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Sample records for agrin binds bmp2

  1. Binding Interactions of Keratin-Based Hair Fiber Extract to Gold, Keratin, and BMP-2.

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    Roche C de Guzman

    Full Text Available Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2 has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD of 1.8 × 10(-4 M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10(-7 M, within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10(-5 M. BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks, suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5, below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the

  2. Enhancement of the Regenerative Potential of Anorganic Bovine Bone Graft Utilizing a Polyglutamate-Modified BMP2 Peptide with Improved Binding to Calcium-Containing Materials.

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    Bain, Jennifer L; Bonvallet, Paul P; Abou-Arraj, Ramzi V; Schupbach, Peter; Reddy, Michael S; Bellis, Susan L

    2015-09-01

    Autogenous bone is the gold standard material for bone grafting in craniofacial and orthopedic regenerative medicine. However, due to complications associated with harvesting donor bone, clinicians often use commercial graft materials that may lose their osteoinductivity due to processing. This study was aimed to functionalize one of these materials, anorganic bovine bone (ABB), with osteoinductive peptides to enhance regenerative capacity. Two peptides known to induce osteoblastic differentiation of mesenchymal stem cells were evaluated: (1) DGEA, an amino acid motif within collagen I and (2) a biomimetic peptide derived from bone morphogenic protein 2 (BMP2pep). To achieve directed coupling of the peptides to the graft surface, the peptides were engineered with a heptaglutamate domain (E7), which confers specific binding to calcium moieties within bone mineral. Peptides with the E7 domain exhibited greater anchoring to ABB than unmodified peptides, and E7 peptides were retained on ABB for at least 8 weeks in vivo. To assess the osteoinductive potential of the peptide-conjugated ABB, ectopic bone formation was evaluated utilizing a rat subcutaneous pouch model. ABB conjugated with full-length recombinant BMP2 (rBMP2) was also implanted as a model for current clinical treatments utilizing rBMP2 passively adsorbed to carriers. These studies showed that E7BMP2pep/ABB samples induced more new bone formation than all other peptides, and an equivalent amount of new bone as compared with rBMP2/ABB. A mandibular defect model was also used to examine intrabony healing of peptide-conjugated ABB. Bone healing was monitored at varying time points by positron emission tomography imaging with (18)F-NaF, and it was found that the E7BMP2pep/ABB group had greater bone metabolic activity than all other groups, including rBMP2/ABB. Importantly, animals implanted with rBMP2/ABB exhibited complications, including inflammation and formation of cataract-like lesions in the eye, whereas

  3. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

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    Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

    2013-12-01

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

  4. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

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    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells. PMID:27306974

  5. Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice

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    Lin, Shuo; Maj, Marcin; Bezakova, Gabriela; Magyar, Josef P.; Brenner, Hans Rudolf; Ruegg, Markus A.

    2008-01-01

    Agrin and its receptor MuSK are required for the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). In the current model the local deposition of agrin by the nerve and the resulting local activation of MuSK are responsible for creating and maintaining the postsynaptic apparatus including clusters of acetylcholine receptors (AChRs). Concomitantly, the release of acetylcholine (ACh) and the resulting depolarization disperses those postsynaptic structures that are not apposed by the nerve and thus not stabilized by agrin-MuSK signaling. Here we show that a miniaturized form of agrin, consisting of the laminin-binding and MuSK-activating domains, is sufficient to fully restore NMJs in agrin mutant mice when expressed by developing muscle. Although miniagrin is expressed uniformly throughout muscle fibers and induces ectopic AChR clusters, the size and the number of those AChR clusters contacted by the motor nerve increase during development. We provide experimental evidence that this is due to ACh, because the AChR agonist carbachol stabilizes AChR clusters in organotypic cultures of embryonic diaphragms. In summary, our results show that agrin function in NMJ development requires only two small domains, and that this function does not depend on the local deposition of agrin at synapses. Finally, they suggest a novel local function of ACh in stabilizing postsynaptic structures. PMID:18685098

  6. Biological activity of a genetically modified BMP-2 variant with inhibitory activity

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    Kübler Alexander C

    2009-02-01

    Full Text Available Abstract Background Alterations of the binding epitopes of bone morphogenetic protein-2 (BMP-2 lead to a modified interaction with the ectodomains of BMP receptors. In the present study the biological effect of a BMP-2 double mutant with antagonistic activity was evaluated in vivo. Methods Equine-derived collagenous carriers were loaded with recombinant human BMP-2 (rhBMP-2 in a well-known dose to provide an osteoinductive stimulus. The study was performed in a split animal design: carriers only coupled with rhBMP-2 (control were implanted into prepared cavities of lower limb muscle of rats, specimens coupled with rhBMP-2 as well as BMP-2 double mutant were placed into the opposite limb in the same way. After 28 days the carriers were explanted, measured radiographically and characterized histologically. Results As expected, the BMP-2 loaded implants showed a typical heterotopic bone formation. The specimens coupled with both proteins showed a significant decreased bone formation in a dose dependent manner. Conclusion The antagonistic effect of a specific BMP-2 double mutant could be demonstrated in vivo. The dose dependent influence on heterotopic bone formation by preventing rhBMP-2 induced osteoinduction suggests a competitive receptor antagonism.

  7. Repressive BMP2 gene regulatory elements near the BMP2 promoter

    International Nuclear Information System (INIS)

    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  8. Repressive BMP2 gene regulatory elements near the BMP2 promoter

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    Jiang, Shan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Chandler, Ronald L. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Fritz, David T. [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Mortlock, Douglas P. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Rogers, Melissa B., E-mail: rogersmb@umdnj.edu [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States)

    2010-02-05

    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  9. Fibrin Hydrogel Based Bone Substitute Tethered with BMP-2 and BMP-2/7 Heterodimers

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    Lindsay S. Karfeld-Sulzer

    2015-03-01

    Full Text Available Current clinically used delivery methods for bone morphogenetic proteins (BMPs are collagen based and require large concentrations that can lead to dangerous side effects. Fibrin hydrogels can serve as osteoinductive bone substitute materials in non-load bearing bone defects in combination with BMPs. Two strategies to even further optimize such a fibrin based system include employing more potent BMP heterodimers and engineering growth factors that can be covalently tethered to and slowly released from a fibrin matrix. Here we present an engineered BMP-2/BMP-7 heterodimer where an N-terminal transglutaminase substrate domain in the BMP-2 portion provides covalent attachment to fibrin together with a central plasmin substrate domain, a cleavage site for local release of the attached BMP-2/BMP-7 heterodimer under the influence of cell-activated plasmin. In vitro and in vivo results revealed that the engineered BMP-2/BMP-7 heterodimer induces significantly more alkaline phosphatase activity in pluripotent cells and bone formation in a rat calvarial model than the engineered BMP-2 homodimer. Therefore, the engineered BMP-2/BMP-7 heterodimer could be used to reduce the amount of BMP needed for clinical effect.

  10. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

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    Evaristus Chibunna Mbanefo

    Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  11. Bmp2 Is Required for Odontoblast Differentiation and Pulp Vasculogenesis

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    Yang, W; Harris, M.A.; Y. Cui; Mishina, Y; Harris, S.E.; Gluhak-Heinrich, J.

    2012-01-01

    Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKOod) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKOod do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation, as reflected by decreased Osterix, Col1a1, and Dspp expression. There is less dentin, and the dentin is hypomineralized and patchy. We also ...

  12. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

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    DeCarlo Arthur A

    2012-09-01

    Full Text Available Abstract Background Many growth factors, such as bone morphogenetic protein (BMP-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS glycosaminoglycans (GAGs, which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS, regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1 expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™. Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid

  13. BMP-2 and titanium particles synergistically activate osteoclast formation

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    Sun, S.X. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedics, Yinchuan, Ningxia Hui Autonomous Region, China, Department of Orthopedics, Affiliated Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Guo, H.H. [Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, China, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Zhang, J. [Institute of Pathology, Xi' an Jiaotong University, Xi' an Shaanxi, China, Institute of Pathology, Xi' an Jiaotong University, Xi' an Shaanxi (China); Yu, B. [Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, China, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Sun, K.N.; Jin, Q.H. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedics, Yinchuan, Ningxia Hui Autonomous Region, China, Department of Orthopedics, Affiliated Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China)

    2014-05-09

    A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.

  14. Plasma Treated High-Density Polyethylene (HDPE Medpor Implant Immobilized with rhBMP-2 for Improving the Bone Regeneration

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    Jin-Su Lim

    2014-01-01

    Full Text Available We investigate the bone generation capacity of recombinant human bone morphogenetic protein-2 (rhBMP-2 immobilized Medpor surface through acrylic acid plasma-polymerization. Plasma-polymerization was carried out at a 20 W at an acrylic acid flow rate of 7 sccm for 5 min. The plasma-polymerized Medpor surface showed hydrophilic properties and possessed a high density of carboxyl groups. The rhBMP-2 was immobilized with covalently attached carboxyl groups using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide. Carboxyl groups and rhBMP-2 immobilization on the Medpor surface were identified by Fourier transform infrared spectroscopy. The activity of Medpor with rhBMP-2 immobilized was examined using an alkaline phosphatase assay on MC3T3-E1 cultured Medpor. These results showed that the rhBMP-2 immobilized Medpor increased the level of MC3T3-E1 cell differentiation. These results demonstrated that plasma surface modification has the potential to immobilize rhBMP-2 on polymer implant such as Medpor and can be used for the binding of bioactive nanomolecules in bone tissue engineering.

  15. BMP-2 Is Involved in Scleral Remodeling in Myopia Development.

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    Honghui Li

    Full Text Available The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2 expression in the sclera of guinea pigs with lens-induced myopia (LIM and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM synthesis in human scleral fibroblasts (HSFs cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1. Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia.

  16. BMP-2 Is Involved in Scleral Remodeling in Myopia Development

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    Li, Honghui; Cui, Dongmei; Zhao, Feng; Huo, Lijun; Hu, Jianmin; Zeng, Junwen

    2015-01-01

    The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups) by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1). Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG) and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia. PMID:25965995

  17. BMP-2 Is Involved in Scleral Remodeling in Myopia Development

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    Honghui Li; Dongmei Cui; Feng Zhao; Lijun Huo; Jianmin Hu; Junwen Zeng

    2015-01-01

    The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced ...

  18. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

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    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-01-01

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  19. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

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    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Kyunghwa [Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangneung 210-702, Gangwondo (Korea, Republic of); Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Jeong-Hwa, E-mail: baekjh@snu.ac.kr [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of)

    2014-05-01

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.

  20. Bmp2 and Bmp4 accelerate alveolar bone development.

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    Ou, Mingming; Zhao, Yibing; Zhang, Fangming; Huang, Xiaofeng

    2015-06-01

    Alveolar bone remodeling is a continuous process that takes place during development and in response to various physiological and pathological stimuli. However, detailed knowledge regarding the underlying mechanisms involved in alveolar bone development is still lacking. This study aims at improving our understanding of alveolar bone formation and the role of bone morphogenetic proteins (Bmps) in this process. Mice at embryonic (E) day 13.5 to postnatal (PN) day 15.5 were selected to observe the process of alveolar bone development. Alveolar bone development was found to be morphologically observable at E14.5. Molar teeth isolated from mice at PN7.5 were pretreated with Bmp2, Bmp4, Noggin, or BSA, and grafted subcutaneously into mice. The subcutaneously implanted tooth germs formed alveolar bone indicating the role of the dental follicle in alveolar bone development. Alveolar bone formation was increased after pretreatment with Bmp2 and Bmp4, but not with Noggin. Gene expression levels in dental follicle cells from murine molars were also determined by real-time RT-PCR. The expression levels of Runx2, Bsp, and Ocn were significantly higher in dental follicle cells cultured with Bmp2 or Bmp4, and significantly lower in those cultured with Noggin when compared with that of the BSA controls. Our results suggest that the dental follicle participates in alveolar bone formation and Bmp2/4 appears to accelerate alveolar bone development.

  1. Matrix-immobilized BMP-2 on microcontact printed fibronectin as in vitro tool to study BMP-mediated signaling and cell migration

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    Kristin eHauff

    2015-05-01

    Full Text Available During development, bone morphogenetic proteins (BMPs exert important functions in several tissues by regulating signaling for cell differentiation and migration. In vivo the extracellular matrix (ECM not only provides a support for adherent cells, but also presents a reservoir of growth factors (GFs. Several constituents of the ECM provide adhesive cues, which serve as binding sites for cell transmembrane receptors, such as integrins, which convey adhesion-mediated signaling to the intracellular compartment. Integrins do not function alone but rather crosstalk and cooperate with other receptors, such as GF receptors, in regulating cell responses to extracellular signals. To this, we present here the immobilization of BMP-2 onto cellular fibronectin (cFN, a key protein of the ECM, to investigate their impact on GF-mediated signaling and migration.Following biotinylation, BMP-2 was linked to biotinylated cFN using NeutrAvidin (NA as cross-linker. Characterization with QCM-D and ELISA confirmed the efficient immobilization of BMP-2 on cFN over a period of 24 h.To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2 we investigated short- and long-term responses of C2C12 myoblasts in comparison to soluble BMP-2 (sBMP-2 or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5 phosphorylation and translocation into the nucleus corresponding to the activation of BMP-mediated Smad-dependent pathway. Additionally, successful suppression of myotube formation was observed after six days.We next implemented this approach to fabricate cFN micro patterned stripes by soft lithography. These stripes only allowed cell-surface interaction on the pattern due to passivation of the surface in between, thus serving as platform for studies on directed cell migration. During a 10 h-period, cells showed an increased migratory activity upon BMP-2 exposure.Thus, this versatile tool retains the GF's bioactivity and allows the presentation of ECM

  2. BMP2 and VEGF promote angiogenesis but retard terminal differentiation of osteoblasts in bone regeneration by up-regulating Id1

    Institute of Scientific and Technical Information of China (English)

    Xiaobin Song; Shaohua Liu; Xun Qu; Yingwei Hu; Xiaoying Zhang; TaoWang; FengcaiWei

    2011-01-01

    Inadequate vascularization limits the repair of bone defects,In order to improve angiogenesis and accelerate osteogenesis,the synergism of co-cultured cells with genetic modification in bone regeneration was investigated in this study.Endothelial progenitor cells (EPCs) and bone marrow stem cells (BMSCs) were transfected with the genes of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) by adenovirus,respectively.The co-cultured cells,designated as four groups including BMSC + EPC,Ad-BMP2-BMSC +EPC,BMSC + Ad-VEGF-EPC,and Ad-BMP2-BMSC + Ad-VEGF-EPC groups,were seeded on an alginate gel and then implanted into rat intramuscularly to evaluate the effects on angiogenesis and osteogenesis.Both VEGF and BMP2 could induce the overexpression of inhibitor of DNA-binding 1(Id1) gene which significantly promoted tube formation in vitro and increase the amount of blood vessels in the Ad-BMP2-BMSC + Ad-VEGF-EPC group after implantation.Nevertheless,overexpression of Id1 retarded the terminal differentiation of osteoblasts and the bone formation.Later,osteogenic gene expression at transcriptional level,calcium nodules,and alkaline phosphatase (ALP) activity showed a gradual decrease and the amount of newly formed osteogenesis area exhibited a small increase in the Ad-BMP2-BMSC + Ad-VEGF-EPC group.This finding suggests that a balanced regulation of Id1 expression in VEGF-EPCs and BMP2-BMSCs may be critical to cell-based and gene-based approaches for bone regeneration.

  3. Complexation and sequestration of BMP-2 from an ECM mimetic hyaluronan gel for improved bone formation.

    Directory of Open Access Journals (Sweden)

    Marta Kisiel

    Full Text Available Bone morphogenetic protein-2 (BMP-2 is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs dermatan sulfate (DS or heparin (HP, prior to loading it into a hyaluronic acid (HA hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.

  4. Transmembrane Agrin Regulates Dendritic Filopodia and Synapse Formation in Mature Hippocampal Neuron Cultures

    OpenAIRE

    McCroskery, Seumas; Bailey, Allison; Lin, Lin; Daniels, Mathew P.

    2009-01-01

    The transmembrane isoform of agrin (Tm-agrin) is the predominant form expressed in the brain but its putative roles in brain development are not well understood. Recent reports have implicated Tm-agrin in the formation and stabilization of filopodia on neurites of immature central and peripheral neurons in culture. In maturing central neurons, dendritic filopodia are believed to facilitate synapse formation. In the present study we have investigated the role of Tm-agrin in regulation of dendr...

  5. Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

    International Nuclear Information System (INIS)

    In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a β-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified by BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals

  6. Signaling Crosstalk between PPARγ and BMP2 in Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Ichiro Takada

    2012-01-01

    Full Text Available Recent studies have revealed that PPARγ’s transactivation function is regulated by extracellular signals. In particular, cytokines and Wnt family proteins suppress the ligand-inducible transactivation function of PPARγ and attenuate adipogenesis/osteoblastogenesis switching in mesenchymal stem cells (MSCs. For example, Wnt5a suppresses PPARγ transcriptional activity through the NLK/SETDB1/CHD7 pathway. Among these factors, BMP2 strongly induces bone formation, but the effect of BMP2 on PPARγ function remains unclear. We examined the effect of BMP2 and PPARγ in ST2 cells and found that PPARγ activation affected BMP2’s signaling pathway through epigenetic regulation. Although BMP2 did not interfere with PPARγ-mediated adipogenesis, BMP2 increased mRNA expression levels of PPARγ target genes (such as Fabp4 and Nr1h3 when cells were first treated with troglitazone (TRO. Moreover, PPARγ activation affected BMP2 through enhancement of histone activation markers (acetylated histone H3 and trimethylated Lys4 of histone H3 on the Runx2 promoter. After TRO treatment for three hours, BMP2 enhanced the levels of active histone marks on the promoter of a PPARγ target gene. These results suggest that the order of treatment with BMP2 and a PPARγ ligand is critical for adipogenesis and osteoblastogenesis switching in MSCs.

  7. BMP-2 induces versican and hyaluronan that contribute to post-EMT AV cushion cell migration.

    Directory of Open Access Journals (Sweden)

    Kei Inai

    Full Text Available Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM components, versican and hyaluronan (HA, and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.

  8. Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

    Science.gov (United States)

    Guo, Feng; Feng, Junsheng; Wang, Feng; Li, Wentong; Gao, Qingping; Chen, Zhuo; Shoff, Lisa; Donly, Kevin J; Gluhak-Heinrich, Jelica; Chun, Yong Hee Patricia; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

    2015-08-01

    Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

  9. Cell saver filtering of extravasated rhBMP-2 after degenerative scoliosis reconstruction

    Directory of Open Access Journals (Sweden)

    Gabriel Liu, MBBCh, MSc, FRCS, FAMS (Orth

    2015-06-01

    Full Text Available RhBMP-2 is a bone fusion enhancer commonly used in scoliosis reconstruction surgery. It is delivered via an absorbable collagen sponge but has been known to migrate away from its delivery site. RhBMP-2 extravasation in surgical drainage has been noted during first two days post-surgery. Cell savers are widely used in scoliosis reconstruction to limit transfusion requirements and are commonly deployed in cases where rhBMP-2 is used for fusion augmentation. It is not known whether rhBMP-2 is present in salvaged blood or filtered away during cell saver recycling. Through this case series of four patients who underwent scoliosis reconstruction, we assess cell saver efficacy in filtering rhBMP-2 molecules by quantifying the amount of rhBMP-2 present in salvaged blood obtained after postoperative drainage recycling by OrthoPAT® cell saver and comparing it to rhBMP-2 leakage in postoperative drainage without cell saver recycling. We report an almost 10-fold reduction of rhBMP-2 concentration in salvaged blood obtained after cell saver recycling of postoperative drainage, suggesting cell saver effectiveness in filtering rhBMP-2 molecules.

  10. BMP2 Regulation of CXCL12 Cellular, Temporal, and Spatial Expression is Essential During Fracture Repair

    Science.gov (United States)

    Myers, Timothy J; Longobardi, Lara; Willcockson, Helen; Temple, Joseph D; Tagliafierro, Lidia; Ye, Ping; Li, Tieshi; Esposito, Alessandra; Moats-Staats, Billie M; Spagnoli, Anna

    2016-01-01

    The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)-dependent fracture healing occurs through a tight control of chemokine C-X-C motif-ligand-12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site-specific response of CXCL12+-BMP2+ endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2cKO/+) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2cKO/+ mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2cKO/+ mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2cKO/cKO endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2-expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12+-BMP2+ perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12+-BMP2+ to osteogenesis while departing their supportive role to angiogenesis. Our findings have far

  11. Expression of active hBMP2 in transgenic tobacco plants.

    Science.gov (United States)

    Suo, Guangli; Chen, Bing; Zhang, Jingyu; Gao, Yuan; Wang, Xia; He, Zhengquan; Dai, Jianwu

    2006-12-01

    Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and "Kozak" sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2. PMID:16819603

  12. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    International Nuclear Information System (INIS)

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  13. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Li-An [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an (China); Yuan, Guohua; Yang, Guobin [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Key Laboratory of Oral Biomedical Engineering Ministry of Education, Wuhan (China); Ortiz-Gonzalez, Iris [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Yang, Wuchen; Cui, Yong [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); MacDougall, Mary [Department of Oral/Maxillofacial Surgery, University of Alabama, Birmingham, AL (United States); Donly, Kevin J. [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Harris, Stephen [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); Chen, Shuo, E-mail: chens0@uthscsa.edu [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States)

    2009-08-14

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  14. Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Jung-Bo Huh

    2015-07-01

    Full Text Available Anorganic bovine bone matrix (Bio-Oss® has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2 has been suggested to overcome this limitation of Bio-Oss®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter were formed in a white rabbit model and then implanted or not (controls with Bio-Oss® or BMP-2/Bio-Oss®. The Bio-Oss® and BMP-2/Bio-Oss® groups had significantly greater new bone areas (expressed as percentages of augmented areas than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss® group (16.50 ± 2.87 (n = 6 had significantly greater new bone areas than the Bio-Oss® group (9.43 ± 3.73 (n = 6 at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss® markedly enhances bone regeneration.

  15. BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

    Science.gov (United States)

    Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

    2015-01-01

    The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration. PMID:25609957

  16. Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration.

    Science.gov (United States)

    Huh, Jung-Bo; Yang, June-Jip; Choi, Kyung-Hee; Bae, Ji Hyeon; Lee, Jeong-Yeol; Kim, Sung-Eun; Shin, Sang-Wan

    2015-01-01

    Anorganic bovine bone matrix (Bio-Oss®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were formed in a white rabbit model and then implanted or not (controls) with Bio-Oss® or BMP-2/Bio-Oss®. The Bio-Oss® and BMP-2/Bio-Oss® groups had significantly greater new bone areas (expressed as percentages of augmented areas) than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss® group (16.50 ± 2.87 (n = 6)) had significantly greater new bone areas than the Bio-Oss® group (9.43 ± 3.73 (n = 6)) at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss® markedly enhances bone regeneration. PMID:26184187

  17. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  18. BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

    Directory of Open Access Journals (Sweden)

    Ming Zhang

    2014-01-01

    Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

  19. Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix.

    Science.gov (United States)

    Ben-David, Dror; Srouji, Samer; Shapira-Schweitzer, Keren; Kossover, Olga; Ivanir, Eran; Kuhn, Gisela; Müller, Ralph; Seliktar, Dror; Livne, Erella

    2013-04-01

    Bone repair strategies utilizing resorbable biomaterial implants aim to stimulate endogenous cells in order to gradually replace the implant with functional repair tissue. These biomaterials should therefore be biodegradable, osteoconductive, osteoinductive, and maintain their integrity until the newly formed host tissue can contribute proper function. In recent years there has been impressive clinical outcomes for this strategy when using osteoconductive hydrogel biomaterials in combination with osteoinductive growth factors such as human recombinant bone morphogenic protein (hrBMP-2). However, the success of hrBMP-2 treatments is not without risks if the factor is delivered too rapidly and at very high doses because of a suboptimal biomaterial. Therefore, the aim of this study was to evaluate the use of a PEGylated fibrinogen (PF) provisional matrix as a delivery system for low-dose hrBMP-2 treatment in a critical size maxillofacial bone defect model. PF is a semi-synthetic hydrogel material that can regulate the release of physiological doses of hrBMP-2 based on its controllable physical properties and biodegradation. hrBMP-2 release from the PF material and hrBMP-2 bioactivity were validated using in vitro assays and a subcutaneous implantation model in rats. Critical size calvarial defects in mice were treated orthotopically with PF containing 8 μg/ml hrBMP-2 to demonstrate the capacity of these bioactive implants to induce enhanced bone formation in as little as 6 weeks. Control defects treated with PF alone or left empty resulted in far less bone formation when compared to the PF/hrBMP-2 treated defects. These results demonstrate the feasibility of using a semi-synthetic biomaterial containing small doses of osteoinductive hrBMP-2 as an effective treatment for maxillofacial bone defects. PMID:23375953

  20. Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

    OpenAIRE

    Jung-Bo Huh; June-Jip Yang; Kyung-Hee Choi; Ji Hyeon Bae; Jeong-Yeol Lee; Sung-Eun Kim; Sang-Wan Shin

    2015-01-01

    Anorganic bovine bone matrix (Bio-Oss®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were fo...

  1. Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

    OpenAIRE

    LI, ZHEN; Hilgenberg, Lutz G. W.; O'Dowd, Diane K.; Smith, Martin A

    1999-01-01

    Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule m...

  2. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Henriksen, Z; Sørensen, O H;

    2004-01-01

    D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP...... activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in...... osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro....

  3. Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats.

