3 microg g(-1). This concentration was significantly greater than in the host muscle, liver and intestine and more than 730 times higher than the exposure concentration. Intraperitoneal P. laevis in goldfish exposed to lead did not accumulate the metal. Thus, it was conclusively shown that metal accumulation in acanthocephalans is associated with the intestinal location and does not occur in the body cavity. PMID:11336747

  7. The Influence of Artificially Introduced N-Glycosylation Sites on the In Vitro Activity of Xenopus laevis Erythropoietin

    PubMed Central

    Nagasawa, Kazumichi; Meguro, Mizue; Sato, Kei; Tanizaki, Yuta; Nogawa-Kosaka, Nami; Kato, Takashi

    2015-01-01

    Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1–3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR. PMID:25898205

  8. Genetic and biochemical biomarkers in the macrophyte Bidens laevis L. exposed to a commercial formulation of endosulfan.

    PubMed

    Pérez, Débora J; Lukaszewicz, Germán; Menone, Mirta L; V Amé, María; Camadro, Elsa L

    2014-09-01

    Previous studies in the wetland macrophyte Bidens laevis L have demonstrated that the insecticide endosulfan induces a high frequency of somatic chromosome aberrations in anaphase-telophase (CAAT) but no DNA changes as determined by the single cell gel electrophoresis (Comet) assay. Thus, cytogenetic biomarkers appear to be more sensitive to the toxic effects of the insecticide than the DNA molecule in the studied species. For this reason, the goals of this study were to use cytogenetic biomarkers--CAAT and abnormal metaphase--and defense biomarkers such as the activity of the antioxidant enzymes--guaiacol peroxidases (POD), glutathione reductase, and microsomal and cytosolic (m- and c-) glutathione-S-transferase (GST)--to evaluate in B. laevis effects caused by a commercial formulation of endosulfan. The frequency of CAAT was increased at 5, 10, 50, and 100 μg/L endosulfan with respect to the negative controls by 3.1, 2.5, 2.5, and 3.2-fold, respectively while the frequency of abnormal metaphases was also increased at the same concentrations by 3.5, 2.8, 3.2, and 11.3-fold, respectively. In addition to these aneugenic effects, other abnormalities such as C-mitosis and chromosome clumping were observed at 10 μg/L endosulfan. On the other hand, POD induction at 0.02, 0.5, 5, and 10 μg/L and m-GST inhibition at 0.5, 10, and 50 μg/L in plants exposed during 24 h to endosulfan were observed but all of these responses were highly variable. In conclusion, only cytogenetic biomarkers like CAAT in B. laevis can serve potentially as early warning systems to detect environmentally relevant concentrations of endosulfan in aquatic ecosystems. PMID:23315857

  9. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

    PubMed

    Bates, Ryan C; Fees, Colby P; Holland, William L; Winger, Courtney C; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J

    2014-02-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  10. Nuclear hourglass technique: An approach that detects electrically open nuclear pores in Xenopus laevis oocyte

    PubMed Central

    Danker, T.; Schillers, H.; Storck, J.; Shahin, V.; Krämer, B.; Wilhelmi, M.; Oberleithner, H.

    1999-01-01

    Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 ± 0.34 S/cm2). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 × 106 NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically “open” state with a mean single NPC electrical conductance of 1.7 ± 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism. PMID:10557355

  11. Embryotoxic effects of environmental chemicals: tests with the South African clawed toad (Xenopus laevis)

    SciTech Connect

    Dumpert, K.

    1987-06-01

    In the course of the investigations reported below, it was shown that p-chloroaniline has a lethal effect on the embryos of Xenopus laevis at a concentration of 100 ppm and is development inhibiting (teratogenic) at concentrations of 1 and 10 ppm, respectively. In the case of aniline, a significant development-inhibiting effect was observed at a concentration as low as 1 ppm. A toxic effect was caused by concentrations between 30 and 40 ppm during embryogenesis and by concentrations above 40 ppm during larval development. A very conspicuous finding was an inhibiting effect of 20 to 40 ppm aniline on pigmentation during embryogenesis and of a concentration as low as 1 ppm on the body size of the young toads. In the case of potassium dichromate, it was possible to barely detect a weak development-inhibiting effect during embryogenesis but no development-retarding effect during larval development. Toxic effects of potassium dichromate occurred during embryogenesis at concentrations of 5 and 7.5 ppm and during the larval development at concentrations above 10 ppm. Sodium dodecylbenzenesulfonic acid at a concentration of 50 ppm was found to have such a strong embryolethal effect that 80% of the eggs showed no cell division at all and the remaining 20% developed to only the bicellular stage. A teratogenic effect of this substance was not observed. Phenol, too, was found to be toxic at a concentration of 50 ppm; in contrast to sodium dodecylbenzenesulfonic acid, however, it did not show any lethal effect on the embryos but it did on the tadpoles, mainly in the first stages of larval development. Lower concentrations of phenol (5 and 10 ppm) had a nonsignificant inhibiting effect on the growth of the larvae. A teratogenic effect of phenol was not detected.

  12. The Pharmacokinetics of Enrofloxacin in Adult African Clawed Frogs (Xenopus laevis)

    PubMed Central

    Howard, Antwain M; Papich, Mark G; Felt, Stephen A; Long, Charles T; McKeon, Gabriel P; Bond, Emmitt S; Torreilles, Stéphanie L; Luong, Richard H; Green, Sherril L

    2010-01-01

    Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 µg/mL, distribution volume was 841.96 mL/kg, and area under the time–concentration curve was 57.59 µg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 µg/mL, 915.85 mL/kg, and 47.42 µg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs’ habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing. PMID:21205443

  13. Intracellular glycosylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis

    SciTech Connect

    Gottlieb, T.A.; Wallace, R.A.

    1982-01-01

    Pulse-chase experiments measuring the rates of incorporation of radiolabeled glucosamine and galactose into intracellular vitellogenin show that glycosylation of this multicomponent protein occurs in a Golgi-enriched fraction isolated from homogenized liver slices. No apparent role of the rough endoplasmic reticulum in this process was demonstrable. Kinetics of the intracellular translocation of glycosylated vitellogenin indicate that the galactosylated intermediate is secreted more rapidly than the glucosamine-labeled precursor. This was corroborated by measuring the rates of accumulation of various pulse-labeled forms of vitellogenin in the chase medium. In addition, a negligible amount of mannose was incorporated into intracellular or secreted vitellogenin. The antibiotic tunicamycin was shown to inhibit (/sup 3/H) glucosamine incorporation into microsomal vitellogenin by 70% without any significant effect on the synthesis of the protein backbone. In addition, nonglycosylated vitellogenin showed normal secretion kinetics. After suitable pretreatment with the antibiotic followed by a labelling period in tunicamycin-free medium, mannose was still not incorporated into vitellogenin, whereas glucosamine behaved in a typical manner. In contrast to this finding, gas-liquid chromatography of the alditol acetate derivatives of the neutral hexoses of vitellogenin showed that mannose was indeed a major component of the vitellogenin oligosaccharide side chain. These preliminary results indicate that the oligosaccharide component of vitellogenin in Xenopus laevis is a ''complex'' type of carbohydrate unit which is linked via an N-glycosidic bond between an asparagine residue and N-acetylglucosamine. With respect to the subcellular localization of glycoprotein assembly in Xenopus liver, there is a significant departure from currently accepted models of glycoprotein synthesis.

  14. Integument structure and function in juvenile Xenopus laevis with disrupted thyroid balance.

    PubMed

    Carvalho, Edison S M; Fuentes, Juan; Power, Deborah M

    2011-12-01

    The skin is the largest organ in the body and is a barrier between the internal and external environment. The present study evaluates how PTU, a goitrogen, that is used to treat hyperthyroidism affects the structure and electrical properties of the frog (Xenopus laevis) skin. The results are considered in the context of the two-membrane model established in the seminal work of Ussing and collegues in the 1940s and 1950s. In vitro experiments with skin from Xenopus adults revealed that PTU can act directly on skin and causes a significant increase (p<0.05, One-way ANOVA) in short circuit current (Isc) via an amiloride-insensitive mechanism. Juvenile Xenopus exposed to waterborne PTU (5 mg/L) had a significantly bigger and more active thyroid gland (p<0.01, Student's t-test) than control Xenopus. The bioelectric properties of skin taken from Xenopus juveniles treated with PTU in vivo had a lower Isc, (3.05±0.4, n=13) and Rt (288.2±39.5) than skin from control Xenopus (Isc, 4.19±1.14, n=14; Rt, 343.3±43.3). A histological assessment of skin from PTU treated Xenopus juveniles revealed the epidermis was significantly thicker (p<0.01, Student's t-test) and had a greater number of modified exocrine glands (p<0.01, Student's t-test) in the dermis compared to control skin. Modifications in skin structure are presumably the basis for its changed bioelectric properties and the study highlights a site of action for environmental chemicals which has been largely neglected. PMID:21963960

  15. FMRP Regulates Neurogenesis In Vivo in Xenopus laevis Tadpoles1,2,3

    PubMed Central

    Faulkner, Regina L.; Wishard, Tyler J.; Thompson, Christopher K.; Liu, Han-Hsuan

    2014-01-01

    Abstract Fragile X Syndrome (FXS) is the leading known monogenic form of autism and the most common form of inherited intellectual disability. FXS results from silencing the FMR1 gene during embryonic development, leading to loss of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates mRNA transport, stability, and translation. FXS is commonly thought of as a disease of synaptic dysfunction; however, FMRP expression is lost early in embryonic development, well before most synaptogenesis occurs. Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable. We investigated whether FMRP affects neurogenesis in Xenopus laevis tadpoles that express a homolog of FMR1. We used in vivo time-lapse imaging of neural progenitor cells and their neuronal progeny to evaluate the effect of acute loss or overexpression of FMRP on neurogenesis in the developing optic tectum. We complimented the time-lapse studies with SYTOX labeling to quantify apoptosis and CldU labeling to measure cell proliferation. Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival. They also have increased neuronal differentiation, but deficient dendritic arbor elaboration. The presence and severity of these defects was highly sensitive to FMRP levels. These data demonstrate that FMRP plays an important role in neurogenesis and suggest that endogenous FMRP levels are carefully regulated. These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS. PMID:25844398