    Directory of Open Access Journals (Sweden)

    Michelle T Poldervaart

    Full Text Available The design of bioactive three-dimensional (3D scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2 influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

  4. Effect of a Novel Nonviral Gene Delivery of BMP-2 on Bone Healing

    Directory of Open Access Journals (Sweden)

    P. Schwabe

    2012-01-01

    Full Text Available Background. Gene therapeutic drug delivery approaches have been introduced to improve the efficiency of growth factors at the site of interest. This study investigated the efficacy and safety of a new nonviral copolymer-protected gene vector (COPROG for the stimulation of bone healing. Methods. In vitro, rat osteoblasts were transfected with COPROG + luciferase plasmid or COPROG + hBMP-2 plasmid. In vivo, rat tibial fractures were intramedullary stabilized with uncoated versus COPROG+hBMP-2-plasmid-coated titanium K-wires. The tibiae were prepared for biomechanical and histological analyses at days 28 and 42 and for transfection/safety study at days 2, 4, 7, 28, and 42. Results. In vitro results showed luciferase expression until day 21, and hBMP-2-protein was measured from day 2 – day 10. In vivo, the local application of hBMP-2-plasmid showed a significantly higher maximum load after 42 days compared to that in the control. The histomorphometric analysis revealed a significantly less mineralized periosteal callus area in the BMP-2 group compared to the control at day 28. The rt-PCR showed no systemic biodistribution of luciferase RNA. Conclusion. A positive effect on fracture healing by nonviral BMP-2 plasmid application from COPROG-coated implants could be shown in this study; however, the effect of the vector may be improved with higher plasmid concentrations. Transfection showed no biodistribution to distant organs and was considered to be safe.

  5. Cell-mediated BMP-2 liberation promotes bone formation in a mechanically unstable implant environment.

    Science.gov (United States)

    Hägi, Tobias T; Wu, Gang; Liu, Yuelian; Hunziker, Ernst B

    2010-05-01

    The flexible alloplastic materials that are used in bone-reconstruction surgery lack the mechanical stability that is necessary for sustained bone formation, even if this process is promoted by the application of an osteogenic agent, such as BMP-2. We hypothesize that if BMP-2 is delivered gradually, in a cell-mediated manner, to the surgical site, then the scaffolding material's lack of mechanical stability becomes a matter of indifference. Flexible discs of Ethisorb were functionalized with BMP-2, which was either adsorbed directly onto the material (rapid release kinetics) or incorporated into a calcium-phosphate coating (slow release kinetics). Unstabilized and titanium-plate-stabilized samples were implanted subcutaneously in rats and retrieved up to 14 days later for a histomorphometric analysis of bone and cartilage volumes. On day 14, the bone volume associated with titanium-plate-stabilized discs bearing an adsorbed depot of BMP-2 was 10-fold higher than that associated with their mechanically unstabilized counterparts. The bone volume associated with discs bearing a coating-incorporated depot of BMP-2 was similar in the mechanically unstabilized and titanium-plate-stabilized groups, and comparable to that associated with the titanium-plate-stabilized discs bearing an adsorbed depot of BMP-2. Hence, if an osteogenic agent is delivered in a cell-mediated manner (via coating degradation), ossification can be promoted even within a mechanically unstable environment.

  6. BMP-2 and BMP-2/7 Heterodimers Conjugated to a Fibrin/Hyaluronic Acid Hydrogel in a Large Animal Model of Mild Intervertebral Disc Degeneration.

    Science.gov (United States)

    Peeters, Mirte; Detiger, Suzanne E L; Karfeld-Sulzer, Lindsay S; Smit, Theo H; Yayon, Avner; Weber, Franz E; Helder, Marco N

    2015-01-01

    Intervertebral disc (IVD) degeneration is etiologically associated with low back pain and is currently only treated in severe cases with spinal fusion. Regenerative medicine attempts to restore degenerated tissue by means of cells, hydrogels, and/or growth factors and can therefore be used to slow, halt, or reverse the degeneration of the IVD in a minimally invasive manner. Previously, the growth factors bone morphogenetic proteins 2 and 7 (BMP-2, -7) were shown to enhance disc regeneration, in vitro and in vivo. Since BMPs have only a short in vivo half-life, and to prevent heterotopic ossification, we evaluated the use of a slow release system for BMP-2 homodimers and BMP-2/7 heterodimers for IVD regeneration. BMP growth factors were conjugated to a fibrin/hyaluronic acid (FB/HA) hydrogel and intradiscally injected in a goat model of mild IVD degeneration to study safety and efficacy. Mild degeneration was induced in five lumbar discs of seven adult Dutch milk goats, by injections with the enzyme chondroitinase ABC. After 12 weeks, discs were treated with either FB/HA-hydrogel only or supplemented with 1 or 5 μg/mL of BMP-2 or BMP-2/7. BMPs were linked to the FB/HA hydrogels using a transglutaminase moiety, to be released through an incorporated plasmin cleavage site. After another 12 weeks, goats were sacrificed and discs were assessed using radiography, MRI T2* mapping, and biochemical and histological analyses. All animals maintained weight throughout the study and no heterotopic bone formation or other adverse effects were noted during follow-up. Radiographs showed significant disc height loss upon induction of mild degeneration. MRI T2* mapping showed strong and significant correlations with biochemistry and histology as shown before. Surprisingly, no differences could be demonstrated in any parameter between intervention groups. To our knowledge, this is the first large animal study evaluating BMPs conjugated to an FB/HA-hydrogel for the treatment of

  7. Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

    Directory of Open Access Journals (Sweden)

    E Ferreira

    2012-07-01

    Full Text Available Transplantation of mesenchymal stem cells (MSCs with electrotransferred bone morphogenetic protein-2 (BMP-2 transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

  8. Regulating the osteogenic function of rhBMP 2 by different titanium surface properties.

    Science.gov (United States)

    Xiao, Ming; Biao, Meina; Chen, Yangmei; Xie, Meiju; Yang, Bangcheng

    2016-08-01

    Bone morphogenetic protein 2 (BMP-2) is important for regulating the osteogenic differentiation of mesenchymal stem cells and the response of bone tissue. It adsorbs on the surface of biomedical implants immediately and plays a role of mediator between the materials surfaces and the host cells. Studies usually connect the material surface properties and the new bone formation directly. However, interaction between the adsorbed BMP-2 on the implant surface and the cells in the tissue is the key to explaining the osteogenic properties of the material. So, in this article, we investigated the conformational and functional changes induced by the surface modified titanium metals. We found that the α-helix and β-sheet structure of rhBMP-2 can be well maintained on the anodic oxidation treated titanium surface. The osteogenic function of rhBMP-2 can sustain for a relatively long time even though there is less amount adhere to the surface compared with that on the acid alkali treated titanium. Surface properties, especially the morphology enable a larger amount of rhBMP-2 to adsorb to the surface of the acid alkali treated titanium, but the conformation of the protein is severely influenced. The percentage of α-helix structure is also significantly decreased so that the efficacy of rhBMP-2 is only maintained in the early time. This study indicated that different surface modification of the surface could regulate the structure of rhBMP-2 and then further influence its osteogenic function. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1882-1893, 2016. PMID:26991341

  9. No advantage to rhBMP-2 in addition to autogenous graft for fracture nonunion.

    Science.gov (United States)

    Takemoto, Richelle; Forman, Jordanna; Taormina, David P; Egol, Kenneth A

    2014-06-01

    Bone morphogenetic proteins are a necessary component of the fracture healing cascade. Few studies have delineated the efficacy of iliac crest bone graft and recombinant human bone morphogenetic protein 2 (rhBMP-2), especially, in comparison with the gold standard treatment of nonunion, which is autogenous bone graft alone. This study compared the outcome of patients with fracture nonunion treated with autogenous bone graft plus rhBMP-2 adjuvant vs patients treated with autogenous bone graft alone. A total of 118 consecutive patients who were to undergo long bone nonunion surgery with autogenous bone graft (50) or autogenous bone graft plus rhBMP-2 (68) were identified. Surgical intervention included either harvested iliac autogenous bone graft or autogenous bone graft plus 1.5 mg/mL of rhBMP-2 placed in and around the site of nonunion. No differences were found in the distribution of nonunion sites included within each group. Twelve-month follow-up was obtained on 100 of 118 patients (84.7%). Analyses of demographic characteristics (including tobacco), medical comorbidities, previous surgeries, and nonunion type (atrophic vs hypertrophic) did not differ. Postoperative complication rates did not differ. The percentage of patients who progressed to union did not differ. Mean time to union in the autogenous bone graft plus rhBMP-2 group was 6.6 months (±3.9) vs 5.4 (±2.7) months in the autogenous bone graft-only group (P=.06). Rates of revision (16.2% for rhBMP-2 plus autogenous bone graft vs 8% for autogenous bone graft) did not differ statistically (P=.19), nor did 12-month scores of pain and functional assessment. Although rhBMP-2 is a safe adjuvant, there was no benefit seen when rhBMP-2 was added to autogenous bone graft in the treatment of long bone nonunion. Given its high cost, rhBMP-2 should be reconsidered as an aid to autogenous bone graft in the treatment of nonunion. PMID:24972432

  10. Effects of codon modification on human BMP2 gene expression in tobacco plants.

    Science.gov (United States)

    Suo, Guangli; Chen, Bing; Zhang, Jingyu; Duan, Ziyuan; He, Zhengquan; Yao, Wei; Yue, Chaoyin; Dai, Jianwu

    2006-07-01

    Bone morphogenetic protein 2 (BMP2) has great potential in therapeutic applications. We are working on generating transgenic plants as a bioreactor to produce BMP2. We have studied the effects of codon optimization on the expression of human BMP2 (hBMP2) in tobacco plants. Three modified hBMP2 genes were transformed into tobacco under the control of either cauliflower mosaic virus 35S (CaMV35S) promoter or double-CaMV35S promoter plus alfalfa mosaic virus (AMV) enhancer. The fused beta-glucuronidase (GUS) reporter gene was used to facilitate the assay of protein expression. The results indicated that codon optimization could increase the protein expression level obviously under CaMV35S promoter. However, under relatively stronger initiation condition (double-CaMV35S promoter plus AMV enhancer), only the gene with the lowest degree of codon optimization could increase the protein expression level. Our findings suggest that the action of codon optimization may be influenced by the factors of promoter strength and A+T content in tobacco plants. PMID:16491379

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

    Science.gov (United States)

    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

  12. Local expression and role of BMP-2/4 in injured spinal cord.

    Science.gov (United States)

    Cui, Z S; Zhao, P; Jia, C X; Liu, H J; Qi, R; Cui, J W; Cui, J H; Peng, Q; Lin, B; Rao, Y J

    2015-08-07

    We investigated local changes in BMP-2/4 expression in rat spinal cords 1 week following injury to study the damage effects of BMP-2/4 in spinal cord injury (SCI). Sprague Dawley rats (45, 4 months old) were randomized into three groups comprising 15 rats each: a SHAM group, an SCI without noggin group (SCIO), and an SCI with noggin group (SCID). The SCIO and SCID groups were subjected to spinal cord hemisection, and motor activity was assessed using the BBB score. Expression of BMP-2/4 in each injured spinal cord section was examined by hematoxylin and eosin staining, immunohistochemistry, and western blot. There were no significant differences in BBB scores among the three groups (P > 0.05). Following hemisection, the BBB score in the SHAM group was significantly higher than in the other two groups on the 1st day after modeling (P 0.05). Seven days after modeling, the BBB score in the SHAM group was significantly higher than in the other two groups (P < 0.05), and the BBB score in the SCID group was obviously higher than in the SCIO group (P < 0.05). The expression of BMP-2/4 was highest in the SCIO group and lowest in the SHAM group (P < 0.05). SCI can cause severe impairment of motor activity in rats. Seven days after SCI, the local expression of BMP-2/4 had obviously increased; noggin can effectively inhibit the expression of BMP-2/4 and reduce impairment.

  13. The p38/MK2/Hsp25 pathway is required for BMP-2-induced cell migration.

    Directory of Open Access Journals (Sweden)

    Cristina Gamell

    Full Text Available BACKGROUND: Bone morphogenetic proteins (BMPs have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. METHODOLOGY/PRINCIPAL FINDINGS: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2's physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38α or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. CONCLUSIONS: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration.

  14. Structural basis of agrin-LRP4-MuSK signaling

    Energy Technology Data Exchange (ETDEWEB)

    Zong, Yinong; Zhang, Bin; Gu, Shenyan; Lee, Kwangkook; Zhou, Jie; Yao, Guorui; Figueiredo, Dwight; Perry, Kay; Mei, Lin; Jin, Rongsheng [Cornell; (GSHU); (Sanford-Burnham)

    2012-06-27

    Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. NMJ formation requires neural agrin, a motoneuron-derived protein, which interacts with LRP4 (low-density lipoprotein receptor-related protein 4) to activate the receptor tyrosine kinase MuSK (muscle-specific kinase). However, little is known of how signals are transduced from agrin to MuSK. Here, we present the first crystal structure of an agrin-LRP4 complex, consisting of two agrin-LRP4 heterodimers. Formation of the initial binary complex requires the z8 loop that is specifically present in neuronal, but not muscle, agrin and that promotes the synergistic formation of the tetramer through two additional interfaces. We show that the tetrameric complex is essential for neuronal agrin-induced acetylcholine receptor (AChR) clustering. Collectively, these results provide new insight into the agrin-LRP4-MuSK signaling cascade and NMJ formation and represent a novel mechanism for activation of receptor tyrosine kinases.

  15. Combination therapy with BMP-2 and a systemic RANKL inhibitor enhances bone healing in a mouse critical-sized femoral defect.

    Science.gov (United States)

    Bougioukli, Sofia; Jain, Ashish; Sugiyama, Osamu; Tinsley, Brian A; Tang, Amy H; Tan, Matthew H; Adams, Douglas J; Kostenuik, Paul J; Lieberman, Jay R

    2016-03-01

    Recombinant human BMP-2 (rhBMP-2) is a potent osteoinductive agent, but has been associated not only with bone formation, but also osteoclastogenesis and bone resorption. Osteoprotegerin (OPG) is a RANKL inhibitor that blocks differentiation and function of osteoclasts. We hypothesized that the combination of local BMP-2 (recombinant protein or a product of gene therapy) plus systemic OPG-Fc is more effective than BMP-2 alone in promoting bone repair. To test this hypothesis we used a mouse critical-sized femoral defect model. Col2.3eGFP (osteoblastic marker) male mice were treated with rhBMP-2 (group I), rhBMP-2 and systemic OPG (group II), rhBMP-2 and delayed administration of OPG (group III), mouse BM cells transduced with a lentiviral vector containing the BMP-2 gene (LV-BMP-2; group IV), LV-BMP-2 and systemic OPG (group V), a carrier alone (group VI) and administration of OPG alone (group VII). All bone defects treated with BMP-2 (alone or combined with OPG) healed, whereas minimal bone formation was noted in animals treated with the carrier alone or OPG alone. MicroCT analysis showed that bone volume (BV) in rhBMP-2+OPG and LV-BMP-2+OPG groups was significantly higher compared to rhBMP-2 alone (p<0.01) and LV-BMP-2 alone (p<0.001). Similar results were observed in histomorphometry, with rhBMP-2 alone defects exhibiting significantly lower bone area (B.Ar) compared to rhBMP-2+OPG defects (p<0.005) and LV-BMP-2 defects having a significantly lower B.Ar compared to all BMP-2+OPG treated groups (p≤0.01). TRAP staining demonstrated a major osteoclast response in the groups that did not receive OPG (rhBMP-2, LV-BMP-2 and sponge alone) beginning as early as 7days post-operatively. In conclusion, we demonstrated that locally delivered BMP-2 (recombinant protein or gene therapy) in combination with systemically administered OPG improved bone healing compared to BMP-2 alone in a mouse critical-sized bone defect. These data indicate that osteoclasts can diminish

  16. Experimental Research on Ectopic Osteogenesis of BMP2-derived Peptide P24 Combined with PLGA Copolymers

    Institute of Scientific and Technical Information of China (English)

    DUAN Zhixia; ZHENG Qixin; GUO Xiaodong; YUAN Quan; CHEN Shunguang

    2007-01-01

    To experimentally evaluate the ectopic osteogenetic capacity of synthesized BMP2-derived peptide P24 combined with poly lactic-co-glycolic acid (PLGA), Wistar rats were divided into two groups: group A, in which BMP2-derived peptide P24/PLGA complex was implanted,and group B which received simple PLGA implant. The complex was respectively implanted into the back muscles of rats. Samples were taken the 1 st, 4 th, 8 th, and the 12 th week after the implantation.Their bone formation was detected by X-ray examination, and tissue response was histologically observed. Western blotting was used for the detection of the expression of collagen Ⅰ (Col- Ⅰ ) and osteopontin (OPN). There was acute inflammation in the tissue around both types of implants at early stage. The cartilage was found around implant areas 4 weeks after the implantation of BMP2-derived peptide p24/PLGA complex, 8 weeks after the implantation, osteoblasts were found, and 12 weeks after the implantation, typical trabecular bone structure was observed. In group B, after 12 weeks, no osteoblasts were found. It is concluded that PLGA is an ideal scaffold material for bone tissue engineering. BMP2-derived peptide can start endochondral ossification and is more effective in inducing ectopic osteogenesis.

  17. Biodegradable Chitosan Nanoparticle Coatings on Titanium for the Delivery of BMP-2

    Directory of Open Access Journals (Sweden)

    Nils Poth

    2015-01-01

    Full Text Available A simple method for the functionalization of a common implant material (Ti6Al4V with biodegradable, drug loaded chitosan-tripolyphosphate (CS-TPP nanoparticles is developed in order to enhance the osseointegration of endoprostheses after revision operations. The chitosan used has a tailored degree of acetylation which allows for a fast biodegradation by lysozyme. The degradability of chitosan is proven via viscometry. Characteristics and degradation of nanoparticles formed with TPP are analyzed using dynamic light scattering. The particle degradation via lysozyme displays a decrease in particle diameter of 40% after 4 days. Drug loading and release is investigated for the nanoparticles with bone morphogenetic protein 2 (BMP-2, using ELISA and the BRE luciferase test for quantification and bioactivity evaluation. Furthermore, nanoparticle coatings on titanium substrates are created via spray-coating and analyzed by ellipsometry, scanning electron microscopy and X-ray photoelectron spectroscopy. Drug loaded nanoparticle coatings with biologically active BMP-2 are obtained in vitro within this work. Additionally, an in vivo study in mice indicates the dose dependent induction of ectopic bone growth through CS-TPP-BMP-2 nanoparticles. These results show that biodegradable CS-TPP coatings can be utilized to present biologically active BMP-2 on common implant materials like Ti6Al4V.

  18. Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Grassi Rici Rose

    2012-02-01

    Full Text Available Abstract Background The bone morphogenetic proteins (BMPs belong to a unique group of proteins that includes the growth factor TGF-β. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p

  19. Improving the osteogenic potential of BMP-2 with hyaluronic acid hydrogel modified with integrin-specific fibronectin fragment

    NARCIS (Netherlands)

    Kisiel, M.; Martino, M.M.; Ventura, M.; Hubbell, J.A.; Hilborn, J.; Ossipov, D.A.

    2013-01-01

    While human bone morphogenetic protein-2 (rhBMP-2) is a promising growth factor for bone regeneration, its clinical efficacy has recently shown to be below expectation. In order to improve the clinical translation of rhBMP-2, there exists strong motivation to engineer better delivery systems. Hyalur

  20. Deficiency of retinaldehyde dehydrogenase 1 induces BMP2 and increases bone mass in vivo.

    Directory of Open Access Journals (Sweden)

    Shriram Nallamshetty

    Full Text Available The effects of retinoids, the structural derivatives of vitamin A (retinol, on post-natal peak bone density acquisition and skeletal remodeling are complex and compartment specific. Emerging data indicates that retinoids, such as all trans retinoic acid (ATRA and its precursor all trans retinaldehyde (Rald, exhibit distinct and divergent transcriptional effects in metabolism. Despite these observations, the role of enzymes that control retinoid metabolism in bone remains undefined. In this study, we examined the skeletal phenotype of mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1, the enzyme responsible for converting Rald to ATRA in adult animals. Bone densitometry and micro-computed tomography (µCT demonstrated that Aldh1a1-deficient (Aldh1a1(-/- female mice had higher trabecular and cortical bone mass compared to age and sex-matched control C57Bl/6 wild type (WT mice at multiple time points. Histomorphometry confirmed increased cortical bone thickness and demonstrated significantly higher bone marrow adiposity in Aldh1a1(-/- mice. In serum assays, Aldh1a1(-/- mice also had higher serum IGF-1 levels. In vitro, primary Aldh1a1(-/- mesenchymal stem cells (MSCs expressed significantly higher levels of bone morphogenetic protein 2 (BMP2 and demonstrated enhanced osteoblastogenesis and adipogenesis versus WT MSCs. BMP2 was also expressed at higher levels in the femurs and tibias of Aldh1a1(-/- mice with accompanying induction of BMP2-regulated responses, including expression of Runx2 and alkaline phosphatase, and Smad phosphorylation. In vitro, Rald, which accumulates in Aldh1a1(-/- mice, potently induced BMP2 in WT MSCs in a retinoic acid receptor (RAR-dependent manner, suggesting that Rald is involved in the BMP2 increases seen in Aldh1a1 deficiency in vivo. Collectively, these data implicate Aldh1a1 as a novel determinant of cortical bone density and marrow adiposity in the skeleton in vivo through modulation of BMP signaling.

  1. Apoptosis inhibitors and mini-agrin have additive benefits in congenital muscular dystrophy mice

    OpenAIRE

    Meinen, Sarina; Lin, Shuo; Thurnherr, Raphael; Erb, Michael; Meier, Thomas; Rüegg, Markus A.

    2011-01-01

    Mutations in LAMA2 cause a severe form of congenital muscular dystrophy, called MDC1A. Studies in mouse models have shown that transgenic expression of a designed, miniaturized form of the extracellular matrix molecule agrin (‘mini-agrin’) or apoptosis inhibition by either overexpression of Bcl2 or application of the pharmacological substance omigapil can ameliorate the disease. Here, we tested whether mini-agrin and anti-apoptotic agents act on different pathways and thus exert additive bene...

  2. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  3. Rescuing Z+ agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing

    OpenAIRE

    Ruggiu, Matteo; Herbst, Ruth; Kim, Natalie; Jevsek, Marko; Fak, John J.; Mann, Mary Anne; Fischbach, Gerald; Burden, Steven J.; Darnell, Robert B.

    2009-01-01

    Synapse formation at the neuromuscular junction (NMJ) requires an alternatively spliced variant of agrin (Z+ agrin) that is produced only by neurons. Here, we show that Nova1 and Nova2, neuron-specific splicing factors identified as targets in autoimmune motor disease, are essential regulators of Z+ agrin. Nova1/Nova2 double knockout mice are paralyzed and fail to cluster AChRs at the NMJ, and breeding them with transgenic mice constitutively expressing Z+ agrin in motor neurons rescued AChR ...

  4. Bone formation of a porous Gelatin-Pectin-biphasic calcium phosphate composite in presence of BMP-2 and VEGF.

    Science.gov (United States)

    Amirian, Jhaleh; Linh, Nguyen Thuy Ba; Min, Young Ki; Lee, Byong-Taek

    2015-05-01

    A composite scaffold of gelatin (Gel)-pectin (Pec)-biphasic calcium phosphate (BCP) was fabricated for the successful delivery of growth factors. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) were coated on the Gel-Pec-BCP surface to investigate of effect of them on bone healing. Surface morphology was investigated by scanning electron microscopy, and BCP dispersion in the hydrogel scaffolds was measured by energy dispersive X-ray spectroscopy. The results obtained from Fourier transform infrared spectroscopy showed that BMP-2 and VEGF were successfully coated on Gel-Pec-BCP hydrogel scaffolds. MC3T3-E1 preosteoblasts were cultivated on the scaffolds to investigate the effect of BMP-2 and VEGF on cell viability and proliferation. VEGF and BMP-2 loaded on Gel-Pec-BCP scaffold facilitated increased cell spreading and proliferation compared to Gel-Pec-BCP scaffolds. In vivo, bone formation was examined using rat models. Bone formation was observed in Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds within 4 weeks, and was greatest with Gel-Pec-BCP/BMP-2 scaffolds. In vitro and in vivo results suggest that Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds could enhance bone regeneration.

  5. Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro

    DEFF Research Database (Denmark)

    Lysdahl, Helle; Baatrup, Anette; Foldager, Casper Bindzus;

    2014-01-01

    treatment strategy in which human bone marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured...... in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of h......MSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided....

  6. Induction of chronic pancreatitis by pancreatic duct ligation activates BMP2, apelin, and PTHrP expression in mice.

    Science.gov (United States)

    Rastellini, Cristiana; Han, Song; Bhatia, Vandanajay; Cao, Yanna; Liu, Ka; Gao, Xuxia; Ko, Tien C; Greeley, George H; Falzon, Miriam

    2015-10-01

    Chronic pancreatitis (CP) is a devastating disease with no treatments. Experimental models have been developed to reproduce the parenchyma and inflammatory responses typical of human CP. For the present study, one objective was to assess and compare the effects of pancreatic duct ligation (PDL) to those of repetitive cerulein (Cer)-induced CP in mice on pancreatic production of bone morphogenetic protein-2 (BMP2), apelin, and parathyroid hormone-related protein (PTHrP). A second objective was to determine the extent of cross talk among pancreatic BMP2, apelin, and PTHrP signaling systems. We focused on BMP2, apelin, and PTHrP since these factors regulate the inflammation-fibrosis cascade during pancreatitis. Findings showed that PDL- and Cer-induced CP resulted in significant elevations in expression and peptide/protein levels of pancreatic BMP2, apelin, and PTHrP. In vivo mouse and in vitro pancreatic cell culture experiments demonstrated that BMP2 stimulated pancreatic apelin expression whereas apelin expression was inhibited by PTHrP exposure. Apelin or BMP2 exposure inhibited PTHrP expression, and PTHrP stimulated upregulation of gremlin, an endogenous inhibitor of BMP2 activity. Transforming growth factor-β (TGF-β) stimulated PTHrP expression. Together, findings demonstrated that PDL- and Cer-induced CP resulted in increased production of the pancreatic BMP2, apelin, and PTHrP signaling systems and that significant cross talk occurred among pancreatic BMP2, apelin, and PTHrP. These results together with previous findings imply that these factors interact via a pancreatic network to regulate the inflammation-fibrosis cascade during CP. More importantly, this network communicated with TGF-β, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans. PMID:26229008

  7. Gene gun transferring-bone morphogenetic protein 2 (BMP-2) gene enhanced bone fracture healing in rabbits

    OpenAIRE

    Li, Wenju; Wei, Haifeng; Xia, Chunmei; Zhu, Xiaomeng; Hou, Guozhu; Xu, Feng; Xinghua SONG; Zhan, Yulin

    2015-01-01

    Purpose: Transferring the bone morphogenetic protein 2 (BMP-2) genes into the tissues or cells can improve the bone healing of the fracture has been widely accepted. We evaluated the efficiency of using gene gun to transfer the BMP-2 gene thereby affected the healing of a fractured bone. Methods: The vector coding for BMP-2 was constructed by a non-replicating encephalo-myocarditis virus (ECMV)-based vector. The segmental bone defect (1.5 cm) model was created by a wire-saw at the middle part...