  16. Prepatterning and patterning of the thalamus along embryonic development of Xenopus laevis

    PubMed Central

    Bandín, Sandra; Morona, Ruth; González, Agustín

    2015-01-01

    Previous developmental studies of the thalamus (alar part of the diencephalic prosomere p2) have defined the molecular basis for the acquisition of the thalamic competence (preparttening), the subsequent formation of the secondary organizer in the zona limitans intrathalamica, and the early specification of two anteroposterior domains (rostral and caudal progenitor domains) in response to inducing activities and that are shared in birds and mammals. In the present study we have analyzed the embryonic development of the thalamus in the anuran Xenopus laevis to determine conserved or specific features in the amphibian diencephalon. From early embryonic stages to the beginning of the larval period, the expression patterns of 22 markers were analyzed by means of combined In situ hybridization (ISH) and immunohistochemical techniques. The early genoarchitecture observed in the diencephalon allowed us to discern the boundaries of the thalamus with the prethalamus, pretectum, and epithalamus. Common molecular features were observed in the thalamic prepatterning among vertebrates in which Wnt3a, Fez, Pax6 and Xiro1 expression were of particular importance in Xenopus. The formation of the zona limitans intrathalamica was observed, as in other vertebrates, by the progressive expression of Shh. The largely conserved expressions of Nkx2.2 in the rostral thalamic domain vs. Gbx2 and Ngn2 (among others) in the caudal domain strongly suggest the role of Shh as morphogen in the amphibian thalamus. All these data showed that the molecular characteristics observed during preparttening and patterning in the thalamus of the anuran Xenopus (anamniote) share many features with those described during thalamic development in amniotes (common patterns in tetrapods) but also with zebrafish, strengthening the idea of a basic organization of this diencephalic region across vertebrates. PMID:26321920

  17. Prolonged vestibular stimulation induces homeostatic plasticity of the vestibulo-ocular reflex in larval Xenopus laevis.

    PubMed

    Dietrich, Haike; Straka, Hans

    2016-07-01

    Vestibulo-ocular reflexes (VOR) stabilise retinal images during head/body motion in vertebrates by generating spatio-temporally precise extraocular motor commands for corrective eye movements. While VOR performance is generally robust with a relatively stable gain, cerebellar circuits are capable of adapting the underlying sensory-motor transformation. Here, we studied cerebellum-dependent VOR plasticity by recording head motion-induced lateral rectus and superior oblique extraocular motor discharge in semi-intact preparations of Xenopus laevis tadpoles. In the absence of visual feedback, prolonged sinusoidal rotation caused either an increase or decrease of the VOR gain depending on the motion stimulus amplitude. The observed changes in extraocular motor discharge gradually saturated after 20 min of constant rotation and returned to baseline in the absence of motion stimulation. Furthermore, plastic changes in lateral rectus and superior oblique motor commands were plane-specific for horizontal and vertical rotations, respectively, suggesting that alterations are restricted to principal VOR connections. Comparison of multi- and single-unit activity indicated that plasticity occurs in all recorded units of a given extraocular motor nucleus. Ablation of the cerebellum abolished motoneuronal gain changes and prevented the induction of plasticity, thus demonstrating that both acquisition and retention of this type of plasticity require an intact cerebellar circuitry. In conclusion, the plane-specific and stimulus intensity-dependent modification of the VOR gain through the feed-forward cerebellar circuitry represents a homeostatic plasticity that likely maintains an optimal working range for the underlying sensory-motor transformation. PMID:27152983

  18. Long-range gap junctional signaling controls oncogene-mediated tumorigenesis in Xenopus laevis embryos

    PubMed Central

    Chernet, Brook T.; Fields, Chris; Levin, Michael

    2015-01-01

    In addition to the immediate microenvironment, long-range signaling may be an important component of cancer. Molecular-genetic analyses have implicated gap junctions—key mediators of cell-cell communication—in carcinogenesis. We recently showed that the resting voltage potential of distant cell groups is a key determinant of metastatic transformation and tumor induction. Here, we show in the Xenopus laevis model that gap junctional communication (GJC) is a modulator of the long-range bioelectric signaling that regulates tumor formation. Genetic disruption of GJC taking place within tumors, within remote host tissues, or between the host and tumors significantly lowers the incidence of tumors induced by KRAS mutations. The most pronounced suppression of tumor incidence was observed upon GJC disruption taking place farther away from oncogene-expressing cells, revealing a role for GJC in distant cells in the control of tumor growth. In contrast, enhanced GJC communication through the overexpression of wild-type connexin Cx26 increased tumor incidence. Our data confirm a role for GJC in tumorigenesis, and reveal that this effect is non-local. Based on these results and on published data on movement of ions through GJs, we present a quantitative model linking the GJC coupling and bioelectrical state of cells to the ability of oncogenes to initiate tumorigenesis. When integrated with data on endogenous bioelectric signaling during left-right patterning, the model predicts differential tumor incidence outcomes depending on the spatial configurations of gap junction paths relative to tumor location and major anatomical body axes. Testing these predictions, we found that the strongest influence of GJ modulation on tumor suppression by hyperpolarization occurred along the embryonic left-right axis. Together, these data reveal new, long-range aspects of cancer control by the host's physiological parameters. PMID:25646081

  19. CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein.

    PubMed

    Palma, Alejandra G; Galizia, Luciano; Kotsias, Basilio A; Marino, Gabriela I

    2016-05-01

    Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases. PMID:26888038

  20. Vestibular lesion-induced developmental plasticity in spinal locomotor networks during Xenopus laevis metamorphosis.

    PubMed

    Beyeler, Anna; Rao, Guillaume; Ladepeche, Laurent; Jacques, André; Simmers, John; Le Ray, Didier

    2013-01-01

    During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance

  1. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

    PubMed Central

    Bates, Ryan C.; Fees, Colby P.; Holland, William L.; Winger, Courtney C.; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J.

    2014-01-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  2. Histochemical Analyses of Biliary Development During Metamorphosis of Xenopus laevis Tadpoles.

    PubMed

    Ueno, Tomoya; Ishihara, Akinori; Yagi, Shinomi; Koike, Toru; Yamauchi, Kiyoshi; Shiojiri, Nobuyoshi

    2015-01-01

    In mammalian liver development, intrahepatic biliary morphogenesis takes place in periportal, but not in pericentral, regions. Liver progenitor cells transiently form epithelial plate structures and then intrahepatic bile ducts around the portal veins under the influence of the mesenchyme. The present study was undertaken to histochemically examine normal biliary development and its dependence on the action of the thyroid hormone triiodothyronine (T3) in Xenopus laevis tadpoles. In these tadpoles, the development of hepatic ducts and intrahepatic biliary ducts commenced along the portal veins at NF stages 48-50 and stages 50-52, respectively, when the blood concentration of thyroid hormone may be still low. Some periportal hepatocytes expressed carbamoylphosphate synthase I and SOX9, which are hepatocyte and biliary cell markers, respectively, suggesting that periportal hepatocytes give rise to biliary epithelial cells. Periportal biliary cells did not form ductal plates, nor was the periportal mesenchyme well developed as seen in fetal mouse livers. jag1 mRNA was moderately expressed in cells of portal veins and biliary epithelial cells, and notch1 and notch2 mRNAs were weakly detectable in biliary epithelial cells during metamorphosis as seen in developing mammalian livers. These results suggest that Notch signaling plays a decisive role in biliary cell differentiation and morphogenesis of Xenopus tadpoles. Anti-thyroid agent treatment of the tadpoles resulted in delayed biliary morphogenesis, suggesting that biliary development may depend on T3. However, T3 treatment of the tadpoles did not enhance biliary development. Thus, T3 may act positively on biliary development at a very low concentration. PMID:25660701

  3. Regional expression of Pax7 in the brain of Xenopus laevis during embryonic and larval development

    PubMed Central

    Bandín, Sandra; Morona, Ruth; Moreno, Nerea; González, Agustín

    2013-01-01

    Pax7 is a member of the highly conserved Pax gene family that is expressed in restricted zones of the central nervous system (CNS) during development, being involved in early brain regionalization and the maintenance of the regional identity. Using sensitive immunohistochemical techniques we have analyzed the spatiotemporal pattern of Pax7 expression in the brain of the anuran amphibian Xenopus laevis, during development. Pax7 expression was first detected in early embryos in the basal plate of prosomere 3, roof and alar plates of prosomere 1 and mesencephalon, and the alar plate of rhombomere 1. As development proceeded, Pax7 cells were observed in the hypothalamus close to the catecholaminergic population of the mammillary region. In the diencephalon, Pax7 was intensely expressed in a portion of the basal plate of prosomere 3, in the roof plate and in scattered cells of the thalamus in prosomere 2, throughout the roof of prosomere 1, and in the commissural and juxtacommissural domains of the pretectum. In the mesencephalon, Pax7 cells were localized in the optic tectum and, to a lesser extent, in the torus semicircularis. The rostral portion of the alar part of rhombomere 1, including the ventricular layer of the cerebellum, expressed Pax7 and, gradually, some of these dorsal cells were observed to populate ventrally the interpeduncular nucleus and the isthmus (rhombomere 0). Additionally, Pax7 positive cells were found in the ventricular zone of the ventral part of the alar plate along the rhombencephalon and the spinal cord. The findings show that the strongly conserved features of Pax7 expression through development shared by amniote vertebrates are also present in the anamniote amphibians as a common characteristic of the brain organization of tetrapods. PMID:24399938

  4. Adaptive plasticity of spino-extraocular motor coupling during locomotion in metamorphosing Xenopus laevis.

    PubMed

    von Uckermann, Géraldine; Lambert, François M; Combes, Denis; Straka, Hans; Simmers, John