  8. Vergleich von BMP-4 versus BMP-2 für die osteogene Differenzierung von Periostzellen

    OpenAIRE

    Klumpp, Florian (Alexander Stephan)

    2010-01-01

    Es ist heute bekannt, dass humane periostale mesenchymale Stammzellen (PMSCs) eine aussichtsreiche Grundlage für ein erfolgreiches Knochen Tissue Engineering darstellen. Dennoch ist die osteogene Differenzierung noch nicht vollständig be-schrieben. Da BMP-2 und BMP-4 nachweislich Regulatoren der Osteogenese sind, bestand die Aufgabe der vorliegenden Arbeit darin, die Wirkung derer auf die osteo-gene Differenzierung humaner PMSCs zu untersuchen. Isolierte humane PMSCs wurden mit Hilfe von o...

  9. BMP-2 in der Therapie der Pseudarthrose langer Röhrenknochen

    OpenAIRE

    Hellriegel, Tom

    2010-01-01

    Effective therapy for long bone non-unions is still a challenge in trauma and orthopedic surgery and treatment is time and cost-intensive. Complications can lead to ensuing health-related problems for the patient and their ability to work can be restricted. An innovative approach to stimulate bone regeneration is the application of growth factors. Bone morphogenetic protein-2 (BMP-2) has a high osteoinductive capacity and might stimulate human non-union healing. The purpose of this stud...

  10. Histone deacetylases control neurogenesis in embryonic brain by inhibition of BMP2/4 signaling.

    Directory of Open Access Journals (Sweden)

    Maya Shakèd

    Full Text Available BACKGROUND: Histone-modifying enzymes are essential for a wide variety of cellular processes dependent upon changes in gene expression. Histone deacetylases (HDACs lead to the compaction of chromatin and subsequent silencing of gene transcription, and they have recently been implicated in a diversity of functions and dysfunctions in the postnatal and adult brain including ocular dominance plasticity, memory consolidation, drug addiction, and depression. Here we investigate the role of HDACs in the generation of neurons and astrocytes in the embryonic brain. PRINCIPAL FINDINGS: As a variety of HDACs are expressed in differentiating neural progenitor cells, we have taken a pharmacological approach to inhibit multiple family members. Inhibition of class I and II HDACs in developing mouse embryos with trichostatin A resulted in a dramatic reduction in neurogenesis in the ganglionic eminences and a modest increase in neurogenesis in the cortex. An identical effect was observed upon pharmacological inhibition of HDACs in in vitro-differentiating neural precursors derived from the same brain regions. A reduction in neurogenesis in ganglionic eminence-derived neural precursors was accompanied by an increase in the production of immature astrocytes. We show that HDACs control neurogenesis by inhibition of the bone morphogenetic protein BMP2/4 signaling pathway in radial glial cells. HDACs function at the transcriptional level by inhibiting and promoting, respectively, the expression of Bmp2 and Smad7, an intracellular inhibitor of BMP signaling. Inhibition of the BMP2/4 signaling pathway restored normal levels of neurogenesis and astrogliogenesis to both ganglionic eminence- and cortex-derived cultures in which HDACs were inhibited. CONCLUSIONS: Our results demonstrate a transcriptionally-based regulation of BMP2/4 signaling by HDACs both in vivo and in vitro that is critical for neurogenesis in the ganglionic eminences and that modulates cortical

  11. Effect of rhBMP-2 on tibial plateau fractures in a canine model.

    Science.gov (United States)

    Schaefer, Susan L; Lu, Yan; Seeherman, Howard; Li, X Jian; Lopez, Mandi J; Markel, Mark D

    2009-04-01

    This study was to determine the efficacy of recombinant human bone morphogenetic protien-2 (rhBMP-2)/calcium phosphate matrix (CPX) paste to accelerate healing in a canine articular fracture model with associated subchondral defect. rhBMP-2/CPX (BMP), CPX alone (CPX) or autogenous bone graft (ABG) was administered to a canine articular tibial plateau osteotomy with a subchondral defect in each of 21 female dogs. The unoperated contralateral limbs served as controls. Ground reaction forces, synovial fluid, radiographic changes, mechanical testing, bone density, and histology of bone and synovium were analyzed at 6 weeks after surgery. Radiographic analysis demonstrated that the BMP and CPX groups showed improved bony healing compared to the ABG group at week 6. Histomorphometric analysis demonstrated that the BMP group had significantly increased trabecular bone volume compared to the CPX and ABG groups. Mechanical testing revealed that the BMP group had significantly greater maximum failure loads than the ABG group. Histological analysis demonstrated that the BMP group had significantly less sub-synovial inflammation than CPX group. This study demonstrated that rhBMP-2/CPX accelerated healing of articular fractures with subchondral defect compared to ABG in most of the parameters evaluated, and had less subsynovial inflammation than the CPX alone in a canine model.

  12. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector

    Science.gov (United States)

    Yue, Jianhui; Wu, Jun; Liu, Di; Zhao, Xiaoli; Lu, William W.

    2015-04-01

    Nanotechnology has made a significant impact on the development of nanomedicine. Nonviral vectors have been attracting more attention for the advantage of biosafety in gene delivery. Polyethylenimine (PEI)-conjugated chitosan (chitosan-g-PEI) emerged as a promising nonviral vector and has been demonstrated in many tumor cells. However, there is a lack of study focused on the behavior of this vector in stem cells which hold great potential in regenerative medicine. Therefore, in this study, in vitro gene delivering effect of chitosan-g-PEI was investigated in bone marrow stem cells. pIRES2-ZsGreen1-hBMP2 dual expression plasmid containing both the ZsGreen1 GFP reporter gene and the BMP2 functional gene was constructed for monitoring the transgene expression level. Chitosan-g-PEI-mediated gene transfer showed 17.2% of transfection efficiency and more than 80% of cell viability in stem cells. These values were higher than that of PEI. The expression of the delivered BMP2 gene in stem cells enhanced the osteogenic differentiation. These results demonstrated that chitosan-g-PEI is capable of applying in delivering gene to stem cells and providing potential applications in stem cell-based gene therapy.

  13. A late role for bmp2b in the morphogenesis of semicircular canal ducts in the zebrafish inner ear.

    Directory of Open Access Journals (Sweden)

    Katherine L Hammond

    Full Text Available The Bone Morphogenetic Protein (BMP genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/- embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

  14. RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: an experiment in dog.

    Science.gov (United States)

    Shi, Shanshan; Cheng, Xiangrong; Wang, Jiawei; Zhang, Wei; Peng, Lin; Zhang, Yufeng

    2009-01-01

    The purpose of this study is to develop a novel recombinant human bone morphogenetic protein-2 (rhBMP-2) sustained release scaffold for dental implant osseointegration, and to evaluate the effect of this scaffold on promoting bone formation. RhBMP-2 was encapsulated in the poly-D,L-lactide-co-glycolide (PLGA) biodegradable microspheres, which were subsequently dispersed in a chitosan/collagen composite scaffold. This rhBMP-2 microspheres-loaded scaffold (S-MB) was compared with a chitosan/collagen scaffold without microspheres that directly encapsulated rhBMP-2 (S-B) in vitro and in vivo. The microstructure of the new scaffold was examined with scanning electron microscopy. The release profile of rhBMP-2 in vitro was measured at interval periods. The effect of rhBMP-2 encapsulated scaffolds on enhancing bone formation through implantation in dogs' mandibles was identified by histological examination of the regenerated bone after 4 weeks of implantation. Due to PLGA microspheres being loaded, the S-MB exhibited lower values at porosity and swelling rate, as well as a higher effective release dose than that of the S-B. Bone density, bone-implant contact, and bone-fill values measured from dog experiments demonstrated that the S-MB induced bone regeneration more quickly and was timely substituted by new bone. It was concluded that this sustained carrier scaffold based on microspheres was more effective to induce implant osseointegration. PMID:18667455

  15. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Xiao Caiwen; Zhou Huifang; Fu Yao; Gu Ping; Fan Xianqun [Department of Ophthalmology, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Liu Guangpeng [Key Laboratory of Tissue Engineering, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Zhang Peng [Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Science (China); Hou Hongliang; Tang Tingting, E-mail: drfanxianqun@126.com [Department of Orthopedics, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China)

    2011-02-15

    Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

  16. 慢性肾脏病患者血清PTH与BMP-2的变化及意义

    Institute of Scientific and Technical Information of China (English)

    范旗; 赵燕凌; 甘西伦

    2008-01-01

    选择慢性肾脏病(CKD)患者65例和健康查体者20例(对照组),根据肾小球滤过率(GFR)将CKD患者分为五组,分别采用化学发光法和酶联免疫吸附法测定血清血清甲状旁腺激素(PTH)及骨形态发生蛋白-2(BMP-2)浓度,分析血清PTH及BMP-2浓度变化的规律.结果 CKD患者血清PTH、BMP-2浓度均明显高于对照组(P<0.01);CKD患者血清BMP-2浓度与PTH呈正相关(r=0.678,P=0.006).认为CKD患者血清PTH及BMP-2浓度随肾功能损害程度加重逐渐升高,提示PTH、BMP-2与CKD患者病情进展有一定关系.

  17. Experimental Comparison of Cranial Particulate Bone Graft, rhBMP-2, and Split Cranial Bone Graft for Inlay Cranioplasty.

    Science.gov (United States)

    Hassanein, Aladdin H; Couto, Rafael A; Kurek, Kyle C; Rogers, Gary F; Mulliken, John B; Greene, Arin K

    2013-05-01

    Background :  Particulate bone graft and recombinant human bone morphogenetic protein-2 (rhBMP-2) are options for inlay cranioplasty in children who have not developed a diploic space. The purpose of this study was to determine whether particulate bone graft or rhBMP-2 has superior efficacy for inlay cranioplasty and to compare these substances to split cranial bone. Methods :  A 17 mm × 17 mm critical-sized defect was made in the parietal bones of 22 rabbits and managed in four ways: Group I (no implant; n=5), Group II (particulate bone graft; n=5), Group III (rhBMP-2; n=7), and Group IV (split cranial bone graft; n=5). Animals underwent microcomputed tomography and histologic analysis 16 weeks after cranioplasty. Results :  Defects without an implant (Group I) demonstrated inferior ossification (41.4%; interquartile range [IQR], 28.9% to 42.5%) compared to those treated with particulate bone graft (Group II: 99.5%; IQR, 97.8% to 100%), rhBMP-2 (Group III: 99.6%; IQR, 99.5% to 100%), or split cranial bone (Group IV: 100%) (P inlay calvarial defect areas equally, although the thickness of bone healed with rhBMP-2 is inferior. Clinically, particulate bone graft or split cranial bone graft may be superior to rhBMP-2 for inlay cranioplasty.

  18. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

    International Nuclear Information System (INIS)

    Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

  19. Does Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Use in Adult Spinal Deformity (ASD) Increase Complications and Are Complications Associated With Location of rhBMP-2 Use?: A Prospective, Multicenter Study of 279 Consecutive Patients.

    Science.gov (United States)

    Bess, Shay; Line, Breton G; Lafarge, Virginie; Schwab, Frank; Shaffrey, Christopher I; Hart, Robert A; Boachie-Adjei, Oheneba; Akbarnia, Behrooz A; Ames, Christopher P; Burton, Douglas C; Deverin, Vedat; Fu, Kai-Ming G; Gupta, Munish; Hostin, Richard; Kebaish, Khaled; Klineberg, Eric; Mundis, Gregory; O'Brien, Michael; Shelokov, Alexis; Smith, Justin S

    2013-11-18

    Study Design. Multi-center, prospective analysis of consecutive ASD patients.Objective. Evaluate complications associated with rhBMP-2 use in ASDSummary of Background Data. Off-label rhBMP-2 use is common, however under-reporting of rhBMP-2 associated complications has been recently scrutinized.Methods. ASD patients consecutively enrolled into a prospective, multicenter database, were evaluated for type and timing of acute perioperative complications. Inclusion criteria: age ≥ 18 years, ASD, spinal arthrodesis >4 levels, and ≥3 months follow-up. Patients divided into those receiving rhBMP-2 (BMP) or no rhBMP-2 (NOBMP). BMP divided into location of use: posterior (PBMP), interbody (IBMP), and interbody + posterior spine (I+PBMP). Correlations between acute perioperative complications and rhBMP-2 use including total dose, dose/level and location of use were evaluated.Results. 279 patients (mean age 57 years, mean spinal levels fused 12.0, mean follow-up 28.8 months) met inclusion criteria. BMP (n = 172; average posterior dose = 2.5 mg/level, average interbody dose = 5 mg/level) had similar age, smoking history, previous spine surgery, total spinal levels fused, estimated blood loss, and duration of hospital stay as NOBMP (n = 107; p>0.05). BMP had greater Charlson Comorbidity Index (1.9 vs. 1.2), greater scoliosis (43° vs. 38°), longer operative time (488.2 vs. 414.6 minutes), more osteotomies/patient (4.0 vs. 1.6) and greater percentage of anteroposterior fusion (APSF; 20.9% vs. 8.4%) than NOBMP, respectively (p0.05). Multivariate analysis demonstrated small to non-existent correlations between rhBMP-2 use and complications.Conclusions. RhBMP-2 use and location of rhBMP-2 use in ASD surgery, at reported doses, does not increase acute major, neurological or wound complications. Research is needed for higher rhBMP-2 dosing and long-term follow-up.

  20. Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

    Science.gov (United States)

    Biroc, S L; Payan, D G; Fisher, J M

    1993-09-17

    The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

  1. BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

    Directory of Open Access Journals (Sweden)

    Finnson Kenneth

    2010-02-01

    Full Text Available Abstract Background Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. Results The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. Conclusion The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has

  2. LRP4 third β-propeller domain mutations cause novel congenital myasthenia by compromising agrin-mediated MuSK signaling in a position-specific manner

    Science.gov (United States)

    Ohkawara, Bisei; Cabrera-Serrano, Macarena; Nakata, Tomohiko; Milone, Margherita; Asai, Nobuyuki; Ito, Kenyu; Ito, Mikako; Masuda, Akio; Ito, Yasutomo; Engel, Andrew G.; Ohno, Kinji

    2014-01-01

    Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Using Sanger and exome sequencing in a CMS patient, we identified two heteroallelic mutations, p.Glu1233Lys and p.Arg1277His, in LRP4 coding for the postsynaptic low-density lipoprotein receptor-related protein 4. LRP4, expressed on the surface of the postsynaptic membrane of the neuromuscular junction, is a receptor for neurally secreted agrin, and LRP4 bound by agrin activates MuSK. Activated MuSK in concert with Dok-7 stimulates rapsyn to concentrate and anchor AChR on the postsynaptic membrane and interacts with other proteins implicated in the assembly and maintenance of the neuromuscular junction. LRP4 also functions as an inhibitor of Wnt/beta-catenin signaling. The identified mutations in LRP4 are located at the edge of its 3rd beta-propeller domain and decrease binding affinity of LRP4 for both MuSK and agrin. Mutations in the LRP4 3rd beta-propeller domain were previously reported to impair Wnt signaling and cause bone diseases including Cenani–Lenz syndactyly syndrome and sclerosteosis-2. By analyzing naturally occurring and artificially introduced mutations in the LRP4 3rd beta-propeller domain, we show that the edge of the domain regulates the MuSK signaling whereas its central cavity governs Wnt signaling. We conclude that LRP4 is a new CMS disease gene and that the 3rd beta propeller domain of LRP4 mediates the two signaling pathways in a position-specific manner. PMID:24234652

  3. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis

    Science.gov (United States)

    Wu, Lian; Wang, Feng; Donly, Kevin J.; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E.; Macdougall, Mary; Chen, Shuo

    2016-01-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2ko/ko dp) cell line by introducing Cre fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2fx/fx dp) cells. iBmp2ko/ko dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2ko/ko dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmpko/ko cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. PMID:26037045

  4. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

    Science.gov (United States)

    Wu, Lian; Wang, Feng; Donly, Kevin J; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

    2015-11-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. PMID:26037045

  5. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.

    Science.gov (United States)

    Wegman, F; Bijenhof, A; Schuijff, L; Oner, F C; Dhert, W J A; Alblas, J

    2011-03-15

    Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

  6. Effects of rhBMP-2 on Mandibular Distraction Osteogenesis and its OPG Expression in Rabbits%rhBMP-2对兔下颌骨牵引成骨区骨保护素表达的影响

    Institute of Scientific and Technical Information of China (English)

    周蕊; 付颖; 李新

    2011-01-01

    目的:通过动物实验,研究应用外源性rhBMP-2对兔下颌骨牵引成骨区骨保护素( osteoprotegerin,OPG)的影响.方法:在48只成年大耳白兔的一侧下颌骨前部行骨切开术,分别将空白胶原、rhBMP-2 1.5 mg胶原复合物植入下颌骨切开处,用牵引器延长一侧下颌骨4 mm,稳定期第1、3、7、14天,分别处死各组动物,取牵引区新生骨痂行组织学及OPG免疫组化染色.结果:下颌牵引延长后牵引间隙均有新骨形成,应用rhBMP-2 1.5mg效果好.免疫组化染色OPG主要定位于成骨细胞的胞浆中.在同一时间内,应用rhBMP-2组较对照组有显著性差异(P<0.05).结论:动物实验表明,rhBMP-2能促进兔下颌骨牵引成骨区新骨的生成.%Objective: To investigate the effects of rhBMP- 2 on mandibular distraction osteogenesis and the expression of OPG in the period of distraction osteogenesis. Methods: Unilateral mandibular osteotomies were performed in 48 mature rabbit. rhBMP - 2 1. 5mg with the collagen carrier was implanted in the osteotomy side of mandibles. Only collagen sponge was implanted in the control group. The mandibles of 48 rabbits were lengthened by 4 mm using a distractor , The animals were killed on 1,3,7,14 days of consolidation period. The distracted calluses were harvested and processed for histological and immunohistochemistry study of OPG. Results: The regenerated bone was found in the distraction gap after mandibular lengthening. But the rhBMP-2 was better. Staining for OPG was localized in osteoblasts of the periosteal region after completion of distraction. The mandibular side treated with rhBMP-2 had greater amount of new bone formation than that treated with collagen sponge. OPG was found in the cytoplasm of osteoblasts and stained brown or dark brown by immunohistochemistry. At the same time, OPG expression indicated that there was siganificant difference between rhBMP-2 group and control group(P<0. 05). Conclusion: rhBMP -2 could accelerate

  7. BMP-2 regulates the formation of oral sulcus in mouse tongue by altering the balance between TIMP-1 and MMP-13.

    Science.gov (United States)

    Fukui, Tadayoshi; Suga, Takeo; Iida, Ryo-Hei; Morito, Mitsuhiko; Luan, Xianghong; Diekwisch, Thomas G H; Nakamura, Yoshiki; Yamane, Akira

    2010-08-01

    The aim of this study is to investigate whether BMP-2 regulates the oral sulcus formation of mouse embryonic tongue by modifying the expression of TIMP and MMP. The BMP-2 siRNA induced a 180% increase in the depth of oral sulcus cavity (P sulcus into the mesenchymal tissues consisting of tongue floor, whereas the recombinant BMP-2 suppressed the process in the organ culture system of mouse embryonic tongue. The BMP-2 siRNA induced a 60% decrease in the expression of TIMP-1 mRNA (P sulcus in the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 220% increases in the expression of TIMP-1 mRNA and the area of the immunostaining for TIMP-1 around the oral sulcus was larger in the mandibles treated with the recombinant BMP-2 than the vehicle. The BMP-2 siRNA induced a 60% increase in the expression of MMP-13 protein and a marked increase in the staining intensity for MMP-13 was observed in the epithelial region of the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 70% decrease in the expression of MMP-13 mRNA and the decrease was mainly observed in the tissues around oral sulcus. The expressions of BMP-2, TIMP-1, and MMP-13 were verified in the tissues around in vivo developing oral sulcus at E11, 12, and 13 by immunohistochemistry. These results suggest that BMP-2 regulates the formation of oral sulcus by altering the balance between TIMP-1 and MMP-13.

  8. BMP2基因修饰犬脂肪源性基质细胞修复自体大段骨缺损%Repairing canine segmental bone defects using BMP2 gene modified adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    李慧武; 戴尅戎; 汤亭亭; 张晓玲; 唐坚; 孙晓江; 张双燕; 楼觉人

    2008-01-01

    Objective To evaluate osteogenetic effectiveness of porous β-tricalcium phosphate(β-TCP) ceramic mixed with human bone morphogenetic protein2 gene(Adv-hBMP2)modified adipose derived stromal cells (ADSCs) in the repair of critical-sized bone defects..Methods The ADSCs taken from the back of beagle dogs were modified by the BMP2 gene.The expression and bone-induction ability of BMP2 was identified by ELISA and ectopic bone formation in nude mice.The cells were applied to a β-tricalcium phosphate (TGP)carrier and implanted into ulnar bone defects in the canine model.18 ulnar bone defects were divided into three groups randomly and filled with granular TCP alone,granular TCP and ADSCs,or TCP and ADSCs transduced with Adv-hBMP2 respectively.All dogs were followed clinically and roentgenographically for 16 weeks and then sacrificed.Results ELISA and ectopic bone formation in nude mice showed the recombinant ADSCs could express BMP2 highly and stably.No bone defects healed after implanting granular TCP alone or granular TCP and ADSCs.In the TCP and ADSCs transduced with AdvhBMP2 group,two defects healed,four partly healed.Histological examination showed woven bone at the both end of the cortices but entirelv fibrous tissue in the middle in which defects filled with TCP alone or TCP and ADSCs.Defects filled with TCP and transduced ADSCs showed substatial new bone formation.Histomorphometry showed TCP combined with ADSCs did not significantly increase new bone area compared with TCP alone.TCP and recombinant ADSCs produced a significant increase in newly formed bone area.Conclusion ADSCs tansduced with BMP2 gene in a TCP carrier can enhance bone regeneratmn to repair the critically-sized bone defect.%目的 评价BMP2基因修饰的犬脂肪源性基质细胞(ADSCs)与β-磷酸三钙(β-TCP)复合修复自体大段骨缺损的疗效.方法 从比格犬背部脂肪组织中提取基质细胞,转染腺病毒介导的人BMP2基因(Adv-hBMP2),通过ELISA和裸鼠体内异位成骨实验鉴定BMP

  9. Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration.

    Science.gov (United States)

    Bouyer, Michael; Guillot, Raphael; Lavaud, Jonathan; Plettinx, Cedric; Olivier, Cécile; Curry, Véronique; Boutonnat, Jean; Coll, Jean-Luc; Peyrin, Françoise; Josserand, Véronique; Bettega, Georges; Picart, Catherine

    2016-10-01

    The rapid and effective bone regeneration of large non-healing defects remains challenging. Bioactive proteins, such as bone morphogenetic protein (BMP)-2, are proved their osteoinductivity, but their clinical use is currently limited to collagen as biomaterial. Being able to deliver BMP-2 from any other biomaterial would broaden its clinical use. This work presents a novel means for repairing a critical size volumetric bone femoral defect in the rat by combining a osteoinductive surface coating (2D) to a polymeric scaffold (3D hollow tube) made of commercially-available PLGA. Using a polyelectrolyte film as BMP-2 carrier, we tune the amount of BMP-2 loaded in and released from the polyelectrolyte film coating over a large extent by controlling the film crosslinking level and initial concentration of BMP-2 in solution. Using microcomputed tomography and quantitative analysis of the regenerated bone growth kinetics, we show that the amount of newly formed bone and kinetics can be modulated: an effective and fast repair was obtained in 1-2 weeks in the best conditions, including complete defect bridging, formation of vascularized and mineralized bone tissue. Histological staining and high-resolution computed tomography revealed the presence of bone regeneration inside and around the tube with spatially distinct organization for trabecular-like and cortical bones. The amount of cortical bone and its thickness increased with the BMP-2 dose. In view of the recent developments in additive manufacturing techniques, this surface-coating technology may be applied in combination with various types of polymeric or metallic scaffolds to offer new perspectives of bone regeneration in personalized medicine. PMID:27454063

  10. Betulinic acid synergically enhances BMP2-induced bone formation via stimulating Smad 1/5/8 and p38 pathways

    OpenAIRE

    Choi, Hyuck; Jeong, Byung-Chul; Kook, Min-Suk; Koh, Jeong-Tae

    2016-01-01

    Background Healing of bone defects is a dynamic and orchestrated process that relies on multiple growth factors and cell types. Bone morphogenetic protein 2 (BMP2) is a key growth factor for bone healing, which stimulates mesenchymal stem cells to differentiate into osteoblasts. Betulinic acid (BetA) is a natural pentacyclic triterpenoid from plants. This study aimed to examine combinatory effects of BetA and BMP2 on ectopic bone generation in mice. Results In MC3T3-E1 preosteoblast culture, ...

  11. 脊柱融合术患者BMP-2基因突变的检测及其意义%Mutational analysis of BMP-2 gene in the patients of spinal fusion

    Institute of Scientific and Technical Information of China (English)

    周传利; 陈晓亮

    2007-01-01

    目的 检测脊柱融合术患者的骨形态发生蛋白-2(BMP-2)基因突变状况.方法 从80例行脊柱融合术患者的术前空腹静脉血中提取DNA,采用聚合酶链反应-单链构象多态性分析(PCR-SSCP)及测序技术,检测其BMP-2基因部分编码区及其侧翼序列的突变.结果 脊柱融合术患者的外周静脉血中BMP-2基因有突变:TCG→GCG,TCA→TCG,并引起相应多肽的结构改变.结论 脊柱融合术患者中存在BMP-2基因突变及多态性分布,并有可能影响植骨融合效果.

  12. Influence of BMP-2 on early follicular development and mRNA expression of oocyte specific genes in bovine preantral follicles cultured in vitro.

    Science.gov (United States)

    Rossi, Rodrigo O D S; da Cunha, Ellen V; Portela, Antonia M L R; Passos, José R S; Costa, José J N; Silva, Anderson W B; Saraiva, Márcia V A; Peixoto, Christina A; Donato, Mariana A M; van den Hurk, Robert; Silva, José R V

    2016-03-01

    This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (PFSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5. PMID:26435174

  13. Expression of Human BMP-2 Gene in Different Tissues of Tobacco Plants%重组人BMP-2在烟草不同组织中的表达

    Institute of Scientific and Technical Information of China (English)

    高原; 索广力; 韩津; 何正权; 姚伟; 戴建武

    2006-01-01

    骨形态发生蛋白(BMPs)是一类调节骨组织发育的生长因子.BMP-2是BMP家族中诱骨活性最强的.在骨组织工程研究和临床应用中需要大量的BMP-2.因此,研究出一种能够有效地大量生产BMP-2的方法是十分必要的.随着植物分子生物学的进展,转基因植物被用作一种生物反应器来生产目的蛋白.以gus作为报告基因,研究了重组人bmp-2基因在烟草中的表达.通过GUS活性检测、半定量PCR和Western blotting分析了根、茎、叶组织中基因表达的水平,结果显示融合蛋白在根和茎组织中表达量显著高于叶组织.由于根和茎组织中蛋白组成与叶组织相比相对简单,提示其更易于进行目的蛋白的纯化.%The bone morphogenetic proteins (BMPs) are a family of growth factors that regulate the development of bone. BMP-2 is the most effective in the induction of bone tissue. A large amount of BMP-2 is needed for both bone tissue engineering research and clinical application. Thus, an effective way is necessary to produce sufficient BMP-2 protein. With the advance in plant biotechnology, transgenic plants have been targeted as a bioreactor to produce desired recombinant proteins. Here, the expression of recombinant human bmp-2 gene (rhbmp-2) was studied in tobacco plants using gus as a reporter gene. The difference of expression levels in root, stem and leaf tissues was analyzed by GUS activity assay, semi-quantitive RT-PCR and westem blotting.The results indicated that the expression levels of fusion protein in root and stem tissues were significantly higher than those in leaf tissue. For the protein compositions in root and stem tissues were simpler than those in leaf tissue,this suggested that the purification process with root and stem tissues would potentially be easier.

  14. Ectopic Bone Formation in vivo Induced by a Novel Synthetic Peptide Derived from BMP-2 Using Porous Collagen Scaffolds

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To investigate the osteoinductive and ectopicly osteogenic effects of a novel peptide P24 derived from bone morphogenetic protein 2 (BMP2), biodegradable collagen scaffolds (CS) were used to load BMP-2-derived peptide solutions with different concentrations (0.4 mg peptide/CS, 0.1 mg peptide/CS and pure CS, respectively), and the implants were implanted into muscular pockets on the back of Wistar rats.Radiographs and histological analysis were performed to evaluate the ectopic bone effects. Active ectopic bone formation was seen in both groups containing the peptide at different concentration (0.4 mg and 0.1 mg),whereas no bone formation and only fibrous tissue was seen in the pure CS group. The new bone formation induced by the peptide P24 displayed a dose-dependent and time-dependent efficiency. The new bone formation in the 0.4 mg peptide/CS group significantly increased than that of the 0.1 mg peptide/CS group. This novel BMP-2-derived peptide had excellent osteoinductive and ectopicly osteogenic properties which were similar to those of BMP2.

  15. Immunohistological Localization of BMP-2, BMP-7, and Their Receptors in Knee Joints with Focal Cartilage Lesions

    Directory of Open Access Journals (Sweden)

    Hagen Schmal

    2012-01-01

    Full Text Available Introduction. Although it is well known that BMP-2 and BMP-7 play significant roles in cartilage metabolism, data about intra-articular expression and localization of these proteins and their receptors in humans are rare. Methods. Biopsies of synovia and debrided cartilage were taken in patients undergoing autologous chondrocyte implantation. Expression of BMP-2, BMP-7, and their receptors BMPR-1A, BMPR-1B and BMPR-2 were semiquantitatively evaluated by immunohistological staining. Results. BMP-7 was equally highly expressed in all cartilage and synovial biopsies. Increased levels of BMPR-1A, but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P=0.002. BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression of the analyzed proteins or Mankin and Pritzker scores. Duration of symptoms and localization of lesions were associated with KOSS (P<0.02, but there was no influence of these parameters on protein expression. Conclusions. BMP-2, BMP-7, and BMPR-1A were expressed in cartilage and synovia of knees with focal cartilage lesions. Although defect localization and duration of symptoms decisively influence KOSS, there was no associated alteration of protein expression observed.

  16. Vitapex can promote the expression of BMP-2 during the bone regeneration of periapical lesions in rats

    Directory of Open Access Journals (Sweden)

    Xianyin Xia

    2013-01-01

    Full Text Available Purpose: To investigate the effect of Vitapex on the healing of periapical lesions and the expression of bone morphogenetic protein (BMP-2 during the periapical bone regeneration. Materials and Methods: Periapical lesions were induced in Sprague-Dawley (S-D rats by an occlusal pulp exposure in the mandibular first molars and were verified by X-ray. Total of 36 rats were randomly divided into three groups, and they were obturated with Zinc Oxide Eugenol (ZOE, or with Vitapex, or non-treated as negative control group. The rats of three groups were randomly killed at week 0, 2, 4, and 8 after root canal therapy, and then the mandibles were processed for histological examination and immunohistochemistry analysis. Results: At week 0, only a few BMP-2 positive cells could be observed in all rats. While the expression of BMP-2 was dramatically increased in case of Vitapex group at week 2 and week 4, and then climaxed at week 8. However, no apparent changes were observed in ZOE group and negative group at week 2, 4, and 8. Conclusion: These observations suggested that Vitapex has a greater ability in inducing bone regeneration than ZOE by the expression of BMP-2 induction in the treatment of rats experimental periapical lesions.

  17. BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway.

    Science.gov (United States)

    Jin, Eun-Jung; Lee, Sun-Young; Choi, Young-Ae; Jung, Jae-Chang; Bang, Ok-Sun; Kang, Shin-Sung

    2006-12-31

    The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells. PMID:17202865

  18. N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

    Science.gov (United States)

    Modarresi, Rozbeh; Lafond, Toulouse; Roman-Blas, Jorge A; Danielson, Keith G; Tuan, Rocky S; Seghatoleslami, M Reza

    2005-05-01

    We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin. PMID:15723280

  19. BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects

    Directory of Open Access Journals (Sweden)

    Xiong L

    2015-01-01

    Full Text Available Long Xiong,1 Jianhua Zeng,1 Aihua Yao,2 Qiquan Tu,3 Jingtang Li,1 Liang Yan,4 Zhiming Tang1 1Department of Osteology, People’s Hospital of Jiangxi Province, Nanchang, Jiangxi, People’s Republic of China; 2School of Materials Science and Engineering, Tongji University, Shanghai, People’s Republic of China; 3Department of Osteology, People’s Hospital of Jiujiang County, Jiujiang, Jiangxi, People’s Republic of China; 4Department of Osteology, The Third Hospital of Nanchang City, Nanchang, Jiangxi, People’s Republic of China Abstract: The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2, a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 µm with a core (60±18 µm and a mesoporous shell (180±42 m2/g surface area were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 µg/mg. There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option

  20. Kinetics and thermodynamics studies on the BMP-2 adsorption onto hydroxyapatite surface with different multi-morphological features

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Zhiwei; Huangfu, Changxin; Wang, Yanying; Ge, Hongwei; Yao, Yao; Zou, Ping; Wang, Guangtu [College of Science, Sichuan Agricultural University, Ya' an 625014 (China); He, Hua [Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Wenjiang, Sichuan 611130 (China); Rao, Hanbing, E-mail: rhbscu@gmail.com [College of Science, Sichuan Agricultural University, Ya' an 625014 (China)

    2015-07-01

    The effect of the surface topography on protein adsorption process is of great significance for designing hydroxyapatite (HA) ceramic material surfaces. In this work, three different topographies of HA materials HA-sheet, HA-rod, and HA-whisker were synthesized and testified by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Brunauer–Emmett–Teller (BET) and a field emission scanning electron microscopy (FE-SEM). We have systematically investigated the adsorption kinetics and thermodynamics of bone morphogenetic proteins (BMP-2) on the three different topography surfaces of HA, respectively. The results showed that the maximum adsorption capacities of HA-sheet, HA-rod and HA-whisker were (219.96 ± 10.18), (247.13 ± 12.35), and (354.67 ± 17.73) μg · g{sup −1}, respectively. Kinetic parameters, rate constants, equilibrium adsorption capacities and related correlation coefficients, for each kinetic model were calculated as well as discussed. It demonstrated that the adsorption of BMP-2 onto HA could be described by the pseudo second-order equation. Adsorption of BMP-2 onto HA followed the Langmuir isotherm. It confirmed that compared with other samples HA-whisker had more adsorption sites for its high specific surface area which could provide more opportunities for protein molecules. The adsorption processes were endothermic (ΔH > 0), spontaneous (ΔG < 0) and entropy increasing (ΔS > 0). A possible adsorption mechanism has been proposed. In addition, the BMP-2 could be adsorbed to the surface which existed slight conformational changes by FT-IR. - Highlights: • A novel protein adsorption studies based on sheet, rod and whisker of HA were designed. • Kinetic and thermodynamics parameters of BMP-2 and HA bonded materials were evaluated. • Surface topographies of the HA effect BMP-2 adsorption • The HA-whisker material had excellent adsorption performance for protein enrichment. • The electrostatic interaction is responsible for the

  1. Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression

    OpenAIRE

    Kawai, Mariko; Bessho, Kazuhisa; Maruyama, Hiroki; Miyazaki, Jun-ichi; Yamamoto, Toshio

    2006-01-01

    Background: Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS.

  2. In silico Mechano-Chemical Model of Bone Healing for the Regeneration of Critical Defects: The Effect of BMP-2.

    Directory of Open Access Journals (Sweden)

    Frederico O Ribeiro

    Full Text Available The healing of bone defects is a challenge for both tissue engineering and modern orthopaedics. This problem has been addressed through the study of scaffold constructs combined with mechanoregulatory theories, disregarding the influence of chemical factors and their respective delivery devices. Of the chemical factors involved in the bone healing process, bone morphogenetic protein-2 (BMP-2 has been identified as one of the most powerful osteoinductive proteins. The aim of this work is to develop and validate a mechano-chemical regulatory model to study the effect of BMP-2 on the healing of large bone defects in silico. We first collected a range of quantitative experimental data from the literature concerning the effects of BMP-2 on cellular activity, specifically proliferation, migration, differentiation, maturation and extracellular matrix production. These data were then used to define a model governed by mechano-chemical stimuli to simulate the healing of large bone defects under the following conditions: natural healing, an empty hydrogel implanted in the defect and a hydrogel soaked with BMP-2 implanted in the defect. For the latter condition, successful defect healing was predicted, in agreement with previous in vivo experiments. Further in vivo comparisons showed the potential of the model, which accurately predicted bone tissue formation during healing, bone tissue distribution across the defect and the quantity of bone inside the defect. The proposed mechano-chemical model also estimated the effect of BMP-2 on cells and the evolution of healing in large bone defects. This novel in silico tool provides valuable insight for bone tissue regeneration strategies.

  3. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  4. rhBMP-2/CPC强化骨质疏松椎体力学强度的实验研究%The experimental study of the effect of rhBMP-2/CPC on biomechanical intensity for osteoporotic vertebrae

    Institute of Scientific and Technical Information of China (English)

    罗志强

    2011-01-01

    Objective To observe the dynamic efficacy of recombinant human bone morphogenetic protein-2/calcium phosphate cement ( rhBMP-2/CPC ) on the augment of biomechanical intensity of the lumbar vertebrae in ovariectomized sheep. Methods Twelve adult female sheep were ovariectomized and raised for 1 year after the surgery. Bone mineral densities ( BMD) of the lumbar vertebrae were measured before and after the surgery. L1-L6 were the experimental objects. The sheep in control group were offered no treatment. The sheep in CPC group were injected with CPC (2.0ml) via the transpedicular of the vertebra. The sheep in rhBMP-2/CPC group were injected with rhBMP-2/CPC (2. 0ml) via the transpedicular of the vertebra. Every 3 sheep were randomly killed on day 1, week 6, week 12, and week 24 after the surgery. The vertebra compression test was performed. The ultimate compressive stress ( σalt ) and energy absorption value ( EAV ) of the vertebrae in every group were measured. The biomechanical indexes were compared and analyzed among different methods at the same time and among different time points using same method. Results BMO of the sheep vertebrae significantly decreased at 1 year after the surgery ( P 0.05). However, aalt in rhBMP-2/CPC group was significantly higher than that in CPC group on week 24 ( P 0. 05). However, σalt in rhBMP-2/ CPC group on week 24 was significantly higher than that on other three time points in the same group ( P < 0. 05). Conclusion rhBMP-2/CPC not only improved the immediate mechanical strength of osteoporotic vertebrae, but also maintained the dynamic mechanical strength and further enhanced the lone-term mechanical strength of osteoporotic vertebrae, which could provide an ideal mechanical condition for firm bone fusion of the spine.%目的 观察人重组骨形态发生蛋白-2复合磷酸钙骨水泥(recombinant human bonemorphogenetic protein-2/calcium phosphate cement,rhBMP-2/CPC)强化骨质疏松绵羊腰椎生物力学

  5. Repair of Rabbit Femoral Defects with a Novel BMP2-derived Oligopeptide P24

    Institute of Scientific and Technical Information of China (English)

    Zhixia DUAN; Qixin ZHENG; Xiaodong GUO; Changwen LI; Bin WU; Weigang WU

    2008-01-01

    In this study, the bioactivity of a novel BMP2-derived oligopeptide P24 was investigated by using the model of rabbit femoral defect after loaded in the biodegradable poly (lactic acid / glycolic acid / asparagic acid-co-polyethylene glycol) (PLGA-[ASP-PEG]). A 1.5-cm unilateral segmental bone defect was created in the left femoral diaphysis in each of the 30 new zealand white rabbits.The defects of 18 legs filled with BMP2-derived peptide P24 combined with PLGA-[ASP-PEG]scaffold serves as the experimental group, and the defects in the rest 12 rabbits filled with(PLGA-[ASP-PEG]) without P24 as control group. The bone-repairing capability in the target region of the two group was grossly, radiologically, histopathologically and biomechanically evaluated 4, 8and 12 weeks after the operation. Our results showed that in each group, primary healing of incision was achieved in the two groups. Radiographically, in experimental group, defects were filled with induced callus within 8 weeks, and a cortical bone-like structure was observed in some animals at the12th week. According to the standardized stage of bone defect repair, 9 (64.28%) achieved grade-4healing. In contrast, little bone formation was seen in the defects even 12 weeks after the operation,and 5 (62.50%) had grade 0 healing in this group. Histologically, tissue engineering material was mostly absorbed and cartilage was found around implants in the experimental group at the 4th week;8 weeks after operation, the engineering material was completely absorbed, and formation of woven bone was observed and typical trabecular bone structure could be seen. In control group, 8 weeks after operation, the defect was filled with fibrous tissues, and no bone-like structure was observed. Statistical analysis showed very significant difference in biomechanical indicators between the two groups (P<0.05). It is concluded that new oligopeptide P24 can induce excellent bone regeneration and promote bone repair.

  6. Agrin and synaptic laminin are required to maintain adult neuromuscular junctions.

    Directory of Open Access Journals (Sweden)

    Melanie A Samuel

    Full Text Available As synapses form and mature the synaptic partners produce organizing molecules that regulate each other's differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ, these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-α4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs.

  7. Fabrication of Core-Shell PEI/pBMP2-PLGA Electrospun Scaffold for Gene Delivery to Periodontal Ligament Stem Cells

    Directory of Open Access Journals (Sweden)

    Qiao Xie

    2016-01-01

    Full Text Available Bone tissue engineering is the most promising technology for enhancing bone regeneration. Scaffolds loaded with osteogenic factors improve the therapeutic effect. In this study, the bioactive PEI (polyethylenimine/pBMP2- (bone morphogenetic protein-2 plasmid- PLGA (poly(D, L-lactic-co-glycolic acid core-shell scaffolds were prepared using coaxial electrospinning for a controlled gene delivery to hPDLSCs (human periodontal ligament stem cells. The pBMP2 was encapsulated in the PEI phase as a core and PLGA was employed to control pBMP2 release as a shell. First, the scaffold characterization and mechanical properties were evaluated. Then the gene release behavior was analyzed. Our results showed that pBMP2 was released at high levels in the first few days, with a continuous release behavior in the next 28 days. At the same time, PEI/pBMP2 showed high transfection efficiency. Moreover, the core-shell electrospun scaffold showed BMP2 expression for a much longer time (more than 28 days compared with the single axial electrospun scaffold, as evaluated by qRT-PCR and western blot after culturing with hPDLSCs. These results suggested that the core-shell PEI/pBMP2-PLGA scaffold fabricated by coaxial electrospinning had a good gene release behavior and showed a prolonged expression time with a high transfection efficiency.

  8. Expression of human bone morphogenetic protein (BMP-2 and BMP-4 genes in transgenic bovine fibroblasts Expressão dos genes bone morphogenetic protein (BMP-2 e BMP-4 em fibroblastos bovinos transgênicos

    Directory of Open Access Journals (Sweden)

    C. Oleskovicz

    2004-08-01

    Full Text Available cDNAs dos genes bone morphogenetic protein-2 (BMP-2 e bone morphogenetic protein-4 (BMP-4 foram sintetizados a partir de RNA total extraído de tecidos ósseos de pacientes que apresentavam trauma facial (fraturas do maxilar entre o 7º e o 10º dia pós-trauma e clonados num vetor para expressão em células mamíferas, sob controle do promotor de citomegalovírus (CMV. Os vetores contendo os genes BMP-2 e o BMP-4 foram utilizados para a transfecção de fibroblastos bovinos. mRNAs foram indiretamente detectados por RT-PCR nas células transfectadas. As proteínas BMP-2 e BMP-4 foram detectadas mediante análises de Western blot. Os resultados demonstram a possibilidade de produção desses fatores de crescimento celular em fibroblastos bovinos. Essas células poderão ser utilizadas como fontes doadoras de material genético para a técnica de transferência nuclear na geração de animais transgênicos.

  9. Changes with Age and the Effect of Recombinant Human BMP-2 on Proteoglycan and Collagen Gene Expression in Rabbit Anulus Fibrosus Cells

    Institute of Scientific and Technical Information of China (English)

    Qin-Ming FEI; Xiao-Xing JIANG; Tong-Yi CHEN; Jun LI; Hideki MURAKAMI; Kai-Jow TSAI; William C. HUTTON

    2006-01-01

    In order to compare the difference between young and old intervertebral disc cells and their responsiveness to recombinant human bone morphogenetic protein-2 (rhBMP-2), disc cells were isolated from the anulus fibrosus (AF) and transition zones of lumbar discs from eight old and eight young New Zealand white rabbits. Compared with the cells from the young rabbits, cells from old rabbits respond less to rhBMP-2 treatment with respect to sulfated-glycosaminoglycan (sGAG) synthesis and aggrecan gene expression. But in collagen Ⅰ and collagen Ⅱ gene expressions, there are no significant differences between the old and the young. When comparing sGAG content, aggrecan, and collagen Ⅱ gene expression of the old AF cells after rhBMP-2 treatment with that of the young AF cells without rhBMP-2 treatment, the old AF cells with rhBMP-2 treatment have a greater capacity to synthesize sGAG bound in the cells and to release sGAG in the media, as well as to express aggrecan and collagen Ⅱ gene. It can be concluded that old AF cells after rhBMP-2 treatment have a greater capacity to synthesize sGAG and express aggrecan and collagen Ⅱ as compared to young AF cells without rhBMP-2 treatment. Thus rhBMP-2 can reverse the decline in the anabolic capacity of the disc cells with ageing. So it seems that rhBMP-2 has potential for use as an agent to retard a key component of disc degeneration and loss of disc matrix.

  10. E. coli-Produced BMP-2 as a Chemopreventive Strategy for Colon Cancer: A Proof-of-Concept Study

    Directory of Open Access Journals (Sweden)

    Saravanan Yuvaraj

    2012-01-01

    Full Text Available Colon cancer is a serious health problem, and novel preventive and therapeutical avenues are urgently called for. Delivery of proteins with anticancer activity through genetically modified bacteria provides an interesting, potentially specific, economic and effective approach here. Interestingly, bone morphogenetic protein 2 (BMP-2 is an important and powerful tumour suppressor in the colon and is thus an attractive candidate protein for delivery through genetically modified bacteria. It has not been shown, however, that BMP production in the bacterial context is effective on colon cancer cells. Here we demonstrate that transforming E. coli with a cDNA encoding an ileal-derived mature human BMP-2 induces effective apoptosis in an in vitro model system for colorectal cancer, whereas the maternal organism was not effective in this respect. Furthermore, these effects were sensitive to cotreatment with the BMP inhibitor Noggin. We propose that prevention and treatment of colorectal cancer using transgenic bacteria is feasible.

  11. Effect of implantation of biodegradable magnesium alloy on BMP-2 expression in bone of ovariectomized osteoporosis rats

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Yue, E-mail: 373073766@qq.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China); Ren, Ling, E-mail: lren@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Liu, Chang, E-mail: meixifan1971@163.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China); Yuan, Yajiang, E-mail: yuan925@163.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China); Lin, Xiao, E-mail: linx@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Tan, Lili, E-mail: lltan@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Chen, Shurui, E-mail: 272146792@qq.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China); Yang, Ke, E-mail: kyang@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Mei, Xifan, E-mail: meixifan1971@163.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China)

    2013-10-01

    The study was focused on the implantation of a biodegradable AZ31 magnesium alloy into the femoral periosteal of the osteoporosis modeled rats. The experimental results showed that after 4 weeks implantation of AZ31 alloy in the osteoporosis modeled rats, the expression of BMP-2 in bone tissues of the rats was much enhanced, even higher than the control group, which should promote the bone formation and be beneficial for reducing the harmful effect of osteoporosis. Results of HE stains showed that the implantation of AZ31 alloy did not have obvious pathological changes on both the liver and kidney of the animal. - Highlights: • Mg alloy greatly increased expression of BMP-2 in osteoporosis modeled rat bone. • Mg alloy showed good biological safety. • Mg alloy is beneficial for reducing the symptom of osteoporosis.

  12. Effect of implantation of biodegradable magnesium alloy on BMP-2 expression in bone of ovariectomized osteoporosis rats

    International Nuclear Information System (INIS)

    The study was focused on the implantation of a biodegradable AZ31 magnesium alloy into the femoral periosteal of the osteoporosis modeled rats. The experimental results showed that after 4 weeks implantation of AZ31 alloy in the osteoporosis modeled rats, the expression of BMP-2 in bone tissues of the rats was much enhanced, even higher than the control group, which should promote the bone formation and be beneficial for reducing the harmful effect of osteoporosis. Results of HE stains showed that the implantation of AZ31 alloy did not have obvious pathological changes on both the liver and kidney of the animal. - Highlights: • Mg alloy greatly increased expression of BMP-2 in osteoporosis modeled rat bone. • Mg alloy showed good biological safety. • Mg alloy is beneficial for reducing the symptom of osteoporosis

  13. Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats.

    OpenAIRE

    Schliephake, Henning; Weich, Herbert A.; Dullin, Christian; Gruber, Rudolf; Frahse, Sarah

    2008-01-01

    The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 4...

  14. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bone marrow mesenchymal stemcells(MSCs)are pluripotential stemcells that have the capacitytodifferentiate into chondrocytes and osteoblasts[1].Ithas been well documented that bone morphogeneticproteins(BMPs),a group of proteins belonging tothe TGF-βsuperfamily,can induce bone for mationbothin vivoandin vitroas well as promote osteo-blastic differentiation of MSC[2].HeterologousBMP2is successfully transferred to MSCs and genetherapy is employed based on repairing bony andcartilage defects,spinal fusion[3-5]....

  15. Repair of rabbit radial bone defects using true bone ceramics combined with BMP-2-related peptide and type I collagen

    International Nuclear Information System (INIS)

    An ideal bone graft material is the one characterized with good biocompatibility, biodegradation, osteoconductivity and osteoinductivity. In this study, a novel synthetic BMP-2-related peptide (designated P24) corresponding to residues of the knuckle epitope of BMP-2 was introduced into a biomimetic scaffold based on sintered bovine bone or true bone ceramics (TBC) and type I collagen (TBC/collagen I) using a simulated body fluid (SBF). Hydroxylapatite crystal mineralization with a Ca/P molar ratio of 1.63 was observed on the surface of P24/TBC/collagen I composite by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) techniques. Cell adhesion rate evaluation of bone marrow stromal cells (BMSCs) seeded on materials in vitro showed that the percentage of cells attached to P24/TBC/collagen I composite was significantly higher than that of the TBC/collagen I composite. A 10 mm unilateral segmental bone defect was created in the radius of New Zealand white rabbits and randomly implanted with three groups of biomaterials (Group A: P24/TBC/collagen I composite; Group B: TBC/collagen I composite and Group C: TBC alone). Based on radiographic evaluation and histological examination, the implants of P24/TBC/collagen I composite significantly stimulated bone growth, thereby confirming the enhanced rate of bone healing compared with that of TBC/collagen I composite and TBC alone. It was concluded that BMP-2-related peptide P24 could induce nucleation of calcium phosphate crystals on the surface of TBC/collagen I composite. The TBC/collagen I composite loaded with the synthetic BMP-2-related peptide is a promising scaffold biomaterial for bone tissue engineering.