    2016-04-15

    During swimming in the amphibian ITALIC! Xenopus laevis, efference copies of rhythmic locomotor commands produced by the spinal central pattern generator (CPG) can drive extraocular motor output appropriate for producing image-stabilizing eye movements to offset the disruptive effects of self-motion. During metamorphosis, ITALIC! X. laevisremodels its locomotor strategy from larval tail-based undulatory movements to bilaterally synchronous hindlimb kicking in the adult. This change in propulsive mode results in head/body motion with entirely different dynamics, necessitating a concomitant switch in compensatory ocular movements from conjugate left-right rotations to non-conjugate convergence during the linear forward acceleration produced during each kick cycle. Here, using semi-intact or isolated brainstem/spinal cord preparations at intermediate metamorphic stages, we monitored bilateral eye motion along with extraocular, spinal axial and limb motor nerve activity during episodes of spontaneous fictive swimming. Our results show a progressive transition in spinal efference copy control of extraocular motor output that remains adapted to offsetting visual disturbances during the combinatorial expression of bimodal propulsion when functional larval and adult locomotor systems co-exist within the same animal. In stages at metamorphic climax, spino-extraocular motor coupling, which previously derived from axial locomotor circuitry alone, can originate from both axial and ITALIC! de novohindlimb CPGs, although the latter's influence becomes progressively more dominant and eventually exclusive as metamorphosis terminates with tail resorption. Thus, adaptive interactions between locomotor and extraocular motor circuitry allows CPG-driven efference copy signaling to continuously match the changing spatio-temporal requirements for visual image stabilization throughout the transitional period when one propulsive mechanism emerges and replaces another. PMID:27103674

  5. Identification and characterization of a novel intelectin in the digestive tract of Xenopus laevis.

    PubMed

    Nagata, Saburo

    2016-06-01

    The intelectin (Intl) family is a group of secretory lectins in chordates that serve multiple functions, including innate immunity, through Ca(2+)-dependent recognition of carbohydrate chains. Although six Intl family lectins have so far been reported in Xenopus laevis, none have been identified in the intestine. Using a monoclonal antibody to the Xenopus embryonic epidermal lectin (XEEL or Intl-1), I identified cross-reactive proteins in the intestines. The proteins were purified by affinity chromatography on a galactose-Sepharose column and found to be oligomers consisting of N-glycosylated 39 kDa and 40.5 kDa subunit peptides. N-terminal amino acid sequencing of these peptides, followed by cDNA cloning, identified two novel Intls (designated Intl-3 and Intl-4) that showed 59-79% amino acid identities with known Xenopus Intl family proteins. From the amino acid sequence, immunoreactivity, and properties of the recombinant protein, Intl-3 was considered the intestinal lectin identified by the anti-XEEL antibody. The purified Intl-3 protein could potentially bind to Escherichia coli and its lipopolysaccharides (LPS), and to Staphylococcus aureus and its peptidoglycans, depending on Ca(2+). In addition, the Intl-3 protein agglutinated E. coli cells in the presence of Ca(2+). Intraperitoneal injection of LPS increased the intestinal and rectal contents of Intl-3 and XCL-1 (or 35K serum lectin) proteins within three days; however, unlike XCL-1, Intl-3 was detectable in neither the sera nor the other tissues regardless of LPS stimulation. Immunohistochemical analyses revealed accumulation of the Intl-3 protein in mucus secretory granules of intestinal goblet cells. The results of this study suggest that Xenopus Intl-3 is involved in the innate immune protection of the digestive tract against bacterial infections. PMID:26855011

  6. Trialkyltin Rexinoid-X Receptor Agonists Selectively Potentiate Thyroid Hormone Induced Programs of Xenopus laevis Metamorphosis.

    PubMed

    Mengeling, Brenda J; Murk, Albertinka J; Furlow, J David

    2016-07-01

    The trialkyltins tributyltin (TBT) and triphenyltin (TPT) can function as rexinoid-X receptor (RXR) agonists. We recently showed that RXR agonists can alter thyroid hormone (TH) signaling in a mammalian pituitary TH-responsive reporter cell line, GH3.TRE-Luc. The prevalence of TBT and TPT in the environment prompted us to test whether they could also affect TH signaling. Both trialkyltins induced the integrated luciferase reporter alone and potentiated TH activation at low doses. Trimethyltin, which is not an RXR agonist, did not. We turned to a simple, robust, and specific in vivo model system of TH action: metamorphosis of Xenopus laevis, the African clawed frog. Using a precocious metamorphosis assay, we found that 1nM TBT and TPT, but not trimethyltin, greatly potentiated the effect of TH treatment on resorption phenotypes of the tail, which is lost at metamorphosis, and in the head, which undergoes extensive remodeling including gill loss. Consistent with these responses, TH-induced caspase-3 activation in the tail was enhanced by cotreatment with TBT. Induction of a transgenic reporter gene and endogenous collagenase 3 (mmp13) and fibroblast-activating protein-α (fap) genes were not induced by TBT alone, but TH induction was significantly potentiated by TBT. However, induction of other TH receptor target genes such as TRβ and deiodinase 3 by TH were not affected by TBT cotreatment. These data indicate that trialkyltins that can function as RXR agonists can selectively potentiate gene expression and resultant morphological programs directed by TH signaling in vivo. PMID:27167774

  7. Vestibular Lesion-Induced Developmental Plasticity in Spinal Locomotor Networks during Xenopus laevis Metamorphosis

    PubMed Central

    Beyeler, Anna; Rao, Guillaume; Ladepeche, Laurent; Jacques, André; Simmers, John; Le Ray, Didier

    2013-01-01

    During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance

  8. Alu sequences transcription in X. laevis oocytes: nuclear-cytoplasmic partitioning and evidence for 3' end processing reactions.

    PubMed Central

    Perlino, E; Paonessa, G; Ciliberto, G

    1985-01-01

    A large Alu-family cluster in the 5' flanking region of a human alpha 1-acid glycoprotein gene has been identified and sequenced. Individual members microinjected into X. laevis oocytes are transcribed only when canonical box A and B components of the split pol III promoter are present. Alu transcripts accumulate in the nucleus. An unusually short Alu transcript, able to assume a stable secondary structure, undergoes a 3' end processing reaction similar to the one required for tRNA 3' end maturation. Images PMID:4080545

  9. Rediscovery and a new record of Hemidactylus laevis (Reptilia: Gekkonidae) from Somaliland, with notes on and resurrection of Hemidactylus fragilis.

    PubMed

    Mazuch, Tomáš; Šmíd, Jiří; Bauer, Aaron M

    2016-01-01

    Hemidactylus laevis, a gekkonid lizard originally described from the Gaan Libah Mountains in Somaliland, is recorded herein from Erigavo, Sanaag Region, in the same state, which represents the second-ever known specimen and locality for this species in more than 100 years since its description. The species is endemic to Somaliland. Data on morphology and natural history, as well as the first life photographs are provided. Based on morphological data we conclude the species to be closely related to H. laticaudatus. Additionally, H. fragilis from southern Somalia and extreme south-east Ethiopia is resurrected from the synonymy of H. frenatus based on morphological data. PMID:27395191

  10. Intracellular microRNA profiles form in the Xenopus laevis oocyte that may contribute to asymmetric cell division.

    PubMed

    Sidova, Monika; Sindelka, Radek; Castoldi, Mirco; Benes, Vladimir; Kubista, Mikael

    2015-01-01

    Asymmetric distribution of fate determinants within cells is an essential biological strategy to prepare them for asymmetric division. In this work we measure the intracellular distribution of 12 maternal microRNAs (miRNA) along the animal-vegetal axis of the Xenopus laevis oocyte using qPCR tomography. We find the miRNAs have distinct intracellular profiles that resemble two out of the three profiles we previously observed for mRNAs. Our results suggest that miRNAs in addition to proteins and mRNAs may have asymmetric distribution within the oocyte and may contribute to asymmetric cell division as cell fate determinants. PMID:26059897

  11. Intracellular microRNA profiles form in the Xenopus laevis oocyte that may contribute to asymmetric cell division

    PubMed Central

    Sidova, Monika; Sindelka, Radek; Castoldi, Mirco; Benes, Vladimir; Kubista, Mikael

    2015-01-01

    Asymmetric distribution of fate determinants within cells is an essential biological strategy to prepare them for asymmetric division. In this work we measure the intracellular distribution of 12 maternal microRNAs (miRNA) along the animal-vegetal axis of the Xenopus laevis oocyte using qPCR tomography. We find the miRNAs have distinct intracellular profiles that resemble two out of the three profiles we previously observed for mRNAs. Our results suggest that miRNAs in addition to proteins and mRNAs may have asymmetric distribution within the oocyte and may contribute to asymmetric cell division as cell fate determinants. PMID:26059897

  12. Early changes in the ultrastructure of the pars intermedia of the pituitary of Xenopus laevis after change of background color.

    PubMed

    Volcanes, B D; Weatherhead, B

    1976-01-01

    Stereological analysis of the secretory cells of the pars intermedia of Xenopus laevis over a period of 3 days following the transfer of animals from a white to a black background has revealed that significant alterations in the ultrastructural appearance of these cells can be detected 8 h after the transfer. In particular, changes in the secretory granules and the rough endoplasmic reticulum were found to correlate well with previous reports concerning the melanocyte-stimulating hormone (MSH) content and the capacity for protein synthesis of the pars intermedia. PMID:1028946

  13. Expression of Exogenous mRNA in Xenopus laevis Embryos for the Study of Cell Cycle Regulation

    NASA Astrophysics Data System (ADS)

    Sible, Jill C.; Wroble, Brian N.

    The microinjection of mRNA that is transcribed and capped in vitro into fertilized eggs and embryos of Xenopus laevis provides a powerful means for discovering the function of proteins during early development. Proteins may be overexpressed for a gain-of-function effect or exogenous protein function may be compromised by the microinjection of mRNA encoding “dominant-negative” proteins. This methodology is particularly suited for the investigation of the regulation of the cell cycle, checkpoints, and apoptosis in early development.

  14. Redescription and new host record of Capsala laevis (Monogenoidea: Capsalidae: Capsalinae) from gill of roundscale spearfish, Tetrapturus georgii (Perciformes: Istiophoridae) in the northwestern Atlantic Ocean.

    PubMed

    Barse, Ann M; Bullard, Stephen A

    2012-08-01

    Specimens of a capsalid collected from the gill arches of 2 roundscale spearfish, Tetrapturus georgii Lowe, 1840, (Perciformes: Istiophoridae), captured in the northwestern Atlantic Ocean were identified as Capsala laevis (Verrill, 1875) Johnston, 1929 by having the combination of papillae on the ventral surface of haptor, dorsomarginal body sclerites in a single column extending the entire body length, haptoral accessory sclerites, conical papillae distributing over the ventral body surface, and an anterior attachment organ with a fimbriated posterior margin. The new specimens plus the holotype were used to conduct a taxonomic redescription of C. laevis using light and scanning electron microscopy. We documented that the holotype (USNPC No. 7179) and the new specimens of C. laevis from roundscale spearfish each had papillae on the ventral surface of the anterior attachment organs and sensory papillae on the dorsal body surface. Although data are insufficient at this time to justify proposal of a new species, the new specimens differed from the holotype and published accounts of C. laevis by having a sinistral dorsomarginal patch comprising 27-35 sclerites whereas the holotype has a dorsomarginal patch comprising 60 sclerites. Capsala laevis morphologically most closely resembles Capsala ovalis (Goto, 1894) Price, 1938 , but can be most easily differentiated from it by having dorsomarginal body sclerites. This represents the first record of any parasite from the recently taxonomically resurrected roundscale spearfish, long considered by some as a junior subjective synonym of white marlin, Tetrapturus albidus Poey, 1860 and, concomitantly, a new host record for Capsalidae Baird, 1853. An updated list of host records for C. laevis is provided. A perusal of that literature reveals that the identity of the type host for C. laevis is indeterminate beyond Istiophoridae species and that subsequent reports of the type host as ' T. albidus ' are presumptuous (originally

  15. Dynamic activation of bone morphogenetic protein signaling in collagen-induced arthritis supports their role in joint homeostasis and disease

    PubMed Central

    Daans, Melina; Lories, Rik JU; Luyten, Frank P

    2008-01-01

    Introduction Rheumatoid arthritis is a chronic systemic autoimmune disease affecting peripheral joints and leading to loss of joint function. The severity and outcome of disease are dependent on the balance between inflammatory/destructive and homeostatic or repair pathways. Increasing evidence suggests a role for bone morphogenetic protein (BMP) signaling in joint homeostasis and disease. Methods Activation of BMP signaling in collagen-induced arthritis as a model of rheumatoid arthritis was studied by immunohistochemistry and Western blot for phosphorylated SMAD1/5 at different time points. Expression of different BMP ligands and noggin, a BMP antagonist, was determined on synovium and cartilage extracts of arthritic knees, at different time points, with quantitative polymerase chain reaction. At the protein level, BMP2 and BMP7 were studied with immunohistochemistry. Finally, the effect of anti-tumor necrosis factor-alpha (TNFα) treatment on the expression of BMP2, BMP7, and growth and differentiation factor-5 (GDF5) in synovium and cartilage of arthritic knees was investigated. Results A time-dependent activation of the BMP signaling pathway in collagen-induced arthritis was demonstrated with a dynamic and characteristic expression pattern of different BMP subfamily members in synovium and cartilage of arthritic knees. As severity increases, the activation of BMP signaling becomes more prominent in the invasive pannus tissue. BMP2 is present in cartilage and the hyperplastic lining layer. BMP7 is found in the sublining zone and inflammatory infiltrate. Treatment with etanercept slowed down progression of disease, but no change in expression of GDF5, BMP2, and BMP7 in synovium was found; in the cartilage, however, blocking of TNFα increased the expression of BMP7. Conclusions BMP signaling is dynamically activated in collagen-induced arthritis and is partly TNFα-independent. TNFα blocking increased the expression of BMP7 in the articular cartilage, possibly

  16. Comparison of the metal accumulation capacity between the acanthocephalan Pomphorhynchus laevis and larval nematodes of the genus Eustrongylides sp. infecting barbel (Barbus barbus)

    PubMed Central

    2013-01-01

    Background Metal uptake and accumulation in fish parasites largely depends on the parasite group with acanthocephalans showing the highest accumulation rates. Additionally, developmental stage (larvae or adult) as well as parasite location in the host are suggested to be decisive factors for metal bioconcentration in parasites. By using barbel (Barbus barbus) simultaneously infected with nematode larvae in the body cavity and adult acanthocephalans in the intestine, the relative importance of all of these factors was compared in the same host. Methods Eleven elements Arsenic (As), Cadmium (Cd), Cobalt (Co), Copper (Cu), Iron (Fe), Manganese (Mn), Lead (Pb), Selenium (Se), Tin (Sn), Vanadium (V) and Zinc (Zn) were analyzed in barbel tissues (muscle, intestine, liver) as well as in their acanthocephalan parasites Pomphorhynchus laevis and the larval nematode Eustrongylides sp. (L4) using inductively coupled plasma mass spectrometry (ICP-MS). Results Nine elements were detected in significantly higher levels in the parasites compared to host tissues. The element composition among parasites was found to be strongly dependent on parasite taxa/developmental stage and localization within the host. Intestinal acanthocephalans accumulated mainly toxic elements (As, Cd, Pb), whereas the intraperitoneal nematodes bioconcentrated essential elements (Co, Cu, Fe, Se, Zn). Conclusion Our results suggest that in addition to acanthocephalans, nematodes such as Eustrongylides sp. can also be applied as bioindicators for metal pollution. Using both parasite taxa simultaneously levels of a wide variety of elements (essential and non essential) can easily be obtained. Therefore this host-parasite system can be suggested as an appropriate tool for future metal monitoring studies, if double infected fish hosts are available. PMID:23332036

  17. Postoperative Cyst Associated with Bone Morphogenetic Protein Use in Posterior and Transforaminal Lumbar Interbody Fusion Managed Conservatively: Report of Two Cases

    PubMed Central

    Mejía, Diana M; Drazin, Doniel; Anand, Neel

    2016-01-01

    Bone morphogenetic protein use in spinal surgery for off-label indications continues to remain popular. One area where its use has known associated radicular complications is posterior or transforaminal lumbar interbody fusion. These complications include radiculitis, cyst development, and heterotopic ossification, amongst others. Typically, cyst development has been treated surgically. We present two cases of bone morphogenetic protein-related cysts treated medically and thus, present medical treatment as an alternative treatment option. PMID:27014519

  18. Studies of Xenopus laevis mitochondrial DNA: D-loop mapping and characterization of DNA-binding proteins

    SciTech Connect

    Cairns, S.S.

    1987-01-01

    In X. laevis oocytes, mitochondrial DNA accumulates to 10/sup 5/ times the somatic cell complement, and is characterized by a high frequency of a triple-stranded displacement hoop structure at the origin of replication. To map the termini of the single strands, it was necessary to correct the nucleotide sequence of the D-loop region. The revised sequence of 2458 nucleotides contains 54 discrepancies in comparison to a previously published sequence. Radiolabeling of the nascent strands of the D-loop structure either at the 5' end or at the 3' end identifies a major species with a length of 1670 nucleotides. Cleavage of the 5' labeled strands reveals two families of ends located near several matches to an element, designated CSB-1, that is conserved in this location in several vertebrate genomes. Cleavage of 3' labeled strands produced one fragment. The unique 3' end maps to about 15 nucleotides preceding the tRNA/sup Pro/ gene. A search for proteins which may bind to mtDNA in this region to regulate nucleic acid synthesis has identified three activities in lysates of X. laevis mitochondria. The DNA-binding proteins were assayed by monitoring their ability to retard the migration of labeled double- or single-stranded DNA fragments in polyacrylamide gels. The DNA binding preference was determined by competition with an excess of either ds- or ssDNA.

  19. Nucleotide sequence of the L1 ribosomal protein gene of Xenopus laevis: remarkable sequence homology among introns.

    PubMed Central

    Loreni, F; Ruberti, I; Bozzoni, I; Pierandrei-Amaldi, P; Amaldi, F

    1985-01-01

    Ribosomal protein L1 is encoded by two genes in Xenopus laevis. The comparison of two cDNA sequences shows that the two L1 gene copies (L1a and L1b) have diverged in many silent sites and very few substitution sites; moreover a small duplication occurred at the very end of the coding region of the L1b gene which thus codes for a product five amino acids longer than that coded by L1a. Quantitatively the divergence between the two L1 genes confirms that a whole genome duplication took place in Xenopus laevis approximately 30 million years ago. A genomic fragment containing one of the two L1 gene copies (L1a), with its nine introns and flanking regions, has been completely sequenced. The 5' end of this gene has been mapped within a 20-pyridimine stretch as already found for other vertebrate ribosomal protein genes. Four of the nine introns have a 60-nucleotide sequence with 80% homology; within this region some boxes, one of which is 16 nucleotides long, are 100% homologous among the four introns. This feature of L1a gene introns is interesting since we have previously shown that the activity of this gene is regulated at a post-transcriptional level and it involves the block of the normal splicing of some intron sequences. Images Fig. 3. Fig. 5. PMID:3841512

  20. Effects of low dose endosulfan exposure on brain neurotransmitter levels in the African clawed frog Xenopus laevis.

    PubMed

    Preud'homme, Valérie; Milla, Sylvain; Gillardin, Virginie; De Pauw, Edwin; Denoël, Mathieu; Kestemont, Patrick

    2015-02-01

    Understanding the impact of pesticides in amphibians is of growing concern to assess the causes of their decline. Among pesticides, endosulfan belongs to one of the potential sources of danger because of its wide use and known effects, particularly neurotoxic, on a variety of organisms. However, the effect of endosulfan was not yet evaluated on amphibians at levels encompassing simultaneously brain neurotransmitters and behavioural endpoints. In this context, tadpoles of the African clawed frog Xenopus laevis were submitted to four treatments during 27 d: one control, one ethanol control, and two low environmental concentrations of endosulfan (0.1 and 1 μg L(-1)). Endosulfan induced a significant increase of brain serotonin level at both concentrations and a significant increase of brain dopamine and GABA levels at the lower exposure but acetylcholinesterase activity was not modified by the treatment. The gene coding for the GABA transporter 1 was up-regulated in endosulfan contaminated tadpoles while the expression of other genes coding for the neurotransmitter receptors or for the enzymes involved in their metabolic pathways was not significantly modified by endosulfan exposure. Endosulfan also affected foraging, and locomotion in links with the results of the physiological assays, but no effects were seen on growth. These results show that low environmental concentrations of endosulfan can induce adverse responses in X. laevis tadpoles. At a broader perspective, this suggests that more research using and linking multiple markers should be used to understand the complex mode of action of pollutants. PMID:25192837

  1. The colloidal thyroxine (T4) ring as a novel biomarker of perchlorate exposure in the African clawed frog Xenopus laevis

    USGS Publications Warehouse

    Hu, F.; Sharma, Bibek; Mukhi, S.; Patino, R.; Carr, J.A.