  16. The expressions of IGF-1, BMP-2 and TGF-β1 in cartilage of condylar hyperplasia.

    Science.gov (United States)

    Meng, Q; Long, X; Deng, M; Cai, H; Li, J

    2011-01-01

    Condylar hyperplasia is a complex post-natal growth abnormality of the mandible and condyle, which leads to facial asymmetry. We investigated the distributions of insulin-like growth factors (IGF-1), bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) in cartilage of condylar hyperplasia and revealed relationships between age and the cartilaginous thickness. Twenty patients with condylar hyperplasia were divided into four histopathological types. The cartilaginous thickness and age in different histological types were analysed, and the localizations of IGF-1, BMP-2 and TGF-β1 were detected by immunohistochemistry analysis. The cartilaginous thickness of condylar hyperplasia significantly increased. The cartilaginous thickness of type III was significantly thicker than type I and type II, Bivariate correlation revealed a significant correlations between age and the cartilaginous thickness (r = 0·68, P = 0·01). However, the expressions of IGF-1, BMP-2 and TGF-β1 were the strongest in type I. In almost all types of condylar hyperplasia, the presence of IGF-1 and BMP-2 was found mainly in the proliferative chondrocyte layer and the hypertrophic chondrocyte layer, and only a few in the calcified chondrocyte layer. The presence of TGF-β1 widely distributed from the fibrous articular surface to the calcified cartilage. These findings suggest that the proliferative activity of cartilage in condylar hyperplasia is strongly associated with age and cartilaginous thickness. Therefore, the four pathological types of condylar hyperplasia seem more likely to be four discontinuous stages. PMID:20626571

  17. Rat aortic smooth muscle cells cultured on hydroxyapatite differentiate into osteoblast-like cells via BMP-2-SMAD-5 pathway.

    Science.gov (United States)

    Nahar-Gohad, Pranjal; Gohad, Neeraj; Tsai, Chen-Chih; Bordia, Rajendra; Vyavahare, Naren

    2015-04-01

    Vascular calcification is an important pathological condition associated with increased risk of cardiovascular mortality. Hydroxyapatite (HA) found in such deposits is the same polymorph of calcium (Ca) found in bone, indicating calcification may involve mechanisms akin to bone formation. Vascular smooth muscle cells (Vsmcs) have been shown to undergo phenotypic change to osteoblast-like cells. However, the mechanisms underlying this phenotypic change are unclear, and whether the stimulus to become osteogenic is a result of loss of mineralization inhibitors or early mineral deposits is not known. Our aim in this study is to identify mechanisms and signal transduction pathways that cause differentiation of Vsmcs into osteoblast-like cells in the presence of HA. We first characterized vascular origin of Vsmcs by studying the expression of smooth muscle cell markers: myosin heavy chain and smooth muscle actin along with SM22α at both mRNA and protein levels. Vsmcs grown on HA exhibited progressive change in cellular morphology at 3-, 7-, and 14-day time points. Culturing of Vsmcs on HA disc resulted in decrease in media Ca levels and increased expression of Ca-sensing receptor (CaSR) on Vsmcs resulting in upregulation of intracellular CaSR signaling leading to increased BMP-2 secretion. BMP-2 pathway mediated differentiation of Vsmcs to osteoblast-like cells shown by expression of osteogenic markers like runt-related transcription factor 2, osteocalcin, and alkaline phosphatase at mRNA and protein levels. Blocking CaSR by NPS-2143 reduced BMP-2 secretion and blocking the BMP-2 pathway by LDN-193189, a BMP inhibitor, modulated expression of osteogenic markers confirming their role in osteogenesis of Vsmcs. PMID:25725805

  18. Full regeneration of segmental bone defects using porous titanium implants loaded with BMP-2 containing fibrin gels

    Directory of Open Access Journals (Sweden)

    J van der Stok

    2015-03-01

    Full Text Available Regeneration of load-bearing segmental bone defects is a major challenge in trauma and orthopaedic surgery. The ideal bone graft substitute is a biomaterial that provides immediate mechanical stability, while stimulating bone regeneration to completely bridge defects over a short period. Therefore, selective laser melted porous titanium, designed and fine-tuned to tolerate full load-bearing, was filled with a physiologically concentrated fibrin gel loaded with bone morphogenetic protein-2 (BMP-2. This biomaterial was used to graft critical-sized segmental femoral bone defects in rats. As a control, porous titanium implants were either left empty or filled with a fibrin gels without BMP-2. We evaluated bone regeneration, bone quality and mechanical strength of grafted femora using in vivo and ex vivo µCT scanning, histology, and torsion testing. This biomaterial completely regenerated and bridged the critical-sized bone defects within eight weeks. After twelve weeks, femora were anatomically re-shaped and revealed open medullary cavities. More importantly, new bone was formed throughout the entire porous titanium implants and grafted femora regained more than their innate mechanical stability: torsional strength exceeded twice their original strength. In conclusion, combining porous titanium implants with a physiologically concentrated fibrin gels loaded with BMP-2 improved bone regeneration in load-bearing segmental defects. This material combination now awaits its evaluation in larger animal models to show its suitability for grafting load-bearing defects in trauma and orthopaedic surgery.

  19. The effects of 3D bioactive glass scaffolds and BMP-2 on bone formation in rat femoral critical size defects and adjacent bones

    International Nuclear Information System (INIS)

    Reconstruction of critical size defects in the load-bearing area has long been a challenge in orthopaedics. In the past, we have demonstrated the feasibility of using a biodegradable load-sharing scaffold fabricated from poly(propylene fumarate)/tricalcium phosphate (PPF/TCP) loaded with bone morphogenetic protein-2 (BMP-2) to successfully induce healing in those defects. However, there is limited osteoconduction observed with the PPF/TCP scaffold itself. For this reason, 13-93 bioactive glass scaffolds with local BMP-2 delivery were investigated in this study for inducing segmental defect repairs in a load-bearing region. Furthermore, a recent review on BMP-2 revealed greater risks in radiculitis, ectopic bone formation, osteolysis and poor global outcome in association with the use of BMP-2 for spinal fusion. We also evaluated the potential side effects of locally delivered BMP-2 on the structures of adjacent bones. Therefore, cylindrical 13-93 glass scaffolds were fabricated by indirect selective laser sintering with side holes on the cylinder filled with dicalcium phosphate dehydrate as a BMP-2 carrier. The scaffolds were implanted into critical size defects created in rat femurs with and without 10 μg of BMP-2. The x-ray and micro-CT results showed that a bridging callus was found as soon as three weeks and progressed gradually in the BMP group while minimal bone formation was observed in the control group. Degradation of the scaffolds was noted in both groups. Stiffness, peak load and energy to break of the BMP group were all higher than the control group. There was no statistical difference in bone mineral density, bone area and bone mineral content in the tibiae and contralateral femurs of the control and BMP groups. In conclusion, a 13-93 bioactive glass scaffold with local BMP-2 delivery has been demonstrated for its potential application in treating large bone defects. (paper)

  20. Experimental and computational investigation of the effect of hydrophobicity on aggregation and osteoinductive potential of BMP-2-derived peptide in a hydrogel matrix.

    Science.gov (United States)

    Moeinzadeh, Seyedsina; Barati, Danial; Sarvestani, Samaneh K; Karimi, Tahereh; Jabbari, Esmaiel

    2015-01-01

    An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73-92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005-0.005 mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the

  1. MRI of transforaminal lumbar interbody fusion: imaging appearance with and without the use of human recombinant bone morphogenetic protein-2 (rhBMP-2)

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Michael G.; Goldberg, Judd M.; Gaskin, Cree M.; Barr, Michelle S.; Alford, Bennett [University of Virginia, Department of Radiology and Medical Imaging, Charlottesville, VA (United States); Patrie, James T. [University of Virginia, Department of Public Health Sciences, Charlottesville, VA (United States); Shen, Francis H. [University of Virginia, Department of Orthopedic Surgery, Charlottesville, VA (United States)

    2014-09-15

    To describe the vertebral endplate and intervertebral disc space MRI appearance following TLIF, with and without the use of rhBMP-2, and to determine if the appearance is concerning for discitis/osteomyelitis. After institutional review board approval, 116 TLIF assessments performed on 75 patients with rhBMP-2 were retrospectively and independently reviewed by five radiologists and compared to 73 TLIF assessments performed on 45 patients without rhBMP-2. MRIs were evaluated for endplate signal, disc space enhancement, disc space fluid, and abnormal paraspinal soft tissue. Endplate edema-like signal was reported when T1-weighted hypointensity, T2-weighted hyperintensity, and endplate enhancement were present. Subjective concern for discitis/osteomyelitis on MRI was graded on a five-point scale. Generalized estimating equation binomial regression model analysis was performed with findings correlated with rhBMP-2 use, TLIF level, graft type, and days between TLIF and MRI. The rhBMP-2 group demonstrated endplate edema-like signal (OR 5.66; 95 % CI [1.58, 20.24], p = 0.008) and disc space enhancement (OR 2.40; 95 % CI [1.20, 4.80], p = 0.013) more often after adjusting for the TLIF level, graft type, and the number of days following TLIF. Both groups had a similar temporal distribution for endplate edema-like signal but disc space enhancement peaked earlier in the rhBMP-2 group. Disc space fluid was only present in the rhBMP-2 group. Neither group demonstrated abnormal paraspinal soft tissue and discitis/osteomyelitis was not considered likely in any patient. Endplate edema-like signal and disc space enhancement were significantly more frequent and disc space enhancement developed more rapidly following TLIF when rhBMP-2 was utilized. The concern for discitis/osteomyelitis was similar and minimal in both groups. (orig.)

  2. MRI of transforaminal lumbar interbody fusion: imaging appearance with and without the use of human recombinant bone morphogenetic protein-2 (rhBMP-2)

    International Nuclear Information System (INIS)

    To describe the vertebral endplate and intervertebral disc space MRI appearance following TLIF, with and without the use of rhBMP-2, and to determine if the appearance is concerning for discitis/osteomyelitis. After institutional review board approval, 116 TLIF assessments performed on 75 patients with rhBMP-2 were retrospectively and independently reviewed by five radiologists and compared to 73 TLIF assessments performed on 45 patients without rhBMP-2. MRIs were evaluated for endplate signal, disc space enhancement, disc space fluid, and abnormal paraspinal soft tissue. Endplate edema-like signal was reported when T1-weighted hypointensity, T2-weighted hyperintensity, and endplate enhancement were present. Subjective concern for discitis/osteomyelitis on MRI was graded on a five-point scale. Generalized estimating equation binomial regression model analysis was performed with findings correlated with rhBMP-2 use, TLIF level, graft type, and days between TLIF and MRI. The rhBMP-2 group demonstrated endplate edema-like signal (OR 5.66; 95 % CI [1.58, 20.24], p = 0.008) and disc space enhancement (OR 2.40; 95 % CI [1.20, 4.80], p = 0.013) more often after adjusting for the TLIF level, graft type, and the number of days following TLIF. Both groups had a similar temporal distribution for endplate edema-like signal but disc space enhancement peaked earlier in the rhBMP-2 group. Disc space fluid was only present in the rhBMP-2 group. Neither group demonstrated abnormal paraspinal soft tissue and discitis/osteomyelitis was not considered likely in any patient. Endplate edema-like signal and disc space enhancement were significantly more frequent and disc space enhancement developed more rapidly following TLIF when rhBMP-2 was utilized. The concern for discitis/osteomyelitis was similar and minimal in both groups. (orig.)

  3. Experimental and computational investigation of the effect of hydrophobicity on aggregation and osteoinductive potential of BMP-2-derived peptide in a hydrogel matrix.

    Science.gov (United States)

    Moeinzadeh, Seyedsina; Barati, Danial; Sarvestani, Samaneh K; Karimi, Tahereh; Jabbari, Esmaiel

    2015-01-01

    An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73-92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005-0.005 mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the

  4. Improved Bone Formation in Osteoporotic Rabbits with the Bone Morphogenetic Protein-2 (rhBMP-2) Coated Titanium Screws Which Were Coated By Using Plasma Polymerization Technique

    OpenAIRE

    Salih Gulsen; Dilek Cokeliler; Hilal Goktas; Aysu Kucukturhan; Bilgehan Ozcil; Hakan Caner

    2014-01-01

    Delaying of bone fusion in osteoporotic patients underwent spinal stabilization surgery leads to screw loosening, and this causes pseudoarticulation, mobility and fibrosis at vertebral segments. To prevent these complications, the screws coated with recombinant bone morphogenetic protein-2 (rhBMP-2) could be used. To verify this hypothesis, we coated 5 Titanium screws with rhBMP-2 using plasma polymerization method, and also used 10 uncoated screws for making comparison between coated and unc...

  5. Medium-Term Function of a 3D Printed TCP/HA Structure as a New Osteoconductive Scaffold for Vertical Bone Augmentation: A Simulation by BMP-2 Activation

    Directory of Open Access Journals (Sweden)

    Mira Moussa

    2015-04-01

    Full Text Available Introduction: A 3D-printed construct made of orthogonally layered strands of tricalcium phosphate (TCP and hydroxyapatite has recently become available. The material provides excellent osteoconductivity. We simulated a medium-term experiment in a sheep calvarial model by priming the blocks with BMP-2. Vertical bone growth/maturation and material resorption were evaluated. Materials and methods: Titanium hemispherical caps were filled with either bare- or BMP-2 primed constructs and placed onto the calvaria of adult sheep (n = 8. Histomorphometry was performed after 8 and 16 weeks. Results: After 8 weeks, relative to bare constructs, BMP-2 stimulation led to a two-fold increase in bone volume (Bare: 22% ± 2.1%; BMP-2 primed: 50% ± 3% and a 3-fold decrease in substitute volume (Bare: 47% ± 5%; BMP-2 primed: 18% ± 2%. These rates were still observed at 16 weeks. The new bone grew and matured to a haversian-like structure while the substitute material resorbed via cell- and chemical-mediation. Conclusion: By priming the 3D construct with BMP-2, bone metabolism was physiologically accelerated, that is, enhancing vertical bone growth and maturation as well as material bioresorption. The scaffolding function of the block was maintained, leaving time for the bone to grow and mature to a haversian-like structure. In parallel, the material resorbed via cell-mediated and chemical processes. These promising results must be confirmed in clinical tests.

  6. Co-delivery of platelet-derived growth factor (PDGF-BB) and bone morphogenic protein (BMP-2) coated onto heparinized titanium for improving osteoblast function and osteointegration.

    Science.gov (United States)

    Kim, Sung Eun; Yun, Young-Pil; Lee, Jae Yong; Shim, June-Sung; Park, Kyeongsoon; Huh, Jung-Bo

    2015-12-01

    The aim of this study was to improve osteoblast function by delivering two growth factors, PDGF-BB and BMP-2, incorporated onto heparinized titanium (Hep-Ti) substrate. To achieve co-delivery of PDGF-BB and BMP-2, the surface of anodized Ti was immobilized with heparin, and then the two growth factors were coated onto the Hep-Ti surface. Incorporation of the two growth factors onto Hep-Ti was evaluated by SEM and XPS. Incorporated PDGF-BB and BMP-2 were released from the Hep-Ti substrate in a sustained manner. In vitro studies revealed that osteoblasts grown on PDGF-BB- and BMP-2-immobilized Hep-Ti increased ALP activity, calcium deposition, osteocalcin and osteopontin levels as compared to those grown on PDGF-BB alone- or BMP-2 alone-immobilized Hep-Ti. These results suggested that co-delivery of PDGF-BB and BMP-2 using Hep-Ti substrate will be a promising material for the enhancement of osteoblast function and osteointegration.

  7. Disequilibrium of BMP2 Levels in the Breast Stem Cell Niche Launches Epithelial Transformation by Overamplifying BMPR1B Cell Response

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    Marion Chapellier

    2015-02-01

    Full Text Available Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

  8. Disequilibrium of BMP2 Levels in the Breast Stem Cell Niche Launches Epithelial Transformation by Overamplifying BMPR1B Cell Response

    Science.gov (United States)

    Chapellier, Marion; Bachelard-Cascales, Elodie; Schmidt, Xenia; Clément, Flora; Treilleux, Isabelle; Delay, Emmanuel; Jammot, Alexandre; Ménétrier-Caux, Christine; Pochon, Gaëtan; Besançon, Roger; Voeltzel, Thibault; Caron de Fromentel, Claude; Caux, Christophe; Blay, Jean-Yves; Iggo, Richard; Maguer-Satta, Véronique

    2015-01-01

    Summary Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer. PMID:25601208

  9. Integrins, muscle agrin and sarcoglycans during muscular inactivity conditions: an immunohistochemical study

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    G Anastasi

    2009-06-01

    Full Text Available Sarcoglycans are transmembrane proteins that seem to be functionally and pathologically as important as dystrophin. Sarcoglycans cluster together to form a complex, which is localized in the cell membrane of skeletal, cardiac, and smooth muscle. It has been proposed that the dystrophin-glycoprotein complex (DGC links the actin cytoskeleton with the extracellular matrix and the proper maintenance of this connection is thought to be crucial to the mechanical stability of the sarcolemma. The integrins are a family of heterodimeric cell surface receptors which play a crucial role in cell adhesion including cell-matrix and intracellular interactions and therefore are involved in various biological phenomena, including cell migration, and differentiation tissue repair. Sarcoglycans and integrins play a mechanical and signaling role stabilizing the systems during cycles of contraction and relaxation.Several studies suggested the possibility that integrins might play a role in muscle agrin signalling. On these basis, we performed an immunohistochemical analyzing sarcoglycans, integrins and agrin, on human skeletal muscle affected by sensitive-motor polyneuropathy, in order to better define the correlation between these proteins and neurogenic atrophy due to peripheral neuropathy. Our results showed the existence of a cascade mechanism which provoke a loss of regulatory effects of muscle activity on costameres, due to loss of muscle and neural agrin.This cascade mechanism could determine a quantitative modification of transmembrane receptors and loss of ?7B could be replaced and reinforced by enhanced expression of the ?7A integrin to restore muscle fiber viability. Second, it is possible that the reduced cycles of contraction and relaxation of muscle fibers, during muscular atrophy, provoke a loss of mechanical stresses transmitted over cell surface receptors that physically couple the cytoskeleton to extracellular matrix. Consequently, these mechanical

  10. Experimental study of human BMP-2 on osteogenic induction in BMSCs of dogs in vitro%人BMP-2体外定向诱导犬BMSCs向成骨方向分化的实验研究

    Institute of Scientific and Technical Information of China (English)

    许蕾; 韩建国; 李家锋

    2015-01-01

    Objective:To provide seed cells for bone tissue engineering in the late establishment by establishing the cul-ture system of bone marrow mesenchymal stem cells( BMSCs)of dogs in vitro,and using human BMP-2 to make them in-duced to differentiate into osteoblasts. Methods:The extraction of BMSCs of adult beagle dogs was made,then the whole marrow adherence method and density gradient centrifugation were used to isolate and culture BMSCs in vitro,and observe the cell growth morphology everyday. The third generation BMSCs with good growth form was divided into two groups. The experimental group were cultured with adding 200ng/ml human BMP-2 containing fetal bovine serum(FBS)while the control group were cultured only with complete medium containing FBS. Then we used the detection of alkaline phosphatase staining after 3 weeks′induction,alizarin red staining and Von-Kossa staining after 4 weeks′induction to identify the differentiation of osteoblasts. Results:After 3 weeks of induction of experimental group with alkaline phosphatase,staining showed the cyto-plasm of positive expression of black particles,and it was negative in the control group;After 4 weeks of induction of experi-mental group with alizarin red staining and Von-Kossa staining showed positive expression of calcium nodules,and it was negative in the control group. All the staining results in the experimental group showed the characteristics of osteoblasts. Conclusion:BMSCs of dogs,which are extracted and cultivated in vitro,can directionally differentiate into osteoblasts under the action of human BMP-2.%目的:通过将犬骨髓间充质干细胞( bone marrow mesenchymal stem cells,BMSCs)建立体外培养体系,运用人骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)体外定向诱导分化为成骨细胞,为后期建立骨组织工程提供种子细胞。方法提取比格犬BMSCs,全骨髓贴壁法结合密度梯度离心法行体外分离培养,每日观察细

  11. A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

    Science.gov (United States)

    Claros, Silvia; Rico-Llanos, Gustavo A.; Becerra, José; Andrades, José A.

    2014-01-01

    Transforming growth factor-beta (TGF-β) is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM) cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of rhTGF (recombinant human TGF)-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein)-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo. PMID:24968268

  12. A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Silvia Claros

    2014-06-01

    Full Text Available Transforming growth factor-beta (TGF-β is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS for 10 days in the presence of rhTGF (recombinant human TGF-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo.

  13. Trehalose maintains bioactivity and promotes sustained release of BMP-2 from lyophilized CDHA scaffolds for enhanced osteogenesis in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Jun Zhao

    Full Text Available Calcium phosphate (Ca-P scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2 loaded calcium-deficient hydroxyapatite (CDHA scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR and enzyme-linked immunosorbent assay (ELISA demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2 promoted osteogenic differentiation of bone marrow stromal cells (bMSCs significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ± 5.32% and area (40.71% ± 7.14% as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ± 0.44%, calcein: 6.08% ± 1.37% and mineral apposition rate (4.13 ± 0.62 µm/day in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17 ± 15.02 Mpa and

  14. Construction and identification of recombinant adenovirus vector co-expressing VEGF121 and BMP2 genes and its expression in HEK293 cells%VEGF121和BMP2双基因共表达重组腺病毒载体的构建及其在HEK293中的表达

    Institute of Scientific and Technical Information of China (English)

    栗刚; 吴秀成; 钟声; 王巍; 李媛; 刘丹平

    2011-01-01

    目的 构建人血管内皮生长因子121(VEGF121)与人骨形态发生蛋白2(BMP2)双基因共表达腺病毒载体Adv-BMP2-IRES-VEGF121,并观察其在人胚肾细胞株(HEK293)中的表达情况.方法 对腺病毒质粒pShuttle-CMV-BMP2的目的基因BMP2进行PCR扩增.腺病毒质粒pShuttle-CMV-VEGF121-IRES-hrGFP-1经Kpn I/Xba I酶切后,将BMP2片段定向导入pShuttle-CMV-VEGF121-IRES,构建pShuttle-CMV-V EGF121-IRES-BMP2,并注入大肠杆菌DH5a中扩增,提取质粒.通过酶切分析、PCR检测和序列分析进行鉴定.将构建所得的质粒转染HEK293,采用RT-PCR法检测HEK293中的BMP2、VEGF121 mRNA,Western blot法检测其蛋白.结果 成功构建了Adv-BMP2-IRES-VEGF121.酶切分析及DNA序列测定证实重组质粒构建正确.质粒转染后的HEK293 BMP2和VEGF121表达阳性.结论 成功构建了Adv-BMP2-IRES-VEGF121,其转染HEK293后,VEGF121、BMP2在HEK293中共表达阳性.%Objective To construct and identify the adenovirus shuttle plasmid pShuttle-CMV-VEGF121-IRES-BMP2 and its express in HEK293 cells.Methods The DNA fragments of human BMP2 gene were changed restriction sites and subcloned by PCR.The human BMP2 genes and pShuttle-CMV-VEGF121-IRES were ligated into the plasmid by directional cloning method.The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing.The correct recombinant express plasmid was transfected to HEK293 cells.The expression of VEGF121, BMP2 mRNA were detected by RT-PCR, the VEGF121, BMP2 protein were detected by Western blotting.Results The adenovirus shuttle plasmid was constructed correctly.The VEGF121, BMP2 mRNA and protein were expressed in HEK293 cells.Conclusion The adenovirus shuttle plasmid is constructed, VEGF121, BMP2 mRNA and protein are successfully expressed in HEK293 cells.

  15. BMP2, 4 and 6 and BMPR1B are altered from early stages of bovine cystic ovarian disease development.

    Science.gov (United States)

    Díaz, Pablo U; Hein, Gustavo J; Belotti, Eduardo M; Rodríguez, Fernanda M; Rey, Florencia; Amweg, Ayelén N; Matiller, Valentina; Baravalle, María E; Ortega, Hugo H; Salvetti, Natalia R

    2016-10-01

    Cystic ovarian disease (COD) is an important cause of subfertility in dairy cattle. Bone morphogenetic proteins (BMPs), mainly BMP2, BMP4 and BMP6, play a key role in female fertility. In this study, we hypothesized that an altered BMP system is associated with ovarian alterations contributing to COD pathogenesis. Therefore, we examined the expression of BMP2, BMP4 and BMP6 and BMP receptor 1B (BMPR1B) in the ovaries of animals with spontaneous or ACTH-induced COD, as well as during the development of the disease, in a model of follicular persistence induced by low doses of progesterone (at 5, 10 and 15 days of follicular persistence). Results showed changes in BMP2, BMP4 and BMP6 expression during folliculogenesis, in granulosa and theca cells in the COD groups, as well as at different stages of follicular persistence. Results also showed changes in BMPR1B expression in developing follicles in animals with COD, and at the initial stages of follicular persistence (P5). Comparison between groups showed significant differences, mainly in BMP4 and BMP6 expression, in granulosa and theca cells of different follicular categories. The expression of these BMPs also increased in cystic and persistent follicles, in relation to antral follicles of the control group. BMPR1B showed high expression in cystic follicles. Together, these results may indicate an alteration in BMPs, especially in BMP4 and BMP6, as well as in BMPR1B, which occurs early in folliculogenesis and incipiently during the development of COD, which could be a major cause of recurrence of this disease in cattle.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/early/2016/08/01/REP-15-0315/suppl/DC1. PMID:27486268

  16. Enhancement of Tendon–Bone Healing for Anterior Cruciate Ligament (ACL Reconstruction Using Bone Marrow-Derived Mesenchymal Stem Cells Infected with BMP-2

    Directory of Open Access Journals (Sweden)

    Shiyi Chen

    2012-10-01

    Full Text Available At present, due to the growing attention focused on the issue of tendon–bone healing, we carried out an animal study of the use of genetic intervention combined with cell transplantation for the promotion of this process. Here, the efficacy of bone marrow stromal cells infected with bone morphogenetic protein-2 (BMP-2 on tendon–bone healing was determined. A eukaryotic expression vector containing the BMP-2 gene was constructed and bone marrow-derived mesenchymal stem cells (bMSCs were infected with a lentivirus. Next, we examined the viability of the infected cells and the mRNA and protein levels of BMP-2-infected bMSCs. Gastrocnemius tendons, gastrocnemius tendons wrapped by bMSCs infected with the control virus (bMSCs+Lv-Control, and gastrocnemius tendons wrapped by bMSCs infected with the recombinant BMP-2 virus (bMSCs+Lv-BMP-2 were used to reconstruct the anterior cruciate ligament (ACL in New Zealand white rabbits. Specimens from each group were harvested four and eight weeks postoperatively and evaluated using biomechanical and histological methods. The bMSCs were infected with the lentivirus at an efficiency close to 100%. The BMP-2 mRNA and protein levels in bMSCs were significantly increased after lentiviral infection. The bMSCs and BMP-2-infected bMSCs on the gastrocnemius tendon improved the biomechanical properties of the graft in the bone tunnel; specifically, bMSCs infected with BMP-2 had a positive effect on tendon–bone healing. In the four-week and eight-week groups, bMSCs+Lv-BMP-2 group exhibited significantly higher maximum loads of 29.3 ± 7.4 N and 45.5 ± 11.9 N, respectively, compared with the control group (19.9 ± 6.4 N and 21.9 ± 4.9 N (P = 0.041 and P = 0.001, respectively. In the eight-week groups, the stiffness of the bMSCs+Lv-BMP-2 group (32.5 ± 7.3 was significantly higher than that of the bMSCs+Lv-Control group (22.8 ± 7.4 or control groups (12.4 ± 6.0 (p = 0.036 and 0.001, respectively. Based on the

  17. BMP-2,3,4,5在颌面部神经鞘瘤中的表达分析%Analysis of the Expression of BMP-2,3,4,5 in Nerve Sheath Tumors of Maxillofacial Region

    Institute of Scientific and Technical Information of China (English)

    金岩; 吕红兵; Tipoe GL; 李媛

    2000-01-01

    目的:探讨BMPs家族成员与外周神经肿瘤的关系。方法:用原位杂交方法对BMP-2,3,4,5在人良、恶性神经鞘瘤中的表达进行观察。结果:显示BMP-2,3,4,5 mRNA在外周神经的良恶性神经鞘瘤中均有分布。良性肿瘤中,呈栅栏状排列,肿瘤细胞聚集的区域BMPs表达升高;恶性肿瘤中BMPs的表达高于良性肿瘤。结论:确定了BMPs在外周神经肿瘤中的表达和分布,揭示部分BMPs可能参与了外周神经肿瘤的发生发展过程。

  18. Identification of a novel agrin-dependent pathway in cell signaling and adhesion within the erythroid niche.

    Science.gov (United States)

    Anselmo, A; Lauranzano, E; Soldani, C; Ploia, C; Angioni, R; D'amico, G; Sarukhan, A; Mazzon, C; Viola, A

    2016-08-01

    Establishment of cell-cell adhesion is crucial in embryonic development as well as within the stem cell niches of an adult. Adhesion between macrophages and erythroblasts is required for the formation of erythroblastic islands, specialized niches where erythroblasts proliferate and differentiate to produce red blood cells throughout life. The Eph family is the largest known family of receptor tyrosine kinases (RTKs) and controls cell adhesion, migration, invasion and morphology by modulating integrin and adhesion molecule activity and by modifying the actin cytoskeleton. Here, we identify the proteoglycan agrin as a novel regulator of Eph receptor signaling and characterize a novel mechanism controlling cell-cell adhesion and red cell development within the erythroid niche. We demonstrate that agrin induces clustering and activation of EphB1 receptors on developing erythroblasts, leading to the activation of α5β1 integrins. In agreement, agrin knockout mice display severe anemia owing to defective adhesion to macrophages and impaired maturation of erythroid cells. These results position agrin-EphB1 as a novel key signaling couple regulating cell adhesion and erythropoiesis. PMID:26990660

  19. Simultaneous gene transfer of bone morphogenetic protein (BMP -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression

    Directory of Open Access Journals (Sweden)

    Miyazaki Jun-ichi

    2006-08-01

    Full Text Available Abstract Background Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS. Methods First, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec. Results In the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a time-dependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP-4-expressing cells resided in the matrix between muscle fibers. Conclusion The simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.