    2006-01-01

    The purpose of this study was to determine if changes in colloidal thyroxine (T4) immunoreactivity can be used as a biomarker of perchlorate exposure in amphibian thyroid tissue. Larval African clawed frogs (Xenopus laevis) were exposed to 0, 1, 8, 93, and 1131 ??g perchlorate/l for 38 and 69 days to cover the normal period of larval development and metamorphosis. The results of this study confirmed the presence of an immunoreactive colloidal T4 ring in thyroid follicles of X. laevis and demonstrated that the intensity of this ring is reduced in a concentration-dependent manner by perchlorate exposure. The smallest effective concentration of perchlorate capable of significantly reducing colloidal T4 ring intensity was 8 ??g perchlorate/l. The intensity of the immunoreactive colloidal T4 ring is a more sensitive biomarker of perchlorate exposure than changes in hind limb length, forelimb emergence, tail resorption, thyrocyte hypertrophy, or colloid depletion. We conclude that the colloidal T4 ring can be used as a sensitive biomarker of perchlorate-induced thyroid disruption in amphibians. ?? Copyright 2006 Oxford University Press.

  2. Endocrine effects of 2,2{prime},4,4{prime}-tetrachlorobiphenyl in the African clawed frog (Xenopus laevis)

    SciTech Connect

    Diana, S.; Hansen, L.; Foley, G.; Beasley, V.

    1995-12-31

    Ortho-substituted polychlorinated biphenyls are known to exhibit estrogenic activity and, in some cases, to enhance excretion of tetraiodothyronine (T4), resulting in hypothyroxinemia in mammals. Since thyroxine activity is essential for amphibian metamorphosis, and amphibian sex determination can be altered or reversed by exposure to exogenous estrogens or androgens, the effects of exposure of larvae of the African clawed frog (Xenopus laevis) to 2,2{prime},4,4{prime}-tetrachlorobiphenyl (CB 47) were investigated. Eggs and larvae of X. laevis were exposed to nominal concentrations of CB 47 of 0.05 or 0.25 ppm (1 ppm was found to result in 100% mortality) throughout the period of larval development, and effects on rates of metamorphosis and body growth and on gonad morphology were determined. Stage of metamorphosis, body length and body weight did not differ between treatment and control groups, following exposure to these sub-lethal concentrations, at any time during larval development. Effects of exposure on gonad morphology will be discussed. The failure of CB 47 to delay or prevent metamorphosis under these conditions may be due to poor responsiveness of hepatic UDP-glucuronyl transferases to induction, or novel systems of thyroxine and/or PCB transport, metabolism and excretion in larval amphibians.

  3. Bone Morphogenetic Proteins in Craniofacial Surgery: Current Techniques, Clinical Experiences, and the Future of Personalized Stem Cell Therapy

    PubMed Central

    Chenard, Kristofer E.; Teven, Chad M.; He, Tong-Chuan; Reid, Russell R.

    2012-01-01

    Critical-size osseous defects cannot heal without surgical intervention and can pose a significant challenge to craniofacial reconstruction. Autologous bone grafting is the gold standard for repair but is limited by a donor site morbidity and a potentially inadequate supply of autologous bone. Alternatives to autologous bone grafting include the use of alloplastic and allogenic materials, mesenchymal stem cells, and bone morphogenetic proteins. Bone morphogenetic proteins (BMPs) are essential mediators of bone formation involved in the regulation of differentiation of osteoprogenitor cells into osteoblasts. Here we focus on the use of BMPs in experimental models of craniofacial surgery and clinical applications of BMPs in the reconstruction of the cranial vault, palate, and mandible and suggest a model for the use of BMPs in personalized stem cell therapies. PMID:23226941

  4. Towards a morphogenetic classification of eskers: Implications for modelling ice sheet hydrology

    NASA Astrophysics Data System (ADS)

    Perkins, Andrew J.; Brennand, Tracy A.; Burke, Matthew J.

    2016-02-01

    Validations of paleo-ice sheet hydrological models have used esker spacing as a proxy for ice tunnel density. Changes in crest type (cross-sectional shape) along esker ridges have typically been attributed to the effect of changing subglacial topography on hydro- and ice-dynamics and hence subglacial ice-tunnel shape. These claims assume that all eskers formed in subglacial ice tunnels and that all major subglacial ice tunnels produced a remnant esker. We identify differences in geomorphic context, sinuosity, cross-sectional shape, and sedimentary architecture by analysing eskers formed at or near the margins of the last Cordilleran Ice Sheet on British Columbia's southern Fraser Plateau, and propose a morphogenetic esker classification. Three morphogenetic types and 2 subtypes of eskers are classified based on differences in geomorphic context, ridge length, sinuosity, cross-sectional shape and sedimentary architecture using geophysical techniques and sedimentary exposures; they largely record seasonal meltwater flows and glacial lake outburst floods (GLOFs) through sub-, en- and supraglacial meltwater channels and ice-walled canyons. General principles extracted from these interpretations are: 1) esker ridge crest type and sinuosity strongly reflect meltwater channel type. Eskers formed in subglacial conduits are likely to be round-crested with low sinuosity (except where controlled by ice structure or modified by surging) and contain faults associated with flank collapse. Eskers formed near or at the ice surface are more likely to be sharp-crested, highly sinuous, and contain numerous faults both under ridge crest-lines and in areas of flank collapse. 2) Esker ridges containing numerous flat-crested reaches formed directly on the land-surface in ice-walled canyons (unroofed ice tunnels) or in ice tunnels at atmospheric pressure, and therefore likely record thin or dead ice. 3) Eskers containing macroforms exhibiting headward and downflow growth likely record

  5. Morphogenetic fields within the human dentition: a new, clinically relevant synthesis of an old concept.

    PubMed

    Townsend, Grant; Harris, Edward F; Lesot, Herve; Clauss, Francois; Brook, Alan

    2009-12-01

    This paper reviews the concept of morphogenetic fields within the dentition that was first proposed by Butler (Butler PM. Studies of the mammalian dentition. Differentiation of the post-canine dentition. Proc Zool Soc Lond B 1939;109:1-36), then adapted for the human dentition by Dahlberg (Dahlberg AA. The changing dentition of man. J Am Dent Assoc 1945;32:676-90; Dahlberg AA. The dentition of the American Indian. In: Laughlin WS, editor. The Physical Anthropology of the American Indian. New York: Viking Fund Inc.; 1951. p. 138-76). The clone theory of dental development, proposed by Osborn (Osborn JW. Morphogenetic gradients: fields versus clones. In: Butler PM, Joysey KA, editors Development, function and evolution of teeth. London: Academic Press, 1978. p. 171-201), is then considered before these two important concepts are interpreted in the light of recent findings from molecular, cellular, genetic and theoretical and anthropological investigation. Sharpe (Sharpe PT. Homeobox genes and orofacial development. Connect Tissue Res 1995;32:17-25) put forward the concept of an odontogenic homeobox code to explain how different tooth classes are initiated in different parts of the oral cavity in response to molecular cues and the expression of specific groups of homeobox genes. Recently, Mitsiadis and Smith (Mitsiadis TA, Smith MM. How do genes make teeth to order through development? J Exp Zool (Mol Dev Evol) 2006; 306B:177-82.) proposed that the field, clone and homeobox code models could all be incorporated into a single model to explain dental patterning. We agree that these three models should be viewed as complementary rather than contradictory and propose that this unifying view can be extended into the clinical setting using findings on dental patterning in individuals with missing and extra teeth. The proposals are compatible with the unifying aetiological model developed by Brook (Brook AH. A unifying aetiological explanation for anomalies of tooth number

  6. Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes.

    PubMed

    Heyer, M; Müller-Berger, S; Romero, M F; Boron, W F; Frömter, E

    1999-08-01

    The rat kidney Na+-HCO3- cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na+ and HCO3- as permeable ions. The current carried by rkNBC (INBC) was identified by its response to changing bath Na+ concentration(s) and quantified as the current blocked by 4, 4'-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO3- to Na+ transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 micromol/l) blocks INBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na+ and HCO3- concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO3- concentration was 25 mmol/l throughout, but the Na+ concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflügers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP

  7. N-Glycans in Xenopus laevis testis characterised by lectin histochemistry.

    PubMed

    Valbuena, Galder; Madrid, Juan Francisco; Martínez de Ubago, María; Gómez-Santos, Laura; Alonso, Edurne; Díaz-Flores, Lucio; Sáez, Francisco J

    2016-03-01

    Analysis of glycan chains of glycoconjugates is difficult because of their considerable variety. Despite this, several functional roles for these glycans have been reported. N-Glycans are oligosaccharides linked to asparagine residues of proteins. They are synthesised in the endoplasmic reticulum (ER) in a unique way, and later modified in both the ER and Golgi apparatus, developing different oligosaccharide chains. An essential role for complex N-glycans in mammalian spermatogenesis has been reported. The aim of the present study was to analyse the N-glycans of the Xenopus laevis testis by means of lectin histochemistry. Five lectins were used that specifically recognise mannose-containing and complex glycans, namely Galanthus nivalis agglutinin (GNA) from snowdrops, concanavalin A (Con A) from the Jack bean, Lens culinaris agglutinin (LCA) from lentils and Phaseolus vulgaris erythroagglutinin (PHA-E) and P. vulgaris leukoagglutinin (PHA-L) from the common bean. GNA and Con A labelled the interstitium and most of the germ cell types, whereas LCA and PHA-E showed affinity only for the interstitium. A granular cytoplasmic region was labelled in spermatogonia and spermatocytes by GNA and PHA-L, whereas GNA and LCA labelled a spermatid region that is probably associated with the centriolar basal body of the nascent flagellum. There was no specific labelling in the acrosome. Some unexpected results were found when deglycosylative pretreatments were used: pre-incubation of tissue sections with peptide N glycosidase F, which removes N-linked glycans, reduced or removed labelling with most lectins, as expected. However, after this pretreatment, the intensity of labelling remained or increased for Con A in the follicle (Sertoli) and post-meiotic germ cells. The β-elimination procedure, which removes O-linked glycans, revealed new labelling patterns with GNA, LCA and PHA-L, suggesting that some N-glycans were masked by O-glycans, and thus they became accessible to these

  8. Moluccella laevis lectin, a marker for cellular differentiation programs in mouse gut epithelium.

    PubMed

    Falk, P; Lorenz, R G; Sharon, N; Gordon, J I

    1995-04-01

    We have assembled a system for testing the hypothesis that changes in glycoconjugate production represent markers for defining developmental, spatial, and environmental influences on the proliferation and differentiation programs of various mouse gut epithelial cell lineages. Multilabel immunohistochemical methods were used to survey the interactions of purified lectins with 1) normal fetal, neonatal, and adult FVB/N mouse gut, 2) gastric and intestinal isografts harvested at various developmental stages, and 3) transgenic mouse models of intestinal epithelial cell hyperplasia, dysplasia, and/or neoplasia. As a demonstration of the system's utility, we used the recently purified, alpha-N-acetyl-D-galactosamine-specific, Moluccella laevis lectin (MLL). In the adult FVB/N mouse stomach, MLL only recognizes glycoconjugates produced by a population of nonproliferating neck and prezymogenic cells that occupy a pivotal point in the complex, migration-associated differentiation program of the zymogenic cell lineage. In the developing FVB/N stomach, MLL binds to members of the zymogenic and pit lineages even before morphogenesis of gastric units is completed. Expression of MLL epitopes in pit cells is restricted to the period before the gastric epithelium has completed its morphoregulatory program. Analysis of gastric isografts indicates that these lineage- and developmental stage-specific patterns of glycoconjugate accumulation are not influenced by normal luminal contents. In the adult FVB/N intestine, MLL binding can be used to operationally define variations in the differentiation programs of 1) members of the enteroendocrine and goblet cell lineages during their migration along the crypt-to-villus axis and 2) cells comprising the follicle-associated epithelium overlying Peyer's patches. Accumulation of MLL epitopes in villus-associated enterocytes does not appear to be affected when these cells are induced to reenter the cell cycle by simian virus 40 large T antigen

  9. Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development

    PubMed Central

    2014-01-01

    Background Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT). Results We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are

  10. Effects of GSM-like radiofrequency irradiation during the oogenesis and spermiogenesis of Xenopus laevis.

    PubMed

    Boga, Ayper; Emre, Mustafa; Sertdemir, Yasar; Uncu, İbrahim; Binokay, Secil; Demirhan, Osman

    2016-07-01

    We aimed to evaluate the effect of GSM-like radiofrequency electromagnetic radiation (RF-EMR) on the oogenesis, and spermiogenesis of Xenopus laevis, and so the development of the embryos obtained from Normal Females+Normal Males (i.e. "N(F)+N(M)"); Normal Females+RF-exposed Males (i.e. "N(F)+RF(M)"); RF-exposed Female+Normal Male (i.e. "RF(F)+N(M)"); and RF-exposed Female+RF-exposed Male (i.e. "RF(F)+RF(M)". Various, assessments were performed to determine potential teratogenic effects and mortality, body growth and behavior on first generation embryos. After exposing adults frogs of both sexes to 900MHz RF-EMR (at 1.0W/kg) for 8h a day over a 5-week period, the embryos' specific energy absorption rate (SAR) was calculated. In our present study (control group; 2.2% abnormal, 0.0% dead); with the N(F)+RF(M) combination, the long-term exposure of adult males to GSM-like radiation at 900MHz (RF: 2W) for 5 week/8h/day resulted in normal, abnormal and dead embryo ratios of 88.3%, 3.3% and 8.3%, respectively (p<0.001). In the RF(F)+N(M) combination, long-term exposure (5 week/8h/day) of adult females led to normal, abnormal and dead embryo ratios of 76.7%, 11.7%, and 11.7%, respectively (p<0.001). And in the RF(F)+RF(M) combination, long-term exposure (5 week/8h/day) of both adult males and females led to normal, abnormal and dead embryo ratios of 73.3%, 11.7%, and 15%, respectively (p<0.001). With the exception RF(F)+RF(M) group (p<0.001), no significant changes were observed on body growth (lengths) in comparison to the control group. It was also observed that the offspring of female adult Xenopus exposed to RF-EMR during oogenesis exhibited a more aggressive behavior compared to the control group. Cell phones radiation can thus lead to detrimental effects in humans' male and female reproductive cells. PMID:27017260

  11. Bone Tissue Engineering Using High Permeability Poly-epsilon-caprolactone Scaffolds Conjugated with Bone Morphogenetic Protein-2

    NASA Astrophysics Data System (ADS)

    Mitsak, Anna Guyer

    Bone is the second most commonly transplanted tissue in the United States. Limitations of current bone defect treatment options include morbidity at the autograft harvest site, mechanical failure, and poorly controlled growth factor delivery. Combining synthetic scaffolds with biologics may address these issues and reduce dependency on autografts. The ideal scaffolding system should promote tissue in-growth and nutrient diffusion, control delivery of biologics and maintain mechanical integrity during bone formation. This dissertation evaluates how scaffold permeability, conjugated bone morphogenetic protein-2 (BMP-2) and differentiation medium affect osteogenesis in vitro and bone growth in vivo.. "High" and "low" permeability polycaprolactone (PCL) scaffolds with regular architectures were manufactured using solid free form fabrication. Bone growth in vivo was evaluated in an ectopic mouse model. High permeability scaffolds promoted better 8 week bone growth, supported tissue penetration into the scaffold core, and demonstrated increased mechanical properties due to newly formed bone. Next, the effects of differentiation medium and conjugated BMP-2 on osteogenesis were compared. Conjugation may improve BMP-2 loading efficiency, help localize bone growth and control release. High permeability scaffolds were conjugated with BMP-2 using the crosslinker, sulfo-SMCC. When adipose-derived and bone marrow stromal cells were seeded onto constructs (with or without BMP-2), BMSC expressed more differentiation markers, and differentiation medium affected differentiation more than BMP-2. In vivo, scaffolds with ADSC pre-differentiated in osteogenic medium (with and without BMP-2) and scaffolds with only BMP-2 grew the most bone. Bone volume did not differ among these groups, but constructs with ADSC had evenly distributed, scaffold-guided bone growth. Analysis of two additional BMP-2 attachment methods (heparin and adsorption) showed highest conjugation efficiency for the

  12. Population structure of the African Clawed Frog (Xenopus laevis) in maize-growing areas with atrazine application versus non-maize-growing areas in South Africa

    USGS Publications Warehouse

    Du Preez, L.H.; Solomon, K.R.; Carr, J.A.; Giesy, J.P.; Gross, T.S.; Kendall, R.J.; Smith, E.E.; Van Der Kraak, G. L.; Weldon, C.

    2005-01-01

    The herbicide atrazine has been suggested to cause gonadal deformities in frogs and could possibly impact on reproduction. Since the early 1960s, atrazine has been used in large amounts in maize production areas of South Africa. These areas overlap with populations of the African Clawed Frog (Xenopus laevis) that has a wide distribution in southern Africa and is found in most water-bodies including those where atrazine residues are detected. The aim of this study was to compare various attributes of individual- and population-level responses of X. laevis from maize-growing and non-maize-growing areas. Xenopus laevis were studied in three reference and five maize-growing sites. Sex ratio, snout-vent length, body-mass and age profiles were found to be similar for populations in maize-growing and non-maize-growing areas. Our mark-recapture data indicated that all sites had robust populations. There were no significant relationships between exposure to atrazine and any of the parameters investigated in populations of X. laevis.

  13. DNaseI-hypersensitive sites at promoter-like sequences in the spacer of Xenopus laevis and Xenopus borealis ribosomal DNA.

    PubMed Central

    La Volpe, A; Taggart, M; McStay, B; Bird, A

    1983-01-01

    We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X. laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAaseI. Unlike DNAaseI, Endo R. MspI digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this "proximal domain" is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function. Images PMID:6310495

  14. In vivo Assessment and Potential Diagnosis of Xenobiotics that Perturb the Thyroid Pathway: Proteomic Analysis of Xenopus laevis Brain Tissue following Exposure to Model T4 Inhibitors

    EPA Science Inventory

    As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression profiles in the brain of an amphibian model (Xenopus laevis) following in vivo exposur...

  15. Friend of GATA (FOG) Interacts with the Nucleosome Remodeling and Deacetylase Complex (NuRD) to Support Primitive Erythropoiesis in Xenopus laevis

    PubMed Central

    Mimoto, Mizuho S.; Christian, Jan L.

    2012-01-01

    Friend of GATA (FOG) plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC) development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD), but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect. PMID:22235346

  16. Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

    PubMed

    Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

    2015-07-15

    Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. PMID:25952563

  17. Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

    SciTech Connect

    Wu, William Ka Kei; Sung, Joseph Jao Yiu; Yu Le; Cho, C.H.

    2008-06-27

    Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21{sup Waf1/Cip1} mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21{sup Waf1/Cip1} induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.