  20. The effect of SDF-1α on low dose BMP-2 mediated bone regeneration by release from heparinized mineralized collagen type I matrix scaffolds in a murine critical size bone defect model.

    Science.gov (United States)

    Zwingenberger, Stefan; Langanke, Robert; Vater, Corina; Lee, Geoffrey; Niederlohmann, Eik; Sensenschmidt, Markus; Jacobi, Angela; Bernhardt, Ricardo; Muders, Michael; Rammelt, Stefan; Knaack, Sven; Gelinsky, Michael; Günther, Klaus-Peter; Goodman, Stuart B; Stiehler, Maik

    2016-09-01

    The treatment of critical size bone defects represents a challenge. The growth factor bone morphogenetic protein 2 (BMP-2) is clinically established but has potentially adverse effects when used at high doses. The aim of this study was to evaluate if stromal derived factor-1 alpha (SDF-1α) and BMP-2 released from heparinized mineralized collagen type I matrix (MCM) scaffolds have a cumulative effect on bone regeneration. MCM scaffolds were functionalized with heparin, loaded with BMP-2 and/or SDF-1α and implanted into a murine critical size femoral bone defect (control group, low dose BMP-2 group, low dose BMP-2 + SDF-1α group, and high dose BMP-2 group). After 6 weeks, both the low dose BMP-2 + SDF-1α group (5.8 ± 0.6 mm³, p = 0.0479) and the high dose BMP-2 group (6.5 ± 0.7 mm³, p = 0.008) had a significantly increased regenerated bone volume compared to the control group (4.2 ± 0.5 mm³). There was a higher healing score in the low dose BMP-2 + SDF-1α group (median grade 8; Q1-Q3 7-9; p = 0.0357) than in the low dose BMP-2 group (7; Q1-Q3 5-9) histologically. This study showed that release of BMP-2 and SDF-1α from heparinized MCM scaffolds allows for the reduction of the applied BMP-2 concentration since SDF-1α seems to enhance the osteoinductive potential of BMP-2. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2126-2134, 2016. PMID:27060915

  1. Effect of rhBMP- 2 on bone regeneration and osseointegration in peri- implant defects- histological observation%rhBMP-2在种植体周围骨缺损修复中应用的组织学观察

    Institute of Scientific and Technical Information of China (English)

    黄元瑾; 章锦才; 刘曙光; 蔡德鸿

    2012-01-01

    目的:研究rhBMP- 2及不同载体在种植体周围骨缺损修复中的应用.方法:在beagle犬下颌骨植入种植体,颊侧形成裂开性骨缺损,置入复合了不同浓度rhBMP- 2的珊瑚羟基磷灰石人造骨(CHA)或可吸收胶原海绵(ACS).种植体植入后2、4、8、12 周,获取含种植体骨标本,进行组织学观察.结果:2 周时,rhBMP- 2组可见极少量的新生骨组织.4 周时,rhBMP- 2/ACS组新骨组织由牙槽骨顶端向缺损区中心方向生长;rhBMP- 2/CHA组人造骨颗粒内部和周围出现呈岛状生长的新生骨组织.8 周时,rhBMP- 2/ACS组的新骨形成大片状结构;rhBMP- 2/CHA组人造骨颗粒周围较多骨岛形成.12 周时,rhBMP- 2组的缺损区内骨量和骨高度进一步增加,与种植体形成骨性结合.浓度为0.05 mg/ml和0.2 mg/ml,载体为CHA或ACS促进骨再生作用差异无统计学意义.结论:以CHA或ACS为载体rhBMP- 2能促进种植体周围骨缺损区内的骨组织再生并与种植体表面较好地结合.%Objective: To investigate the effects of rhBMP-2 in bone formation and osseointegration in peri-implant defects. Methods: 8 implants were placed in each of 8 beagle dogs. Dehiscence defects were surgically created on the buccal side of each implant. rhBMP-2 at 0. 05 mg/ml or 0. 2 mg/ml was combined into absorbable collagen sponge( ACS ) or coraline hydroxyapatite( CHA ) respectively and applied into the defects. ACS or CHA alone were used as the control. Animals were sacrificed 2,4,8 and 12 weeks after implantation respectively, non- decalcified ground sections of the bone samples with the implants were made and observed under microscope. Results: 2 weeks after implantation, the newly generated bone was minimal. 4 weeks after implantation, in rhBMP-2/ACS group formations of trabeculae of woven bone could be seen in the defects, in rhBMP-2/CHA the CHA particles were surrounded by newly formed bone. 8 weeks after implantation, in rhBMP-2/ACS group the newly formed bone

  2. Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression

    Science.gov (United States)

    Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H.; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

    2016-01-01

    Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2f/f;Bmp4f/fameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling. PMID:27146352

  3. Cardiogenic induction of pluripotent stem cells streamlined through a conserved SDF-1/VEGF/BMP2 integrated network.

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    Anca Chiriac

    Full Text Available BACKGROUND: Pluripotent stem cells produce tissue-specific lineages through programmed acquisition of sequential gene expression patterns that function as a blueprint for organ formation. As embryonic stem cells respond concomitantly to diverse signaling pathways during differentiation, extraction of a pro-cardiogenic network would offer a roadmap to streamline cardiac progenitor output. METHODS AND RESULTS: To resolve gene ontology priorities within precursor transcriptomes, cardiogenic subpopulations were here generated according to either growth factor guidance or stage-specific biomarker sorting. Innate expression profiles were independently delineated through unbiased systems biology mapping, and cross-referenced to filter transcriptional noise unmasking a conserved progenitor motif (55 up- and 233 down-regulated genes. The streamlined pool of 288 genes organized into a core biological network that prioritized the "Cardiovascular Development" function. Recursive in silico deconvolution of the cardiogenic neighborhood and associated canonical signaling pathways identified a combination of integrated axes, CXCR4/SDF-1, Flk-1/VEGF and BMP2r/BMP2, predicted to synchronize cardiac specification. In vitro targeting of the resolved triad in embryoid bodies accelerated expression of Nkx2.5, Mef2C and cardiac-MHC, enhanced beating activity, and augmented cardiogenic yield. CONCLUSIONS: Transcriptome-wide dissection of a conserved progenitor profile thus revealed functional highways that coordinate cardiogenic maturation from a pluripotent ground state. Validating the bioinformatics algorithm established a strategy to rationally modulate cell fate, and optimize stem cell-derived cardiogenesis.

  4. Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

    Science.gov (United States)

    Keeney, Michael; Chung, Michael T; Zielins, Elizabeth R; Paik, Kevin J; McArdle, Adrian; Morrison, Shane D; Ransom, Ryan C; Barbhaiya, Namrata; Atashroo, David; Jacobson, Gunilla; Zare, Richard N; Longaker, Michael T; Wan, Derrick C; Yang, Fan

    2016-08-01

    Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016. PMID:27059085

  5. Transcriptional repression of Bmp2 by p21(Waf1/Cip1) links quiescence to neural stem cell maintenance.

    Science.gov (United States)

    Porlan, Eva; Morante-Redolat, José Manuel; Marqués-Torrejón, María Ángeles; Andreu-Agulló, Celia; Carneiro, Carmen; Gómez-Ibarlucea, Esther; Soto, Atenea; Vidal, Anxo; Ferrón, Sacri R; Fariñas, Isabel

    2013-11-01

    Relative quiescence and self renewal are defining features of adult stem cells, but their potential coordination remains unclear. Subependymal neural stem cells (NSCs) lacking cyclin-dependent kinase (CDK) inhibitor (CKI) 1a (p21) exhibit rapid expansion that is followed by their permanent loss later in life. Here we demonstrate that transcription of the gene encoding bone morphogenetic protein 2 (Bmp2) in NSCs is under the direct negative control of p21 through actions that are independent of CDK. Loss of p21 in NSCs results in increased levels of secreted BMP2, which induce premature terminal differentiation of multipotent NSCs into mature non-neurogenic astrocytes in an autocrine and/or paracrine manner. We also show that the cell-nonautonomous p21-null phenotype is modulated by the Noggin-rich environment of the subependymal niche. The dual function that we describe here provides a physiological example of combined cell-autonomous and cell-nonautonomous functions of p21 with implications in self renewal, linking the relative quiescence of adult stem cells to their longevity and potentiality.

  6. Gelatin Tight-Coated Poly(lactide-co-glycolide Scaffold Incorporating rhBMP-2 for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Juan Wang

    2015-03-01

    Full Text Available Surface coating is the simplest surface modification. However, bioactive molecules can not spread well on the commonly used polylactone-type skeletons; thus, the surface coatings of biomolecules are typically unstable due to the weak interaction between the polymer and the bioactive molecules. In this study, a special type of poly(lactide-co-glycolide (PLGA-based scaffold with a loosened skeleton was fabricated by phase separation, which allowed gelatin molecules to more readily diffuse throughout the structure. In this application, gelatin modified both the internal substrate and external surface. After cross-linking with glutaraldehyde, the surface layer gelatin was tightly bound to the diffused gelatin, thereby preventing the surface layer gelatin coating from falling off within 14 days. After gelatin modification, PLGA scaffold demonstrated enhanced hydrophilicity and improved mechanical properties (i.e., increased compression strength and elastic modulus in dry and wet states. Furthermore, a sustained release profile of recombinant human bone morphogenetic protein-2 (rhBMP-2 was achieved in the coated scaffold. The coated scaffold also supported the in vitro attachment, proliferation, and osteogenesis of rabbit bone mesenchymal stem cells (BMSCs, indicating the bioactivity of rhBMP-2. These results collectively demonstrate that the cross-linked-gelatin-coated porous PLGA scaffold incorporating bioactive molecules is a promising candidate for bone tissue regeneration.

  7. Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors.

    Science.gov (United States)

    Chen, Jiaxuan; Dosier, Christopher R; Park, Jung Hwa; De, Subhendu; Guldberg, Robert E; Boyan, Barbara D; Schwartz, Zvi

    2016-01-01

    1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein-2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three-dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild-type (WT), Pdia3-silenced (Sh-Pdia3) and VDR-silenced (Sh-VDR) pre-osteoblastic MC3T3-E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε-caprolactone)-collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro-computed tomography (μCT) showed that the mineralized volume of untreated Sh-Pdia3 and Sh-VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh-VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3.

  8. Interplay between self-assembled structure of bone morphogenetic protein-2 (BMP-2) and osteoblast functions in three-dimensional titanium alloy scaffolds: Stimulation of osteogenic activity.

    Science.gov (United States)

    Nune, K C; Kumar, A; Murr, L E; Misra, R D K

    2016-02-01

    Three-dimensional cellular scaffolds are receiving significant attention in bone tissue engineering to treat segmental bone defects. However, there are indications of lack of significant osteoinductive ability of three-dimensional cellular scaffolds. In this regard, the objective of the study is to elucidate the interplay between bone morphogenetic protein (BMP-2) and osteoblast functions on 3D mesh structures with different porosities and pore size that were fabricated by electron beam melting. Self-assembled dendritic microstructure with interconnected cellular-type morphology of BMP-2 on 3D scaffolds stimulated osteoblast functions including adhesion, proliferation, and mineralization, with prominent effect on 2-mm mesh. Furthermore, immunofluorescence studies demonstrated higher density and viability of osteoblasts on lower porosity mesh structure (2 mm) as compared to 3- and 4-mm mesh structures. Enhanced filopodia cellular extensions with extensive cell spreading was observed on BMP-2 treated mesh structures, a behavior that is attributed to the unique self-assembled structure of BMP-2 that effectively communicates with the cells. The study underscores the potential of BMP-2 in imparting osteoinductive capability to the 3D printed scaffolds.

  9. Enhancement of tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells genetically modified with bFGF/BMP2

    Science.gov (United States)

    Chen, Biao; Li, Bin; Qi, Yong-Jian; Ni, Qu-Bo; Pan, Zheng-Qi; Wang, Hui; Chen, Liao-Bin

    2016-01-01

    Many strategies, including various growth factors and gene transfer, have been used to augment healing after anterior cruciate ligament (ACL) reconstruction. The biological environment regulated by the growth factors during the stage of tendon-bone healing was considered important in controlling the integrating process. The purpose of this study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) genetically modified with bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) on healing after ACL reconstruction. BMSCs were infected with an adenoviral vector encoding BMP2 (AdBMP2) or bFGF (AdbFGF). Then, the infected BMSCs were surgically implanted into the tendon-bone interface. At 12 weeks postoperatively, the formation of abundant cartilage-like cells, smaller tibial bone tunnel and significantly higher ultimate load and stiffness levels, through histological analysis, micro-computed tomography and biomechanical testing, were observed. In addition, the AdBMP2-plus-AdbFGF group had the smallest bone tunnel and the best mechanical properties among all the groups. The addition of BMP2 or bFGF by gene transfer resulted in better cellularity, new bone formation and higher mechanical property, which contributed to the healing process after ACL reconstruction. Furthermore, the co-application of these two genes was more powerful and efficient than either single gene therapy. PMID:27173013

  10. Sustained release of VH and rhBMP-2 from nanoporous magnesium-zinc-silicon xerogels for osteomyelitis treatment and bone repair.

    Science.gov (United States)

    Li, Fengqian; Wu, Wen; Xiang, Li; Weng, Gan; Hong, Hua; Jiang, Hong; Qian, Jun

    2015-01-01

    Nanoporous magnesium-zinc-silicon (n-MZS) xerogels with a pore size ∼4 nm, a surface area of 718 cm(2)/g, and a pore volume of 1.24 cm(3)/g were synthesized by a sol-gel method. The n-MZS xerogels had high capacity to load vancomycin hydrochloride (VH) and human bone morphogenetic protein-2 (rhBMP-2), after soaking in phosphate buffered saline (PBS) for 24 hours (1.5 and 0.8 mg/g, respectively). Moreover, the n-MZS xerogels exhibited the sustained release of VH and rhBMP-2 as compared with magnesium-zinc-silicon (MZS) xerogels without nanopores (showing a burst release). The VH/rhBMP-2/n-MZS system not only exhibited a good antibacterial property but also promoted the MG63 cell proliferation and differentiation demonstrating good bactericidal activity and cytocompatibility. The results suggested that n-MZS with larger surface area and high pore volume might be a promising carrier for loading and sustained release of VH and rhBMP-2. Hence, the VH/rhBMP-2/n-MZS system might be one of the promising biomaterials for osteomyelitis treatment and bone repair.

  11. Enhancement of tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells genetically modified with bFGF/BMP2.

    Science.gov (United States)

    Chen, Biao; Li, Bin; Qi, Yong-Jian; Ni, Qu-Bo; Pan, Zheng-Qi; Wang, Hui; Chen, Liao-Bin

    2016-01-01

    Many strategies, including various growth factors and gene transfer, have been used to augment healing after anterior cruciate ligament (ACL) reconstruction. The biological environment regulated by the growth factors during the stage of tendon-bone healing was considered important in controlling the integrating process. The purpose of this study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) genetically modified with bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) on healing after ACL reconstruction. BMSCs were infected with an adenoviral vector encoding BMP2 (AdBMP2) or bFGF (AdbFGF). Then, the infected BMSCs were surgically implanted into the tendon-bone interface. At 12 weeks postoperatively, the formation of abundant cartilage-like cells, smaller tibial bone tunnel and significantly higher ultimate load and stiffness levels, through histological analysis, micro-computed tomography and biomechanical testing, were observed. In addition, the AdBMP2-plus-AdbFGF group had the smallest bone tunnel and the best mechanical properties among all the groups. The addition of BMP2 or bFGF by gene transfer resulted in better cellularity, new bone formation and higher mechanical property, which contributed to the healing process after ACL reconstruction. Furthermore, the co-application of these two genes was more powerful and efficient than either single gene therapy. PMID:27173013

  12. β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner.

    Science.gov (United States)

    Fourel, Laure; Valat, Anne; Faurobert, Eva; Guillot, Raphael; Bourrin-Reynard, Ingrid; Ren, Kefeng; Lafanechère, Laurence; Planus, Emmanuelle; Picart, Catherine; Albiges-Rizo, Corinne

    2016-03-14

    Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin-dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2-induced Smad signaling through a Cdc42-Src-FAK-ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine.

  13. β3 integrin–mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner

    Science.gov (United States)

    Fourel, Laure; Valat, Anne; Faurobert, Eva; Guillot, Raphael; Bourrin-Reynard, Ingrid; Ren, Kefeng; Lafanechère, Laurence; Planus, Emmanuelle; Albiges-Rizo, Corinne

    2016-01-01

    Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin–dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2–induced Smad signaling through a Cdc42–Src–FAK–ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine. PMID:26953352

  14. Injection of a soluble fragment of neural agrin (NT-1654 considerably improves the muscle pathology caused by the disassembly of the neuromuscular junction.

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    Stefan Hettwer

    Full Text Available Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders.

  15. Cell adhesion to agrin presented as a nanopatterned substrate is consistent with an interaction with the extracellular matrix and not transmembrane adhesion molecules

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    Wolfram Tobias

    2008-12-01

    Full Text Available Abstract Background Molecular spacing is important for cell adhesion in a number of ways, ranging from the ordered arrangement of matrix polymers extracellularly, to steric hindrance of adhesion/signaling complexes intracellularly. This has been demonstrated using nanopatterned RGD peptides, a canonical extracellular matrix ligand for integrin interactions. Cell adhesion was greatly reduced when the RGD-coated nanoparticles were separated by more than 60 nm, indicating a sharp spacing-dependent threshold for this form of cell adhesion. Results Here we show a similar dependence of cell adhesion on the spacing of agrin, a protein that exists as both a secreted, matrix-bound form and a type-2 transmembrane form in vivo. Agrin was presented as a substrate for cell adhesion assays by anchoring recombinant protein to gold nanoparticles that were arrayed at tunable distances onto glass coverslips. Cells adhered well to nanopatterned agrin, and when presented as uniformly coated substrates, adhesion to agrin was comparable to other well-studied adhesion molecules, including N-Cadherin. Adhesion of both mouse primary cortical neurons and rat B35 neuroblastoma cells showed a spacing-dependent threshold, with a sharp drop in adhesion when the space between agrin-coated nanoparticles increased from 60 to 90 nm. In contrast, adhesion to N-Cadherin decreased gradually over the entire range of distances tested (uniform, 30, 60, 90, and 160 nm. The spacing of the agrin nanopattern also influenced cell motility, and peptide competition suggested adhesion was partially integrin dependent. Finally, differences in cell adhesion to C-terminal agrin fragments of different lengths were detected using nanopatterned substrates, and these differences were not evident using uniformly coated substrates. Conclusion These results suggest nanopatterned substrates may provide a physiological presentation of adhesive substrates, and are consistent with cells adhering to agrin

  16. Gene delivery nanocarriers of bioactive glass with unique potential to load BMP2 plasmid DNA and to internalize into mesenchymal stem cells for osteogenesis and bone regeneration

    Science.gov (United States)

    Kim, Tae-Hyun; Singh, Rajendra K.; Kang, Min Sil; Kim, Joong-Hyun; Kim, Hae-Won

    2016-04-01

    The recent development of bioactive glasses with nanoscale morphologies has spurred their specific applications in bone regeneration, for example as drug and gene delivery carriers. Bone engineering with stem cells genetically modified with this unique class of nanocarriers thus holds great promise in this avenue. Here we report the potential of the bioactive glass nanoparticle (BGN) system for the gene delivery of mesenchymal stem cells (MSCs) targeting bone. The composition of 15% Ca-added silica, proven to be bone-bioactive, was formulated into surface aminated mesoporous nanospheres with enlarged pore sizes, to effectively load and deliver bone morphogenetic protein-2 (BMP2) plasmid DNA. The enlarged mesopores were highly effective in loading BMP2-pDNA with an efficiency as high as 3.5 wt% (pDNA w.r.t. BGN), a level more than twice than for small-sized mesopores. The BGN nanocarriers released the genetic molecules in a highly sustained manner (for as long as 2 weeks). The BMP2-pDNA/BGN complexes were effectively internalized to rat MSCs with a cell uptake level of ~73%, and the majority of cells were transfected to express the BMP2 protein. Subsequent osteogenesis of the transfected MSCs was demonstrated by the expression of bone-related genes, including bone sialoprotein, osteopontin, and osteocalcin. The MSCs transfected with BMP2-pDNA/BGN were locally delivered inside a collagen gel to the target calvarium defects. The results showed significantly improved bone regeneration, as evidenced by the micro-computed tomographic, histomorphometric and immunohistochemical analyses. This study supports the excellent capacity of the BGN system as a pDNA-delivery nanocarrier in MSCs, and the engineered system, BMP2-pDNA/BGN with MSCs, may be considered a new promising candidate to advance the therapeutic potential of stem cells through genetic modification, targeting bone defects and diseases.The recent development of bioactive glasses with nanoscale morphologies has

  17. Effect of rhBMP2 and rhTGF-β1 on alkaline phosphatase activity of human dental pulp cells in vitro%rhBMP2和rhTGF-β1联合应用对人牙髓细胞碱性磷酸酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    刘嘉利; 张郁; 王晓方; 汪平

    2000-01-01

    目的:探讨rhBMP2和rhTGF-β1联合应用对人牙髓细胞ALPase活性的影响.方法:采用酶动力学的方法,比较不同浓度rhBMP2和rhTGF-β1单独或联合应用对人牙髓细胞的ALPase活性的影响.结果 :rhBMP2对人牙髓细胞的ALPase活性呈浓度依赖性增强,rhTGF-β1对人牙髓细胞ALPase活性的影响与其本身浓度有关 .当rhTGF-β1浓度为1μg/L与rhBMP2联合应用时, ALPa se活性比rhBMP2单独应用明显增高.结论:rhBMP2、rhTGF-β1单独及联合应用于人牙髓细胞时,对人牙髓细胞的ALPase活性作用不同,其对人牙髓细胞的生理功能可能有调节作用.