  18. Morphogenetic and cellular movements that shape the mouse cerebellum; insights from genetic fate mapping.

    PubMed

    Sgaier, Sema K; Millet, Sandrine; Villanueva, Melissa P; Berenshteyn, Frada; Song, Christian; Joyner, Alexandra L

    2005-01-01

    We used the cerebellum as a model to study the morphogenetic and cellular processes underlying the formation of elaborate brain structures from a simple neural tube, using an inducible genetic fate mapping approach in mouse. We demonstrate how a 90 degrees rotation between embryonic days 9 and 12 converts the rostral-caudal axis of dorsal rhombomere 1 into the medial-lateral axis of the wing-like bilateral cerebellar primordium. With the appropriate use of promoters, we marked specific medial-lateral domains of the cerebellar primordium and derived a positional fate map of the murine cerebellum. We show that the adult medial cerebellum is produced by expansion, rather than fusion, of the thin medial primordium. Furthermore, ventricular-derived cells maintain their original medial-lateral coordinates into the adult, whereas rhombic lip-derived granule cells undergo lateral to medial posterior transverse migrations during foliation. Thus, we show that progressive changes in the axes of the cerebellum underlie its genesis. PMID:15629700

  19. A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2.

    PubMed

    Thanyaphoo, Suphannee; Kaewsrichan, Jasadee

    2016-09-01

    Silicon-substituted calcium phosphate (Si-CaP) was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP) cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2) was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future. PMID:27383886

  20. The reaction of the dura to bone morphogenetic protein (BMP) in repair of skull defects.

    PubMed Central

    Takagi, K; Urist, M R

    1982-01-01

    Trephine defects in the adult rat skull 0.8 cm in diameter, which do not spontaneously heal, were filled with a bovine bone morphogenetic protein (BMP) fraction. The defects healed not only by bony ingrowth from the trephine rim, but also by proliferation of pervascular mesenchymal-type cells (pericytes) of the dura mater. Under the influence of BMP, dural pericytes differentiated into chondroid and woven bone. Between three and four weeks postimplantation, sinusoids formed and the woven bone remodelled into lamellar bone. Concurrently, blood-borne bone marrow cells colonized the bone deposits, and the diploe were restored. Demonstrating that it is soluble in interstitial fluid, and diffusible across a nucleopore membrane (which isolated the bony margins of the skull), BMP induced new bone formation in the underlying dura and complete repair of the defect. The response of the dura to the BMP fraction produced more new bone than the response to allogeneic bone matrix. The BMP-induced repair was dose dependent; the quantity of new bone was proportional to the dose of the implanted BMP. Images Fig. 1a. Fig. 1b. Fig. 1c. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. Fig. 9. PMID:7092346

  1. Immobilization of Bone Morphogenetic Protein on DOPA- or Dopamine-Treated Titanium Surfaces to Enhance Osseointegration

    PubMed Central

    Kang, Jeonghwa; Tada, Seiichi; Kitajima, Takashi; Son, Tae Il; Aigaki, Toshiro; Ito, Yoshihiro

    2013-01-01

    Titanium was treated with 3,4-dihydroxy-L-phenylalanine (DOPA) or dopamine to immobilize bone morphogenetic protein-2 (BMP2), a biomolecule. DOPA and dopamine solutions turned into suspensions, and precipitates were produced at high pH. Both treatments produced a brown surface on titanium that was thicker at high pH than low pH. Dopamine produced a thicker layer than DOPA. The hydrophobicity of the surfaces increased after treatment with dopamine independent of pH. Furthermore, there were more amino groups in the layers formed at pH 8.5 than pH 4.5 in both treatments. Dopamine treatment produced more amino groups in the layer than DOPA. BMP2 was immobilized on the treated surfaces via a coupling reaction using carbodiimide. More BMP2 was immobilized on surfaces treated at pH 8.5 than pH 4.5 in both treatments. The immobilized BMP induced specific signal transduction and alkali phosphatase, a differentiation marker. Thus, the present study demonstrates that titanium treated with DOPA or dopamine can become bioactive via the surface immobilization of BMP2, which induces specific signal transduction. PMID:24459666

  2. Bone morphogenetic protein 2 inhibits the proliferation and growth of human colorectal cancer cells

    PubMed Central

    ZHANG, YUNYUAN; CHEN, XIAN; QIAO, MIN; ZHANG, BING-QIANG; WANG, NING; ZHANG, ZHONGLIN; LIAO, ZHAN; ZENG, LIYI; DENG, YOULIN; DENG, FANG; ZHANG, JUNHUI; YIN, LIANGJUN; LIU, WEI; ZHANG, QIAN; YAN, ZHENGJIAN; YE, JIXING; WANG, ZHONGLIANG; ZHOU, LAN; LUU, HUE H.; HAYDON, REX C.; HE, TONG-CHUAN; ZHANG, HONGYU

    2014-01-01

    Colorectal cancer (CRC) is one of the most deadly cancers worldwide. Significant progress has been made in understanding the molecular pathogenesis of CRC, which has led to successful early diagnosis, surgical intervention and combination chemotherapy. However, limited therapeutic options are available for metastatic and/or drug-resistant CRC. While the aberrantly activated Wnt/β-catenin pathway plays a critical initiating role in CRC development, disruption of the bone morphogenetic protein (BMP) pathway causes juvenile polyposis syndrome, suggesting that BMP signaling may play a role in CRC development. However, conflicting results have been reported concerning the possible roles of BMP signaling in sporadic colon cancer. Here, we investigated the effect of BMP2 on the proliferation, migration, invasiveness and tumor growth capability of human CRC cells. Using an adenovirus vector that overexpresses BMP2 and the piggyBac transposon-mediated stable BMP2 overexpression CRC line, we found that exogenous BMP2 effectively inhibited HCT116 cell proliferation and colony formation. BMP2 was shown to suppress colon cancer cell migration and invasiveness. Under a low serum culture condition, forced expression of BMP2 induced a significantly increased level of apoptosis in HCT116 cells. Using a xenograft tumor model, we found that forced expression of BMP2 in HCT116 cells suppressed tumor growth, accompanied by decreased cell proliferation activity. Taken together, our results strongly suggest that BMP2 plays an important inhibitory role in governing the proliferation and aggressive features of human CRC cells. PMID:24993644

  3. Nigrostriatal alterations in bone morphogenetic protein receptor II dominant negative mice

    PubMed Central

    Chou, J.; Harvey, B. K.; Ebendal, T.; Hoffer, B.; Wang, Y.

    2008-01-01

    Summary Background We previously demonstrated that exogenous application of bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson’s disease. The purpose of this study is to examine the endogenous neurotrophic properties of BMP Receptor II in dopaminergic neurons of the nigrostriatal pathway. Methods Adult male BMPRII dominant negative (BMPRIIDN) mice and their wild type controls (WT) were placed in the activity chambers for 3 days to monitor locomotor activity. Animals were sacrificed for tyrosine hydroxylase (TH) immunostaining. A subgroup of BMPRIIDN and WT mice were injected with high doses of methamphetamine (MA) and were sacrificed for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) histochemistry at 4 days after injection. Results BMPRIIDN mice had lower locomotor activity than the WT. There is a significant decrease in TH neuronal number in substantia nigra compacta, TH fiber density in the substantia nigra reticulata, and TH immunoreactivity in striatum in the BMPRIIDN mice, suggesting that deficiency in endogenous BMP signaling reduces dopaminergic innervation and motor function in the nigrostriatal pathway. Administration of MA increased TUNEL labeling in the substantia nigra in the BMPRIIDN mice. Conclusions Endogenous BMPs have trophic effects on nigrostriatal dopaminergic neurons. Deficiency in BMP signaling increases vulnerability to insults induced by high doses of MA. PMID:18642641

  4. Effects of Osseointegration by Bone Morphogenetic Protein-2 on Titanium Implants In Vitro and In Vivo

    PubMed Central

    Teng, Fu-Yuan; Chen, Wen-Cheng; Wang, Yin-Lai; Hung, Chun-Cheng; Tseng, Chun-Chieh

    2016-01-01

    This study designed a biomimetic implant for reducing healing time and achieving early osseointegration to create an active surface. Bone morphogenetic protein-2 (BMP-2) is a strong regulator protein in osteogenic pathways. Due to hardly maintaining BMP-2 biological function and specificity, BMP-2 efficient delivery on implant surfaces is the main challenge for the clinic application. In this study, a novel method for synthesizing functionalized silane film for superior modification with BMP-2 on titanium surfaces is proposed. Three groups were compared with and without BMP-2 on modified titanium surfaces in vitro and in vivo: mechanical grinding; electrochemical modification through potentiostatic anodization (ECH); and sandblasting, alkali heating, and etching (SMART). Cell tests indicated that the ECH and SMART groups with BMP-2 markedly promoted D1 cell activity and differentiation compared with the groups without BMP-2. Moreover, the SMART group with a BMP-2 surface markedly promoted early alkaline phosphatase expression in the D1 cells compared with the other surface groups. Compared with these groups in vivo, SMART silaning with BMP-2 showed superior bone quality and created contact areas between implant and surrounding bones. The SMART group with BMP-2 could promote cell mineralization in vitro and osseointegration in vivo, indicating potential clinical use. PMID:26977141

  5. Mechanical testing of recombinant human bone morphogenetic protein-7 regenerated bone in sheep mandibles.

    PubMed

    Kontaxis, A; Abu-Serriah, M; Ayoub, A F; Barbenel, J C

    2004-01-01

    A new method was developed in this study for testing excised sheep mandibles as a cantilever. The method was used to determine the strength and stiffness of sheep hemi-mandibles including a 35 mm defect bridged by regenerated bone. Recombinant human bone morphogenetic protein-7 (rhBMP-7) in a bovine collagen type-I carrier was used for the bone regeneration. Initial tests on ten intact sheep mandibles confirmed that the strength, stiffness and area beneath the load-deformation curves of the right and left hemi-mandibles were not significantly different, confirming the validity of using the contra-lateral hemi-mandible as a control side. Complete bone regeneration occurred in six hemi-mandibles treated with rhBMP, but the quality and mechanical properties of the bone were very variable. The new bone in three samples contained fibrous tissue and was weaker and less stiff than the contra-lateral side (strength, 10-20 per cent; stiffness, 6-15 per cent). The other half had better-quality bone and was significantly stiffer and stronger (p < 0.05), with strength 45-63 per cent and stiffness 35-46 per cent of the contra-lateral side. Hemi-mandibles treated with collagen alone had no regenerated bone bridge suggesting that 35 mm is a critical-size bone defect. PMID:15648662

  6. Repulsive Guidance Molecule is a structural bridge between Neogenin and Bone Morphogenetic Protein

    PubMed Central

    Healey, Eleanor G.; Bishop, Benjamin; Elegheert, Jonathan; Bell, Christian H.; Padilla-Parra, Sergi; Siebold, Christian

    2015-01-01

    Repulsive guidance molecules (RGMs) control crucial processes spanning cell motility, adhesion, immune cell regulation and systemic iron metabolism. RGMs signal via two fundamental signaling cascades: the Neogenin (NEO1) and the Bone Morphogenetic Protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a novel protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the ternary BMP2–RGM–NEO1 complex crystal structure, which combined with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM–NEO1 complex. Our results show how RGM acts as the central hub linking BMP and NEO1 and physically connecting these fundamental signaling pathways. PMID:25938661