  18. Off-label innovation: characterization through a case study of rhBMP-2 for spinal fusion.

    Science.gov (United States)

    Schnurman, Zane; Smith, Michael L; Kondziolka, Douglas

    2016-09-01

    OBJECTIVE Off-label therapies are widely used in clinical practice by spinal surgeons. Some patients and practitioners have advocated for increased regulation of their use, and payers have increasingly questioned reimbursment for off-label therapies. In this study, the authors applied a model that quantifies publication data to analyze the developmental process from initial on-label use to off-label innovation, using as an example recombinant human bone morphogenetic protein 2 (rhBMP-2) because of its wide off-label use. METHODS As a case study of off-label innovation, the developmental patterns of rhBMP-2 from FDA-approved use for anterior lumbar interbody fusion to several of its off-label uses, including posterolateral lumbar fusion, anterior cervical discectomy and fusion, and posterior lumbar interbody fusion/transforaminal lumbar interbody fusion, were evaluated using the "progressive scholarly acceptance" (PSA) model. In this model, PSA is used as an end point indicating acceptance of a therapy or procedure by the relevant scientific community and is reached when the total number of peer-reviewed studies devoted to refinement or improvement of a therapy surpasses the total number assessing initial efficacy. Report characteristics, including the number of patients studied and study design, were assessed in addition to the time to and pattern of community acceptance, and results compared with previous developmental study findings. Disclosures and reported conflicts of interest for all articles were reviewed, and these data were also used in the analysis. RESULTS Publication data indicated that the acceptance of rhBMP-2 off-label therapies occurred more rapidly and with less evidence than previously studied on-label therapies. Additionally, the community appeared to respond more robustly (by rapidly changing publication patterns) to reports of adverse events than to new questions of efficacy. CONCLUSIONS The development of off-label therapies, including the

  19. Effects of BMP-2 and dexamethasone on osteogenic differentiation of rat dental follicle progenitor cells seeded on three-dimensional beta-TCP

    Energy Technology Data Exchange (ETDEWEB)

    Xu Lulu; Jin Zuolin; Duan Yinzhong [Department of Orthodontics, Stomatological College, Fourth Military Medical University, Xi' an 710032 (China); Liu Hongchen; Wang Dongsheng; E Lingling [Department of Stomatology, China PLA General Hospital, Beijing 100853 (China); Xu Lin, E-mail: jinzuolin88@yahoo.com.c, E-mail: duanyinzhong@yahoo.com.c [Department of Stomatology, the First Hospital of PLA, Lanzhou 730000 (China)

    2009-12-15

    The aim of this study was to investigate the effects of BMP-2 and dexamethasone (Dex) on osteogenic differentiation of rat dental follicle progenitor cells (RDFCs) seeded on three-dimensional beta-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial beta-TCP after 1-3, 3-7, 7-14 days of culture were examined respectively. The growth of cells on the scaffolds was observed by scanning electron microscope (SEM) after 3, 7 days of culture and by implanting in the backs of severe combined immunodeficient (SCID) mice for bone regeneration. The third passage RDFCs could be seen adhered, extended and proliferated on the beta-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10{sup -8} M) or/and BMP-2 (100 ng ml{sup -1}) were significantly higher than their existence in the control group. They were the significantly highest among four groups after joint application of BMP-2 and dexamethasone. After 8 weeks of implantation, the percentage of the new bones formed area in the RDFCs+beta-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+beta-TCP+BMP-2 group. In contrast, beta-TCP, RDFCs+beta-TCP+Dex and control constructs lacked new bone formation by histological staining and histomorphometric analysis. The BMP-2+Dex could significantly promote osteogenic differentiation of RDFCs on beta-TCP. beta-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

  20. Effects of BMP-2 and dexamethasone on osteogenic differentiation of rat dental follicle progenitor cells seeded on three-dimensional β-TCP

    International Nuclear Information System (INIS)

    The aim of this study was to investigate the effects of BMP-2 and dexamethasone (Dex) on osteogenic differentiation of rat dental follicle progenitor cells (RDFCs) seeded on three-dimensional β-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial β-TCP after 1-3, 3-7, 7-14 days of culture were examined respectively. The growth of cells on the scaffolds was observed by scanning electron microscope (SEM) after 3, 7 days of culture and by implanting in the backs of severe combined immunodeficient (SCID) mice for bone regeneration. The third passage RDFCs could be seen adhered, extended and proliferated on the β-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10-8 M) or/and BMP-2 (100 ng ml-1) were significantly higher than their existence in the control group. They were the significantly highest among four groups after joint application of BMP-2 and dexamethasone. After 8 weeks of implantation, the percentage of the new bones formed area in the RDFCs+β-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+β-TCP+BMP-2 group. In contrast, β-TCP, RDFCs+β-TCP+Dex and control constructs lacked new bone formation by histological staining and histomorphometric analysis. The BMP-2+Dex could significantly promote osteogenic differentiation of RDFCs on β-TCP. β-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

  1. Diabetes mellitus affects the biomechanical function of the callus and the expression of TGF-beta1 and BMP2 in an early stage of fracture healing

    OpenAIRE

    Xu, M. T.; Sun, S.; Zhang, L.; Xu, F.; Du, S.L.; Zhang, X. D.; Wang, D. W.

    2015-01-01

    Transforming growth factor beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) are important regulators of bone repair and regeneration. In this study, we examined whether TGF-β1 and BMP-2 expressions were delayed during bone healing in type 1 diabetes mellitus. Tibial fractures were created in 95 diabetic and 95 control adult male Wistar rats of 10 weeks of age. At 1, 2, 3, 4, and 5 weeks after fracture induction, five rats were sacrificed from each group. The expressions of TGF-β1 and ...

  2. Regulació de la migració cel·lular induïda per BMP-2

    OpenAIRE

    Gamell Fullà, Cristina

    2009-01-01

    EN CATALÀ :Les proteïnes morfogenètiques òssies (BMPs) són membres de la superfamília del TGF-beta i s'ha demostrat que participen en la determinació i especificació de varis teixits i òrgans durant el desenvolupament dels vertebrats i que regulen la proliferació, l'apoptosi i la diferenciació de múltiples tipus cel·lulars. Les BMPs van ser originàriament identificades per a la seva habilitat d'induir la formació ectòpica d'os i entre ells, BMP-2, -4 and -7 resulten essencials perquè tingui l...

  3. Differential expression of Bmp2, Bmp4 and Bmp3 in embryonic development of mouse anterior and posterior palate

    Institute of Scientific and Technical Information of China (English)

    NIE Xu-guang

    2005-01-01

    Background The palate is differently regulated and developed along the anterior-posterior axis. The Bmp signal pathway plays a crucial role in palatogenesis. Conditioned-inactivation of Bmp type I receptor Alk2 or Alk3 in the neural crest or craniofacial region leads to palatal cleft in mice. However, how different Bmp members are involved in palatogenesis remains to be elucidated. In the present study, mRNA expression patterns of Bmp2, Bmp3 and Bmp4 in the developing anterior and posterior palates were examined and compared, focusing on the fusion stage. Methods To detect the expression of Bmp mRNA, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagital and coronal sections of mice head from E13 to E18. Results The expression of these Bmps were developmentally regulated in the anterior and posterior palates prior to, during and after palatal fusion. During palatal fusion, Bmp4 expression shifted from the anterior to the posterior palate, Bmp2 was highly expressed in both the anterior and posterior palates in this process, whereas Bmp3 was only localized in the posterior palate. They showed generally non-overlapping pattern in their expression domains. Thereafter, their expression was detected in both the anterior and posterior palates regulating osteogenesis and myogenesis respectively. Conclusions Bmp signalling is involved in palatogenesis in multiple stages and has multiple roles in regulating anterior and posterior palatal development. Disturbances of Bmp signalling during palatogenesis might be a possible mechanism of cleft palate.

  4. The origin of bmp16, a novel Bmp2/4 relative, retained in teleost fish genomes

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    Meyer Axel

    2009-12-01

    Full Text Available Abstract Background Whole genome sequences have allowed us to have an overview of the evolution of gene repertoires. The target of the present study, the TGFβ superfamily, contains many genes involved in vertebrate development, and provides an ideal system to explore the relationships between evolution of gene repertoires and that of developmental programs. Results As a result of a bioinformatic survey of sequenced vertebrate genomes, we identified an uncharacterized member of the TGFβ superfamily, designated bmp16, which is confined to teleost fish species. Our molecular phylogenetic study revealed a high affinity of bmp16 to the Bmp2/4 subfamily. Importantly, further analyses based on the maximum-likelihood method unambiguously ruled out the possibility that this teleost-specific gene is a product of teleost-specific genome duplication. This suggests that the absence of a bmp16 ortholog in tetrapods is due to a secondary loss. In situ hybridization showed embryonic expression of the zebrafish bmp16 in the developing swim bladder, heart, tail bud, and ectoderm of pectoral and median fin folds in pharyngula stages, as well as gut-associated expression in 5-day embryos. Conclusion Comparisons of expression patterns revealed (1 the redundancy of bmp16 expression with its homologs in presumably plesiomorphic expression domains, such as the fin fold, heart, and tail bud, which might have permitted its loss in the tetrapod lineage, and (2 the loss of craniofacial expression and gain of swim bladder expression of bmp16 after the gene duplication between Bmp2, -4 and -16. Our findings highlight the importance of documenting secondary changes of gene repertoires and expression patterns in other gene families.

  5. Transforming growth factor β1 inhibits bone morphogenic protein (BMP-2 and BMP-7 signaling via upregulation of Ski-related novel protein N (SnoN: possible mechanism for the failure of BMP therapy?

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    Ehnert Sabrina

    2012-09-01

    Full Text Available Abstract Background Bone morphogenic proteins (BMPs play a key role in bone formation. Consequently, it was expected that topical application of recombinant human (rhBMP-2 and rhBMP-7 would improve the healing of complex fractures. However, up to 36% of fracture patients do not respond to this therapy. There are hints that a systemic increase in transforming growth factor β1 (TGFβ1 interferes with beneficial BMP effects. Therefore, in the present work we investigated the influence of rhTGFβ1 on rhBMP signaling in primary human osteoblasts, with the aim of more specifically delineating the underlying regulatory mechanisms. Methods BMP signaling was detected by adenoviral Smad-binding-element-reporter assays. Gene expression was determined by reverse transcription polymerase chain reaction (RT-PCR and confirmed at the protein level by western blot. Histone deacetylase (HDAC activity was determined using a test kit. Data sets were compared by one-way analysis of variance. Results Our findings showed that Smad1/5/8-mediated rhBMP-2 and rhBMP-7 signaling is completely blocked by rhTGFβ1. We then investigated expression levels of genes involved in BMP signaling and regulation (for example, Smad1/5/8, TGFβ receptors type I and II, noggin, sclerostin, BMP and activin receptor membrane bound inhibitor (BAMBI, v-ski sarcoma viral oncogene homolog (Ski, Ski-related novel protein N (SnoN and Smad ubiquitination regulatory factors (Smurfs and confirmed the expression of regulated genes at the protein level. Smad7 and SnoN were significantly induced by rhTGFβ1 treatment while expression of Smad1, Smad6, TGFβRII and activin receptor-like kinase 1 (Alk1 was reduced. Elevated SnoN expression was accompanied by increased HDAC activity. Addition of an HDAC inhibitor, namely valproic acid, fully abolished the inhibitory effect of rhTGFβ1 on rhBMP-2 and rhBMP-7 signaling. Conclusions rhTGFβ1 effectively blocks rhBMP signaling in osteoblasts. As possible

  6. Effects of rhBMP-2 on Sandblasted and Acid Etched Titanium Implant Surfaces on Bone Regeneration and Osseointegration: Spilt-Mouth Designed Pilot Study

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    Nam-Ho Kim

    2015-01-01

    Full Text Available This study was conducted to evaluate effects of rhBMP-2 applied at different concentrations to sandblasted and acid etched (SLA implants on osseointegration and bone regeneration in a bone defect of beagle dogs as pilot study using split-mouth design. Methods. For experimental groups, SLA implants were coated with different concentrations of rhBMP-2 (0.1, 0.5, and 1 mg/mL. After assessment of surface characteristics and rhBMP-2 releasing profile, the experimental groups and untreated control groups (n = 6 in each group, two animals in each group were placed in split-mouth designed animal models with buccal open defect. At 8 weeks after implant placement, implant stability quotients (ISQ values were recorded and vertical bone height (VBH, mm, bone-to-implant contact ratio (BIC, %, and bone volume (BV, % in the upper 3 mm defect areas were measured. Results. The ISQ values were highest in the 1.0 group. Mean values of VBH (mm, BIC (%, and BV (% were greater in the 0.5 mg/mL and 1.0 mg/mL groups than those in 0.1 and control groups in buccal defect areas. Conclusion. In the open defect area surrounding the SLA implant, coating with 0.5 and 1.0 mg/mL concentrations of rhBMP-2 was more effective, compared with untreated group, in promoting bone regeneration and osseointegration.

  7. Improved Bone Formation in Osteoporotic Rabbits with the Bone Morphogenetic Protein-2 (rhBMP-2 Coated Titanium Screws Which Were Coated By Using Plasma Polymerization Technique

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    Salih Gulsen

    2014-06-01

    Full Text Available Delaying of bone fusion in osteoporotic patients underwent spinal stabilization surgery leads to screw loosening, and this causes pseudoarticulation, mobility and fibrosis at vertebral segments. To prevent these complications, the screws coated with recombinant bone morphogenetic protein-2 (rhBMP-2 could be used. To verify this hypothesis, we coated 5 Titanium screws with rhBMP-2 using plasma polymerization method, and also used 10 uncoated screws for making comparison between coated and uncoated screws in different groups. And 15 skeletally mature white New Zealand female rabbits were assigned into three different groups: Group 1(N = 5: No osteoporosis induction and insertion of uncoated Titanium screw into right sacrum of each rabbit in group 1; group 2 (N = 5: Osteoporosis induction and insertion of uncoated Titanium screw into right sacrum of each rabbit in group 2; group 3 (N = 5 rhBMP-2 coated Titanium screw inserted into right sacrum of each rabbit in group 3. In summary, using of these coated screws provides new bone formation, but causes less fibrosis and less inflammation than uncoated screws at the interface between the coated screw and bone. Then the plasma polymerization technique provides controlled releasing of rhBMP-2 from the screw to the bone tissue in osteoporotic rabbits.

  8. Intestinal Mucosal Barrier Is Injured by BMP2/4 via Activation of NF-κB Signals after Ischemic Reperfusion

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    Kang Chen

    2014-01-01

    Full Text Available Intestinal ischemic reperfusion (I/R can cause dysfunction of the intestinal mucosal barrier; however, the mechanism of the intestinal mucosal barrier dysfunction caused by I/R remains unclear. In this study, using intestinal epithelial cells under anaerobic cultivation and an in vivo rat intestinal I/R model, we found that hypoxia and I/R increased the expression of BMP2/4 and upregulated BMP type Ia receptor and BMP type II receptor expression. We also found that exogenous BMP2/4 can activate the ERK and AKT signaling pathways in rat small intestine (IEC-6 cells, thereby activating NF-κB signaling, which leads to increased levels of inflammatory factors, such as TNF-α and IL-6. Furthermore, recombinant BMP2/4 decreased the expression of the tight junction protein occludin via the activation of the NF-κB pathway; these effects were abolished by treatment with the BMP-specific antagonist noggin or the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC. All these factors can destroy the intestinal mucosal barrier, thereby leading to weaker barrier function. On the basis of these data, we conclude that BMP2/4 may act as the pathogenic basis for intestinal mucosal barrier dysfunction when the intestines suffer an I/R injury. Our results provide background for the development pharmacologic interventions in the management of I/R injury.

  9. Effects of rhBMP-2 on Sandblasted and Acid Etched Titanium Implant Surfaces on Bone Regeneration and Osseointegration: Spilt-Mouth Designed Pilot Study

    Science.gov (United States)

    Kim, Nam-Ho; Lee, So-Hyoun; Ryu, Jae-Jun; Choi, Kyung-Hee; Huh, Jung-Bo

    2015-01-01

    This study was conducted to evaluate effects of rhBMP-2 applied at different concentrations to sandblasted and acid etched (SLA) implants on osseointegration and bone regeneration in a bone defect of beagle dogs as pilot study using split-mouth design. Methods. For experimental groups, SLA implants were coated with different concentrations of rhBMP-2 (0.1, 0.5, and 1 mg/mL). After assessment of surface characteristics and rhBMP-2 releasing profile, the experimental groups and untreated control groups (n = 6 in each group, two animals in each group) were placed in split-mouth designed animal models with buccal open defect. At 8 weeks after implant placement, implant stability quotients (ISQ) values were recorded and vertical bone height (VBH, mm), bone-to-implant contact ratio (BIC, %), and bone volume (BV, %) in the upper 3 mm defect areas were measured. Results. The ISQ values were highest in the 1.0 group. Mean values of VBH (mm), BIC (%), and BV (%) were greater in the 0.5 mg/mL and 1.0 mg/mL groups than those in 0.1 and control groups in buccal defect areas. Conclusion. In the open defect area surrounding the SLA implant, coating with 0.5 and 1.0 mg/mL concentrations of rhBMP-2 was more effective, compared with untreated group, in promoting bone regeneration and osseointegration. PMID:26504807

  10. Effects of local delivery of BMP2, zoledronate and their combination on bone microarchitecture, biomechanics and bone turnover in osteoporotic rabbits.

    Science.gov (United States)

    Jing, Da; Hao, Xuguang; Xu, Fang; Liu, Jian; Xu, Fei; Luo, Erping; Meng, Guolin

    2016-01-01

    The hip fracture is one major clinical challenge associated with osteoporosis, resulting in heavy socioeconomic burdens and high mortality. Systemic therapies of anti-osteoporosis drugs are expensive, time-consuming and also evoke substantial side effects, which fails to provide early protection from fractures. Accumulating evidence demonstrates the high bioavailability and therapeutic efficacy of local drug delivery in accelerating facture healing and bone defect repair. This study aims at investigating the effects of local delivery of BMP2 and zoledronate (two promising anabolic/anti-catobolic reagents) encapsulated by fibrin sealants into femoral necks on regulating bone quality and remodeling in osteoporotic rabbits subjected to combined ovariectomy and glucocorticoid injection. We show that 6-week BMP2 delivery exhibited more prominent effect on mitigating trabecular bone microarchitecture deterioration and mechanical strength reduction of femoral necks than local zoledronate treatment. BMP2 plus zoledronate showed more significant improvement of bone microstructure, mechanical strength and bone formation rate at 12 weeks post injection than single BMP2 or zoledronate delivery via μCT, biomechanical, histomorphometric and serum biochemical analyses. This study enriches our knowledge for understanding the availability of local drug delivery for improving bone quantity and quality, which may lead to earlier, safer and more efficient protection from osteoporosis-induced fractures in clinics. PMID:27329730

  11. Mesoporous calcium–silicon xerogels with mesopore size and pore volume influence hMSC behaviors by load and sustained release of rhBMP-2

    Directory of Open Access Journals (Sweden)

    Song W

    2015-03-01

    Full Text Available Wenhua Song,1,* Xiangde Li,1,* Jun Qian,1 Guoyu Lv,2 Yonggang Yan,2 Jiacan Su,3 Jie Wei1 1Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai, People’s Republic of China; 2College of Physical Science and Technology, Sichuan University, Chengdu, People’s Republic of China; 3Changhai Hospital, Second Military Medical University, Shanghai, People’s Republic of China *These authors contributed equally to this paper Abstract: Mesoporous calcium–silicon xerogels with a pore size of 15 nm (MCS-15 and pore volume of 1.43 cm3/g were synthesized by using 1,3,5-mesitylene (TMB as the pore-expanding agent. The MCS-15 exhibited good degradability with the weight loss of 50 wt% after soaking in Tris-HCl solution for 56 days, which was higher than the 30 wt% loss shown by mesoporous calcium–silicon xerogels with a pore size of 4 nm (MCS-4. The pore size and pore volume of MCS-15 had significant influences on load and release of recombinant human bone morphogenetic protein-2 (rhBMP-2. The MCS-15 had a higher capacity to encapsulate a large amount of rhBMP-2; it could adsorb 45 mg/g of rhBMP-2 in phosphate-buffered saline after 24 hours, which was more than twice that with MCS-4 (20 mg/g. Moreover, the MCS-15 system exhibited sustained release of rhBMP-2 as compared with MCS-4 system (showing a burst release. The MCS-15/rhBMP-2 system could promote the proliferation and differentiation of human mesenchymal stem cells, showing good cytocompatibility and bioactivity. The results indicated that MCS-15, with larger mesopore size and higher pore volume, might be a promising carrier for loading and sustained release of rhBMP-2, which could be used as bone repair material with built-in osteoinduction function in bone reconstruction. Keywords: mesoporous calcium–silicon xerogels, pore size, pore volume, load-release, rhBMP-2

  12. Dual Delivery of BMP-2 and bFGF from a New Nano-Composite Scaffold, Loaded with Vascular Stents for Large-Size Mandibular Defect Regeneration

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    Hang Zhao

    2013-06-01

    Full Text Available The aim of this study was to investigate the feasibility and advantages of the dual delivery of bone morphogenetic protein-2 (BMP-2 and basic fibroblast growth factor (bFGF from nano-composite scaffolds (PLGA/PCL/nHA loaded with vascular stents (PLCL/Col/nHA for large bone defect regeneration in rabbit mandibles. Thirty-six large bone defects were repaired in rabbits using engineering bone composed of allogeneic bone marrow mesenchymal stem cells (BMSCs, bFGF, BMP-2 and scaffolds composed of PLGA/PCL/nHA loaded with PLCL/Col/nHA. The experiments were divided into six groups: BMSCs/bFGF/BMP-2/scaffold, BMSCs/BMP-2/scaffold, BMSCs/bFGF/scaffold, BMSCs/scaffold, scaffold alone and no treatment. Sodium alginate hydrogel was used as the carrier for BMP-2 and bFGF and its features, including gelling, degradation and controlled release properties, was detected by the determination of gelation and degradation time coupled with a controlled release study of bovine serum albumin (BSA. AlamarBlue assay and alkaline phosphatase (ALP activity were used to evaluate the proliferation and osteogenic differentiation of BMSCs in different groups. X-ray and histological examinations of the samples were performed after 4 and 12 weeks post-implantation to clarify new bone formation in the mandible defects. The results verified that the use of sodium alginate hydrogel as a controlled release carrier has good sustained release ability, and the combined application of bFGF and BMP-2 could significantly promote the proliferation and osteogenic differentiation of BMSCs (p < 0.05 or p < 0.01. In addition, X-ray and histological examinations of the samples exhibited that the dual release group had significantly higher bone formation than the other groups. The above results indicate that the delivery of both growth factors could enhance new bone formation and vascularization compared with delivery of BMP-2 or bFGF alone, and may supply a promising way of repairing large

  13. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Si, Lina; Shi, Jin; Gao, Wenqun [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Zheng, Min [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Liu, Lingjuan; Zhu, Jing [Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Tian, Jie, E-mail: jietian@cqmu.edu.cn [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China)

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  14. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    International Nuclear Information System (INIS)

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  15. Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats.

    Science.gov (United States)

    Schliephake, Henning; Weich, Herbert A; Dullin, Christian; Gruber, Rudolf; Frahse, Sarah

    2008-01-01

    The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 48 microg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 microg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation. PMID:17936352

  16. Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2

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    Vincent Thea

    2007-12-01

    Full Text Available Abstract Background Articular cartilage has little capacity for repair in vivo, however, a small number of studies have shown that, in vitro, a damage/repair response can be induced. Recent work by our group has shown that cartilage can respond to single impact load and culture by producing repair cells on the articular surface. The purpose of this study was to identify whether chondrocyte outgrowth into a 3D scaffold could be observed following single impact load and culture. The effect of bone morphogenic-2 (BMP-2 on this process was investigated. Methods Cartilage explants were single impact loaded, placed within a scaffold and cultured for up to 20 days +/- BMP-2. Cell numbers in the scaffold, on and extruding from the articular surface were quantified and the immunohistochemistry used to identify the cellular phenotype. Results Following single impact load and culture, chondrocytes were observed in a 3D gelatin scaffold under all culture conditions. Chondrocytes were also observed on the articular surface of the cartilage and extruding out of the parent cartilage and on to the cartilage surface. BMP-2 was demonstrated to quantitatively inhibit these events. Conclusion These studies demonstrate that articular chondrocytes can be stimulated to migrate out of parent cartilage following single impact load and culture. The addition of BMP-2 to the culture medium quantitatively reduced the repair response. It may be that the inhibitory effect of BMP-2 in this experimental model provides a clue to the apparent inability of articular cartilage to heal itself following damage in vivo.