  7. Effects of Osseointegration by Bone Morphogenetic Protein-2 on Titanium Implants In Vitro and In Vivo.

    PubMed

    Teng, Fu-Yuan; Chen, Wen-Cheng; Wang, Yin-Lai; Hung, Chun-Cheng; Tseng, Chun-Chieh

    2016-01-01

    This study designed a biomimetic implant for reducing healing time and achieving early osseointegration to create an active surface. Bone morphogenetic protein-2 (BMP-2) is a strong regulator protein in osteogenic pathways. Due to hardly maintaining BMP-2 biological function and specificity, BMP-2 efficient delivery on implant surfaces is the main challenge for the clinic application. In this study, a novel method for synthesizing functionalized silane film for superior modification with BMP-2 on titanium surfaces is proposed. Three groups were compared with and without BMP-2 on modified titanium surfaces in vitro and in vivo: mechanical grinding; electrochemical modification through potentiostatic anodization (ECH); and sandblasting, alkali heating, and etching (SMART). Cell tests indicated that the ECH and SMART groups with BMP-2 markedly promoted D1 cell activity and differentiation compared with the groups without BMP-2. Moreover, the SMART group with a BMP-2 surface markedly promoted early alkaline phosphatase expression in the D1 cells compared with the other surface groups. Compared with these groups in vivo, SMART silaning with BMP-2 showed superior bone quality and created contact areas between implant and surrounding bones. The SMART group with BMP-2 could promote cell mineralization in vitro and osseointegration in vivo, indicating potential clinical use. PMID:26977141

  8. Experimental study of osteoinduction using a new material as a carrier for bone morphogenetic protein-2.

    PubMed

    Koyama, Noriaki; Okubo, Yasunori; Nakao, Kazumasa; Osawa, Kenji; Bessho, Kazuhisa

    2011-06-01

    We evaluated the usefulness of artificial collagen as a new carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2) by comparing it with that of atelopeptide collagen, which is derived from porcine skin, and which we have previously shown to be useful for the induction of bone. rhBMP-2 5μg with either atelopeptide collagen 3mg or artificial collagen 3mg was implanted into the calf muscle of 10-week-old Wistar rats (n=3 in each group). Three rats were given artificial collagen alone and acted as controls (n=3). Radiographic evaluation, histological analysis, and biochemical examinations were made on day 21 after implantation. Soft radiographs (wavelength 10-0.10nm) showed opaque shadows in both groups. Histological analysis showed that new bone had formed in both experimental groups. Endochondral ossification was found at the outermost edge of the implanted collagen in the atelopeptide group. However, there was less ossification in the implanted collagen in the artificial collagen group. On biochemical examination, alkaline phosphatase activity and calcium concentrations in both experimental groups were higher than in the control group, and were higher in the atelopeptide group than in the artificial collagen group. Our results suggest that artificial collagen is useful as a carrier for rhBMP-2 designed to promote the formation of new bone. PMID:20554359

  9. [Morphogenetic study of human adenovirus type 41 in 293TE cells].

    PubMed

    Song, Jing-Dong; Wang, Min; Zou, Xiao-Hui; Qu, Jian-Guo; Lu, Zhuo-Zhuang; Hong, Tao

    2014-03-01

    To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41. PMID:24923169

  10. Tracheal cartilage regeneration and new bone formation by slow release of bone morphogenetic protein (BMP)-2.

    PubMed

    Igai, Hitoshi; Chang, Sung Soo; Gotoh, Masashi; Yamamoto, Yasumichi; Yamamoto, Masaya; Tabata, Yasuhiko; Yokomise, Hiroyasu

    2008-01-01

    We investigated the efficiency of bone morphogenetic protein (BMP)-2 released slowly from gelatin sponge for tracheal cartilage regeneration. A 1-cm gap was made in the mid-ventral portion of each of 10 consecutive tracheal cartilages. In the control group (n = 4), the resulting gap was left untreated. In the gelatin group (n = 4), plain gelatin was implanted in the gap. In the BMP-2 group (n = 4), gelatin containing 100 microg BMP-2 was implanted. We euthanatized all dogs in each group at 1, 3, 6, and 12 months after the implantation, respectively, and then examined the implant site macro- and microscopically. In the BMP-2 group, regenerated fibrous cartilage and newly formed bone were observed at 1 and 12 months. Regenerated cartilage was observed at the ends of the host cartilage stumps, with newly formed bone in the middle portion. The gaps were filled with regenerated cartilage and newly formed bone. At 3 and 6 months, regenerated cartilage, but not newly formed bone, was evident. The regenerated cartilage was covered with perichondrium and showed continuity with the host cartilage. We succeeded in inducing cartilage regeneration and new bone formation in canine trachea by slow release of 100 microg BMP-2 from gelatin. PMID:18204324

  11. Bone morphogenetic proteins, eye patterning, and retinocollicular map formation in the mouse

    PubMed Central

    Plas, Daniel T.; Dhande, Onkar; Lopez, Joshua E.; Murali, Deepa; Thaller, Christina; Henkemeyer, Mark; Furuta, Yasuhide; Overbeek, Paul; Crair, Michael C.

    2009-01-01

    Patterning events during early eye formation determine retinal cell fate and can dictate the behavior of retinal ganglion cell (RGC) axons as they navigate toward central brain targets. The temporally and spatially regulated expression of bone morphogenetic proteins (BMPs) and their receptors in the retina are thought to play a key role in this process, initiating gene expression cascades that distinguish different regions of the retina, particularly along the dorsoventral axis. Here, we examine the role of BMP and a potential downstream effector, EphB, in retinotopic map formation in the lateral geniculate nucleus (LGN) and superior colliculus (SC). RGC axon behaviors during retinotopic map formation in wild type mice are compared with those in several strains of mice with engineered defects of BMP and EphB signaling. Normal RGC axon sorting produces axon order in the optic tract that reflects the dorsoventral position of the parent RGCs in the eye. A dramatic consequence of disrupting BMP signaling is a missorting of RGC axons as they exit the optic chiasm. This sorting is not dependent on EphB. When BMP signaling in the developing eye is genetically modified, RGC order in the optic tract and targeting in the LGN and SC are correspondingly disrupted. These experiments show that BMP signaling regulates dorsoventral RGC cell fate, RGC axon behavior in the ascending optic tract and retinotopic map formation in the LGN and SC through mechanisms that are in part distinct from EphB signaling in the LGN and SC. PMID:18614674

  12. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2.

    PubMed

    Zhang, Zheng; Wang, Guoxian; Li, Chen; Liu, Danping

    2013-08-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2(+) gene) was cloned into an adenovirus shuttle vector to obtain pShuttle cytomegalovirus (CMV)-BMP2(+)-internal ribosome entry site (IRES)-hrGFP-1. The adenovirus plasmid pAd CMV-BMP2(+)-IRES-hrGFP-1 was constructed by homologous recombination and was transfected into HEK293A cells, followed by adenovirus packaging. pAd CMV-BMP2 was used as the control. The two types of adenovirus were transfected into marrow stromal cells (MSCs). The expression of BMP2 and GFP, as well as the alkaline phosphatase (ALP) activity of expressed BMP2 were detected. Following mutagenesis, the BMP2 gene sequence and recombinant adenovirus vector were as predicted. The novel adenovirus vector expressed both BMP2 and GFP, indicating that a novel recombinant human adenovirus vector expressing BMP2 had been successfully constructed. PMID:24137184

  13. Resveratrol improves bone repair by modulation of bone morphogenetic proteins and osteopontin gene expression in rats.

    PubMed

    Casarin, R C; Casati, M Z; Pimentel, S P; Cirano, F R; Algayer, M; Pires, P R; Ghiraldini, B; Duarte, P M; Ribeiro, F V

    2014-07-01

    This study investigated the effect of resveratrol on bone healing and its influence on the gene expression of osteogenic markers. Two calvarial defects were created and one screw-shaped titanium implant was inserted in the tibia of rats that were assigned to daily administration of placebo (control group, n=15) or 10mg/kg of resveratrol (RESV group, n=15) for 30 days. The animals were then sacrificed. One of the calvarial defects was processed for histomorphometric analysis and the tissue relative to the other was collected for mRNA quantification of bone morphogenetic protein (BMP)-2, BMP-7, osteopontin (OPN), bone sialoprotein (BSP), osteoprotegrin (OPG), and receptor activator of NF-κB ligand (RANKL). Implants were removed by applying a counter-torque force. Histomorphometric analysis revealed higher remaining defect in the calvarial defects of the control group than the RESV group (P=0.026). Resveratrol increased the counter-torque values of implant removal when compared to control therapy (P=0.031). Gene expression analysis showed a higher expression of BMP-2 (P=0.011), BMP-7 (P=0.049), and OPN (P=0.002) genes in the RESV group than in the control group. In conclusion, resveratrol improved the repair of critical-sized bone defects and the biomechanical retention of implants. Indeed, this natural agent may up-regulate the gene expression of important osteogenic markers. PMID:24530035

  14. Osteoinduction by combining bone morphogenetic protein (BMP)-2 with a bioactive novel nanocomposite

    PubMed Central

    Sharma, A.; Meyer, F.; Hyvonen, M.; Best, S. M.; Cameron, R. E.; Rushton, N.

    2012-01-01

    Objectives There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite. Methods BMP-2 was synthesised using an Escherichia coli expression system and purified. In vitro bioactivity was confirmed using C2C12 cells and an alkaline phosphatase assay. The modified solution-evaporation method was used to fabricate α-TCP/PLGA nanocomposite and this was characterised using X-ray diffraction and scanning electron microscopy. Functionalisation of α-TCP/PLGA nanocomposite by adsorption of BMP-2 was performed and release of BMP-2 was characterised using an enzyme-linked immunosorbent assay (ELISA). Results Alkaline phosphatase activity of C2C12 cells was increased by the presence of all BMP-2/nanocomposite discs compared with the presence of a blank disc (p = 0.0022), and increased with increasing incubation concentrations of BMP-2, showing successful adsorption and bioactivity of BMP-2. A burst release profile was observed for BMP-2 from the nanocomposite. Conclusions Functionalisation of α-TCP/PLGA with BMP-2 produced osteoinduction and was dose-dependent. This material therefore has potential application as an osteoinductive agent in regenerative medicine. PMID:23610684

  15. Stimulation of bone healing by sustained bone morphogenetic protein 2 (BMP-2) delivery.