  17. Sustained release of VH and rhBMP-2 from nanoporous magnesium–zinc–silicon xerogels for osteomyelitis treatment and bone repair

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    Li FQ

    2015-06-01

    Full Text Available Fengqian Li,1,* Wen Wu,2,* Li Xiang,1 Gan Weng,1 Hua Hong,3 Hong Jiang,4 Jun Qian31Department of Pharmacy, Shanghai Xuhui Dahua Hospital, 2Department of Orthopaedics, Ninth People’s Hospital, School of Medicine, Shanghai Jiaotong University, 3Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, 4School of Materials Science and Engineering, University of Shanghai for Science and Technology, Shanghai, People’s Republic of China*Co-first authors contributed equally to this workAbstract: Nanoporous magnesium–zinc–silicon (n-MZS xerogels with a pore size of ~4 nm, a surface area of 718 cm2/g, and a pore volume of 1.24 cm3/g were synthesized by a sol–gel method. The n-MZS xerogels had high capacity to load vancomycin hydrochloride (VH and human bone morphogenetic protein-2 (rhBMP-2, after soaking in phosphate buffered saline (PBS for 24 hours (1.5 and 0.8 mg/g, respectively. Moreover, the n-MZS xerogels exhibited the sustained release of VH and rhBMP-2 as compared with magnesium–zinc–silicon (MZS xerogels without nanopores (showing a burst release. The VH/rhBMP-2/n-MZS system not only exhibited a good antibacterial property but also promoted the MG63 cell proliferation and differentiation demonstrating good bactericidal activity and cytocompatibility. The results suggested that n-MZS with larger surface area and high pore volume might be a promising carrier for loading and sustained release of VH and rhBMP-2. Hence, the VH/rhBMP-2/n-MZS system might be one of the promising biomaterials for osteomyelitis treatment and bone repair.Keywords: nanoporous xerogels, sustained release, drugs, osteomyelitis, bone regeneration, bactericidal activity, cytocompatibility

  18. BMP-2基因修饰自体BMSCs移植促进兔下颌骨牵张成骨新骨形成的实验研究%EXPERIMENTAL STUDY ON TRANSPLANTATION OF BMP-2 GENE TRANSFECTED AUTOGENOUS BONE MESENCHYMAL STEM CELLS FOR PROMOTING BONE REGENERATION IN RABBIT MANDIBULAR DISTRACTION OSTEOGENESIS

    Institute of Scientific and Technical Information of China (English)

    黄旋平; 周诺; 江献芳; 杨媛媛; 李华; 谢庆条

    2012-01-01

    目的:检测骨形态发生蛋白-2(BMP-2)基因mRNA及其蛋白的表达,探讨BMP-2基因修饰自体骨髓间充质干细胞(BMSCs)移植对兔下颌骨牵张成骨新骨形成的促进作用.方法:取新西兰白兔36只随机分为3组,每组12只.建立牵张成骨动物模型,在固定期第2天,实验组于牵张间隙注射200 μL的BMP-2基因修饰的自体BMSCs液;对照组注射等量自体BMSCs液;空白组注射等量生理盐水.分别于固定2,6周通过逆转录多聚酶链式反应(RT-PCR)、免疫组化等手段检测BMP-2基因mRNA及其蛋白的表达情况.结果:实验组牵张间隙新生骨组织均可见BMP-2基因mRNA和其蛋白强阳性表达.结论:BMP-2基因修饰的自体BMSCs移植能有效促进兔下颌骨牵张成骨新骨形成.%Objective:To examine the expressions of both bone morphogenetic protein-2 (BMP-2) gene mR-NA and related proteins and investigate the promotive effect of transplantation of BMP-2 gene transfected autogenous bone mesenchymal stem cells on bone regeneration in rabbit mandibular distraction osteogene-sis. Methods: Thirty-six New Zealand's white rabbits were randomly divided into three groups with twelve in each. All objects -were prepared into distraction osteogenesis surgical model on right mandibles. On the 2nd day of consolidation, experimental, control, and blank groups -were injected -with the same amount of 200 juL of the solution with BMP-2 gene transfected autogenous bone mesenchymal stem cells, the solution with autogenous bone mesenchymal stem cells, and physiological saline at distraction gap, respectively. The expressions of BMP-2 mRNA and related proteins -were examined by RT-PCR and immunohistochem-istry at the ends of the 2nd and 6th -week consolidations, respectively. Results: Strongly positive expression of both BMP-2 gene mRNA and related proteins were confirmed on regenerated bone in distraction gap. Conclusion: The transplantation of BMP-2 gene transfected autogenous bone

  19. Wnt1 and BMP2: two factors recruiting multipotent neural crest progenitors isolated from adult bone marrow.

    Science.gov (United States)

    Glejzer, A; Laudet, E; Leprince, P; Hennuy, B; Poulet, C; Shakhova, O; Sommer, L; Rogister, B; Wislet-Gendebien, S

    2011-06-01

    Recent studies have shown that neural crest-derived progenitor cells can be found in diverse mammalian tissues including tissues that were not previously shown to contain neural crest derivatives, such as bone marrow. The identification of those "new" neural crest-derived progenitor cells opens new strategies for developing autologous cell replacement therapies in regenerative medicine. However, their potential use is still a challenge as only few neural crest-derived progenitor cells were found in those new accessible locations. In this study, we developed a protocol, based on wnt1 and BMP2 effects, to enrich neural crest-derived cells from adult bone marrow. Those two factors are known to maintain and stimulate the proliferation of embryonic neural crest stem cells, however, their effects have never been characterized on neural crest cells isolated from adult tissues. Using multiple strategies from microarray to 2D-DIGE proteomic analyses, we characterized those recruited neural crest-derived cells, defining their identity and their differentiating abilities. PMID:20976520

  20. Evaluation of a Novel HA/ZrO2-Based Porous Bioceramic Artificial Vertebral Body Combined with a rhBMP-2/Chitosan Slow-Release Hydrogel

    Science.gov (United States)

    Shi, Yihui; Quan, Renfu; Xie, Shangju; Li, Qiang; Cao, Guoping; Zhuang, Wei; Zhang, Liang; Shao, Rongxue; Yang, Disheng

    2016-01-01

    A new HA/ZrO2-based porous bioceramic artificial vertebral body (AVB), carried a recombinant human bone morphogenetic protein-2 (rhBMP-2)/chitosan slow-release hydrogel was prepared to repair vertebral bone defect in beagles. An ionic cross-linking was used to prepare the chitosan hydrogel (CS gel) as the rhBMP-2 slow-release carrier. The vertebral body defects were implanted with the rhBMP-2-loaded AVB in group A, or a non-drug-loaded AVB in group B, or autologous iliac in group C. The encapsulation rate of rhBMP-2 in rhBMP-2-loaded CS gel was 91.88±1.53%, with a drug load of 39.84±2.34 ng/mg. At 6, 12, 24 weeks postoperatively, radiography showed that the bone calluses gradually increased with time in group A, where the artificial vertebral body had completely fused with host-bone at 24 weeks after surgery. In group C, an apparent bone remodeling was occurred in the early stages, and the graft-bone and host-bone had also fused completely at 24 weeks postoperatively. In group B, fusion occurred less than in groups A and C. At 24 weeks after surgery, micro-computed tomography (Micro-CT) revealed that the volume of newly-formed bone in group A was significantly more than in group B (p<0.05). At 24 weeks after surgery, ultra-compressive strengths of the operated segments were 14.03±1.66 MPa in group A, 8.62±1.24 MPa in group B, and 13.78±1.43 MPa in group C. Groups A and C were both significantly higher than group B (p < 0.05). At 24 weeks postoperatively, the hard tissue sections showed that the AVB of group A had tightly fused with host bone, and that pores of the AVB had been filled with abundant nearly mature bone, and that the new bone structured similarly to a trabecular framework, which was similar to that in group C. In contrast, implant fusion of the AVB in group B was not as apparent as group A. In conclusion, the novel HA/ZrO2-based porous bioceramic AVB carried the rhBMP-2-loaded CS gel can promote the repair of bony defect, and induce bone tissue to

  1. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy.

    Directory of Open Access Journals (Sweden)

    Nahoko Shintani

    Full Text Available BACKGROUND: Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2 induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2 and transforming growth factor beta 1 (TGF-ß1 were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml for 4 (or 6 weeks. FGF-2 (10 ng/ml or TGF-ß1 (10 ng/ml was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2, but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. CONCLUSIONS/SIGNIFICANCE: TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of

  2. Multifunctional Thin Film Biomatrice Biosensor in a Degradable Scaffold Containing Bone Morphogenetic Protein-2 (BMP-2) for Controlled Release in Skeletal Tissue Engineering

    Science.gov (United States)

    McDaniel, Harvey; Lomax, Linda

    2001-03-01

    Bone morphonogenetic proteins (BMP-2) have been under investigation for three decades. Deminerialized bone and extracts of deminerialized bone are o steoinductive with a temporal sequence of bone induction. Native and recombi nant BMP's have shown the ability, thru growth and differentiative factors t o induce de novo bone formation both invitro and invivo. Their principle fun ction is to induce transformation of undifferentiated mesenchymal cells into osteoblasts. Native and recombinant BMP's, when purified and used without carrier disp erse after implantation and exert no effect on bone induction. The delivery system provides the missing component to successsfully applying osteogenic p roteins for clinical need. Biological and physio-chemical properties are str ictly adhered tofor a successful delivery system. The BMP delivery system ca rrier for osteo inductive payload provided; 1)non tumorgenic genecity, 2) no n immunogenecity, 3) water insoluble, 4) biosorbability with predictable enz ymatic degradation, and 5) an optimized surface for compatibility, cell migr ation and attachment with a negative surface change that encouraged target c ell attachment. Being a controlled Release System, it binded the proteins wi th predictible BMP released kinetics. Porosity with interconnecting voids pr otected the BMP from noon specific proteolysis and promoted rapid vascular a nd mesenchymal invasion. Far wide ranging clinical applications of mechanica l and biofunctional requirements were met with the BMP delivery system. Cohe sion and malleability were reqiured forcontour augmentation, and reconstruct ion of the discontinuity defects, prevented dislocation and retained the sha pe and bone replaced the system. Biological systems have elastic activity associated with them. The activi ty was current associated with a time dependant biological/biochemical react ion (enzymic activity). Bioelectric phoenomena associated with charged molec ules in a biologic structure caused

  3. Efficacy of rhBMP-2 loaded PCL/PLGA/β-TCP guided bone regeneration membrane fabricated by 3D printing technology for reconstruction of calvaria defects in rabbit

    International Nuclear Information System (INIS)

    We successfully fabricated a three-dimensional (3D) printing-based PCL/PLGA/β-TCP guided bone regeneration (GBR) membrane that slowly released rhBMP-2. To impregnate the GBR membrane with intact rhBMP-2, collagen solution encapsulating rhBMP-2 (5 µg ml−1) was infused into pores of a PCL/PLGA/β-TCP membrane constructed using a 3D printing system with four dispensing heads. In a release profile test, sustained release of rhBMP-2 was observed for up to 28 d. To investigate the efficacy of the GBR membrane on bone regeneration, PCL/PLGA/β-TCP membranes with or without rhBMP-2 were implanted in an 8 mm calvaria defect of rabbits. Bone formation was evaluated at weeks 4 and 8 histologically and histomorphometrically. A space making ability of the GBR membrane was successfully maintained in both groups, and significantly more new bone was formed at post-implantation weeks 4 and 8 by rhBMP-2 loaded GBR membranes. Interestingly, implantation with rhBMP-2 loaded GBR membranes led to almost entire healing of calvaria defects within 8 weeks. (paper)

  4. Efficacy of rhBMP-2 loaded PCL/PLGA/β-TCP guided bone regeneration membrane fabricated by 3D printing technology for reconstruction of calvaria defects in rabbit.

    Science.gov (United States)

    Shim, Jin-Hyung; Yoon, Min-Chul; Jeong, Chang-Mo; Jang, Jinah; Jeong, Sung-In; Cho, Dong-Woo; Huh, Jung-Bo

    2014-11-10

    We successfully fabricated a three-dimensional (3D) printing-based PCL/PLGA/β-TCP guided bone regeneration (GBR) membrane that slowly released rhBMP-2. To impregnate the GBR membrane with intact rhBMP-2, collagen solution encapsulating rhBMP-2 (5 µg ml(-1)) was infused into pores of a PCL/PLGA/β-TCP membrane constructed using a 3D printing system with four dispensing heads. In a release profile test, sustained release of rhBMP-2 was observed for up to 28 d. To investigate the efficacy of the GBR membrane on bone regeneration, PCL/PLGA/β-TCP membranes with or without rhBMP-2 were implanted in an 8 mm calvaria defect of rabbits. Bone formation was evaluated at weeks 4 and 8 histologically and histomorphometrically. A space making ability of the GBR membrane was successfully maintained in both groups, and significantly more new bone was formed at post-implantation weeks 4 and 8 by rhBMP-2 loaded GBR membranes. Interestingly, implantation with rhBMP-2 loaded GBR membranes led to almost entire healing of calvaria defects within 8 weeks.

  5. The effects of substrate-streching strain on the BMP-2 mRNA expression in three kinds of mouse cell lines%基底拉伸应变对小鼠三种骨组织细胞BMP-2 mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    闫玉仙; 宋梅; 郭春; 郭勇; 宫元伟; 李瑞欣; 张西正

    2010-01-01

    目的 研究基底拉伸应变对小鼠成骨细胞系MC3T3-E1、破骨细胞系RAW264.7及骨细胞MLO-Y4三种细胞BMP-2 mRNA表达的影响.方法 三种细胞随机分为0 με、1 000 με、1 500 με、2 000 με、2 500 με和5 000 με组,最佳拉伸时间和周期为1次/d,每次1 h,连续3 d,频率为0.5 Hz.采用卫生装备研究所自行设计研制的四点弯曲装置对小鼠三种细胞进行拉伸加载.采用RT-PCR技术分别研究不同应变对小鼠三种细胞BMP-2 mRNA表达.结果 MC3T3-E1细胞RT-PCR结果显示:1 500 με、2 000 με组和2 500 με组与0 με组相比BMP-2 mRNA表达显著增强(P<0.01);5 000 με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);RAW264.7细胞RT-PCR结果显示:1 500 με、2 000 με组和2 500 με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);5 000με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);MLO-Y4细胞BMP2基因表达结果与MC3T3-E1一致.结论 ①BMP-2在成骨细胞系MC3T3-E1、破骨细胞系RAW-264.7及骨细胞系MLO-Y4三种细胞中均有表达;②1 500 με、2 000 με、2 500 με三种生理剂量的拉伸应变可以显著增加MC3T3-E1、MLO-Y4 细胞BMP-2的表达,并呈剂量依赖性,超生理剂量5 000 με可以显著降低MC3T3-E1、MLO-Y4细胞BMP-2的表达;③相同的力学拉伸作用条件下,BMP-2在RAW-264.7细胞中表达与MC3T3-E1、MLO-Y4细胞的表达趋势相反.

  6. Posterior tooth replacement with dental implants in sites augmented with rhBMP-2 at time of extraction--a case series.

    Science.gov (United States)

    Levin, Barry P; Tawil, Peter

    2012-02-01

    This case series demonstrates seven molar-site implants placed in six consecutively treated patients. All sites were augmented with rhBMP-2 (1.50 mg/cc)/ACS (recombinant human Bone Morphogenetic Protein-2/Absorbable Collagen Sponge) at extraction to regenerate bone-facilitating implant placement. In four patients, osteotomies were initiated with trephines to evaluate qualitatively for native bone and for the absence of residual ACS. All sites facilitated implant placement after augmentation. All seven implants achieved primary stabilization and were functionally loaded. No implants were lost or developed complications. It can be concluded that augmenting molar extraction sockets with rhBMP-2/ACS can allow standard implant placement in the posterior dentition that is capable of withstanding a functional load.

  7. Self-assembled Biodegradable Nanoparticles and Polysaccharides as Biomimetic ECM Nanostructures for the Synergistic effect of RGD and BMP-2 on Bone Formation.

    Science.gov (United States)

    Wang, Zhenming; Dong, Li; Han, Lu; Wang, Kefeng; Lu, Xiong; Fang, Liming; Qu, Shuxin; Chan, Chun Wai

    2016-01-01

    Producing biomimetic extracellular matrix (ECM) is an effective approach to improve biocompatibility of medical devices. In this study, biomimetic ECM nanostructures are constructed through layer-by-layer self-assembling positively charged chitosan (Chi), negatively charged oxidized sodium alginate (OAlg), and positively charged bovine serum albumin (BSA)-based nanoparticles. The BSA-based nanoparticles in the self-assembled films not only result in porous nanostructures similar to natural ECM, but also preserve the activity and realize the sustained release of Bone morphogenetic protein-2 (BMP-2). The results of bone marrow stem cells (BMSCs) culture demonstrate that the penta-peptide glycine-arginine-glycine-aspartate-serine (GRGDS) grafted Chi/OAlg films favor cell adhesion and proliferation. GRGDS and BMP-2 in biomimetic ECM nanostructures synergistically promote BMSC functions and new bone formation. The RGD and BMP incorporated biomimetic ECM coatings could be applied on a variety of biomedical devices to improve the bioactivity and biocompatibility. PMID:27121121

  8. Treatment of critically sized femoral defects with recombinant BMP-2 delivered by a modified mPEG-PLGA biodegradable thermosensitive hydrogel

    OpenAIRE

    Peng, Kuo-Ti; Hsieh, Meng-Yow; Lin, Carl T.; Chen, Chin-Fu; Lee, Mel S.; Huang, Yi-You; CHANG, PEY-JIUM

    2016-01-01

    Background Reconstruction of a segmental fracture with massive bone loss is still a challenge for orthopaedic surgeons. The aim of our study was to develop a suitable biodegradable thermosensitive hydrogel system as a carrier for bone morphogenetic protein (BMP)-2 delivery in the treatment of critical-sized femoral defects. Methods A block copolymer composed of monomethoxypoly(ethylene glycol) (mPEG), poly(lactic-co-glycolic acid) (PLGA) and 2, 2’-Bis (2-oxazolin) (Box) was synthesized by rin...

  9. Preparation of porous bioceramics using reverse thermo-responsive hydrogels in combination with rhBMP-2 carriers: in vitro and in vivo evaluation.

    Science.gov (United States)

    Fu, Yin-Chih; Chen, Chung-Hwan; Wang, Chau-Zen; Wang, Yan-Hsiung; Chang, Je-Ken; Wang, Gwo-Jaw; Ho, Mei-Ling; Wang, Chih-Kuang

    2013-11-01

    Porous biphasic calcium phosphates (BCP) were fabricated using reverse thermo-responsive hydrogels with hydroxyapatite (HAp) and β-tricalcium (β-TCP) powder and planetary centrifugal mixer. This hydrogel mixture slurry will shrink and compress the HAp powder during the sintering process. The porous bioceramics are expected to have good mechanical properties after sintering at 1200°C. Reverse thermo-responsive hydrogels of poly[(N-isopropylacrylamide)-co-(methacrylic acid)] p(NiPAAm-MAA) were synthesized by free-radical cross-linking copolymerization, and their chemical properties were evaluated by nuclear magnetic resonance spectroscopy, infrared spectroscopy, and electrospray-ionization mass spectrometry. The lower critical solution temperature (LCST) of the hydrogel was determined using turbidity measurements. A thermogravimetric analysis was used to examine the thermal properties. The porous bioceramic properties were analyzed by X-ray diffraction, scanning electron microscopy, bulk density, compressive strength testing and cytotoxicity. The compressive strength and average porosity of the porous bioceramics were examined at approximately 6.8MPa and 66% under 10wt% p(NiPAAm-MAA)=99:1 condition. The ratio of HAp/β-TCP can adjust two different compositional behaviors during the 1200°C sintering process without resulting in cell toxicity. The (rhBMP-2)-HAp-PLGA carriers were fabricated as in our previous study of the double emulsion and drop-coating technique. Results of animal study included histological micrographs of the 1-mm defect in the femurs, with the rhBMP-2 carrier group, the bioceramic spacer group and the bioceramic spacer with rhBMP-2 carriers group showing better callus formation around the femur defect site than the control group. The optimal dual effects of the bone growth factors from osteoconductive bioceramics and osteoinductive rhBMP-2 carriers produced better bone formation. PMID:23880039

  10. The Value of SPECT/CT in Monitoring Prefabricated Tissue-Engineered Bone and Orthotopic rhBMP-2 Implants for Mandibular Reconstruction.

    Directory of Open Access Journals (Sweden)

    Miao Zhou

    Full Text Available Bone tissue engineering shows good prospects for mandibular reconstruction. In recent studies, prefabricated tissue-engineered bone (PTEB by recombinant human bone morphogenetic proteins (rhBMPs applied in vivo has found to be an effective alternative for autologous bone grafts. However, the optimal time to transfer PTEB for mandibular reconstruction is still not elucidated. Thus, here in an animal experiment of rhesus monkey, the suitable transferring time for PTEB to reconstruct mandibular defects was evaluated by 99mTc-MDP SPECT/CT, and its value in monitoring orthotopic rhBMP-2 implants for mandibular reconstruction was also evaluated. The result of SPECT/CT showed higher 99mTc-MDP uptake, indicating osteoinductivity, in rhBMP-2 incorporated demineralized freeze-dried bone allograft (DFDBA and coralline hydroxyapatite (CHA implants than those without BMP stimulation. 99mTc-MDP uptake of rhBMP-2 implant peaked at 8 weeks following implantation while CT showed the density of these implants increased after 13 weeks' prefabrication. Histology confirmed that mandibular defects were repaired successfully with PTEB or orthotopically rhBMP-2 incorporated CHA implants, in accordance with SPECT/CT findings. Collectively, data shows 99mTc-MDP SPECT/CT is a sensitive and noninvasive tool to monitor osteoinductivity and bone regeneration of PTEB and orthotopic implants. The PTEB achieved peak osteoinductivity and bone density at 8 to 13 weeks following ectopic implantation, which would serve as a recommendable time frame for its transfer to mandibular reconstruction.

  11. Effect of recombinant human bone morphogenetic protein 2/polylactide-co-glycolic acid (rhBMP-2/PLGA) with core decompression on repair of rabbit femoral head necrosis

    Institute of Scientific and Technical Information of China (English)

    Zhao-Xun; Pan; Hong-Xin; Zhang; Ye-Xin; Wang; Long-Di; Zhai; Wei; Du

    2014-01-01

    Objective:To observe the effect of recombinant human bone morphogenetic protein 2/polylactide-co-glycolic acid(rhBMP-2/PLGA) with core decompression on repair of rabbit femoral head necrosis.Methods:Bilateral femoral head necrosis models of rabbit were established by steroid injection.A total of 48 rabbits(96 femoral head necrosis) were randomly divided into 4groups:Group A,control group with12 rabbits,24 femoral head necrosis;Group B,treated with rhBMP-2/PLCA implantation after core depression,with 12 rabbits,24 femoral head necrosis;Group C,treated with rhBMP-2 implantation after core depression,with 12 rabbits,24 femoral head necrosis;Croup D treated with core depression group without implantation,with 12 rabbits,24 femoral head necrosis.All animals were sacrificed after 12 weeks.The ability of repairing bone defect was evaluated by X-ray radiograph.Bone mineral density analysis of the defect regions were used to evaluate the level of ossification.The morphologic change and bone formation was assessed by HE staining.The angiogenesis was evaluated by VEGF immunohistochemistry.Results:The osteogenetic ability and quality of femoral head necrosis in group B were better than those of other groups after 12 weeks by X-ray radiograph and morphologic investigation.And the angiogenesis in group B was better than other groups.Group C had similar osteogenetic quality of femoral head necrosis and angiogenesis with group D.Conclusions:The treatment of rhBMP-2/PLCA implantation after core depression can promote the repair of rabbit femoral head necrosis.It is a promising and efficient synthetic bone material to treat the femoral head necrosis.

  12. The effect of a slow mode of BMP-2 delivery on the inflammatory response provoked by bone-defect-filling polymeric scaffolds.

    Science.gov (United States)

    Wu, Gang; Liu, Yuelian; Iizuka, Tateyuki; Hunziker, Ernst Bruno

    2010-10-01

    We investigated the inflammatory response to, and the osteoinductive efficacies of, four polymers (collagen, Ethisorb, PLGA and Polyactive) that bore either an adsorbed (fast-release kinetics) or a calcium-phosphate-coating-incorporated (slow-release kinetics) depot of BMP-2. Titanium-plate-supported discs of each polymer (n = 6 per group) were implanted at an ectopic (subcutaneous) ossification site in rats (n = 48). Five weeks later, they were retrieved for a histomorphometric analysis of the volumes of ectopic bone and foreign-body giant cells (a gauge of inflammatory reactivity), and the degree of polymer degradation. For each polymer, the osteoinductive efficacy of BMP-2 was higher when it was incorporated into a coating than when it was directly adsorbed onto the material. This mode of BMP-2 carriage was consistently associated with an attenuation of the inflammatory response. For coated materials, the volume density of foreign-body giant cells was inversely correlated with the volume density of bone (r(2) = 0.96), and the volume density of bone was directly proportional to the surface-area density of the polymer (r(2) = 0.97). Following coating degradation, other competitive factors, such as the biocompatibility and the biodegradability of the polymer itself, came into play.

  13. High-purity magnesium interference screws promote fibrocartilaginous entheses regeneration in the anterior cruciate ligament reconstruction rabbit model via accumulation of BMP-2 and VEGF.

    Science.gov (United States)

    Cheng, Pengfei; Han, Pei; Zhao, Changli; Zhang, Shaoxiang; Wu, Hongliu; Ni, Jiahua; Hou, Peng; Zhang, Yuanzhuang; Liu, Jingyi; Xu, Haidong; Liu, Shen; Zhang, Xiaonong; Zheng, Yufeng; Chai, Yimin

    2016-03-01

    Interference screw in the fixation of autologous tendon graft to the bone tunnel is widely accepted for the reconstruction of anterior cruciate ligament (ACL), but the regeneration of fibrocartilaginous entheses could hardly be achieved with the traditional interference screw. In the present work, biodegradable high-purity magnesium (HP Mg) showed good cytocompatibility and promoted the expression of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), fibrocartilage markers (Aggrecan, COL2A1 and SOX-9), and glycosaminoglycan (GAG) production in vitro. The HP Mg screw was applied to fix the semitendinosus autograft to the femoral tunnel in a rabbit model of ACL reconstruction with titanium (Ti) screw as the control. The femur-tendon graft-tibia complex was retrieved at 3, 6, 9 and 12 weeks. Gross observation and range of motion (ROM) of the animal model reached normal levels at 12 weeks. No sign of host reaction was found in the X-ray scanning. The HP Mg group was comparable to the Ti group with respect to biomechanical properties of the reconstructed ACL, and the ultimate load to failure and stiffness increased 12 weeks after surgery. In the histological analysis, the HP Mg group formed distinct fibrocartilage transition zones at the tendon-bone interface 12 weeks after surgery, whereas a disorganized fibrocartilage layer was found in the Ti group. In the immunohistochemical analysis, highly positive staining of BMP-2, VEGF and the specific receptor for BMP-2 (BMPR1A) was shown at the tendon-bone interface of the HP Mg group compared with the Ti group. Furthermore, the HP Mg group had significantly higher expression of BMP-2 and VEGF than the Ti group in the early phase of tendon-bone healing, followed by enhanced expression of fibrocartilage markers and GAG production. Therefore we proposed that the stimulation of BMP-2 and VEGF by Mg ions was responsible for the fibrochondrogenesis of Mg materials. HP Mg was promising as a

  14. SPECIFIC BINDING OF HUMAN BONE MORPHOGENETIC PROTEIN (2A) WITH MOUSE OSTEOBLASTIC CELLS

    Institute of Scientific and Technical Information of China (English)

    刘新平; 陈苏民; 陈南春; 高磊; 赵忠良

    1996-01-01

    Human bone morphogenetic protein 2A (hBMP2A) cDNA terminal 567 nucleotides were cloned and expressed in a phage display vector pCSM2I. Hulnata BMP2A C-terminal peptide displayed on the surface of the phage can bind specifically to the sttrface of mouse osteoblastie cell (MC3T3) membrane. ELISA assay showed a positive signal of the binding by using antibody against M13 phage gene 8 protein. After labeling with 3HTdR,the counts of the binding groups were 3 to 10 times higher than the control groups. It suggests that the'surface of MC3T3 cells exist the recepzor for hBMP2A.

  15. Effects of recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) in grade III open tibia fractures treated with unreamed nails-A clinical and health-economic analysis.

    Science.gov (United States)

    Alt, Volker; Borgman, Benny; Eicher, Alexander; Heiss, Christian; Kanakaris, Nikolaos K; Giannoudis, Peter V; Song, Fujian

    2015-11-01

    Recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) is licensed in Europe for open tibia fractures treated with unreamed nails. However, there is limited data available on the specific use of rhBMP-2 in combination with unreamed nails for open tibia fractures. The intention of the current study was to evaluate the medical and health-economic effects of rhBMP-2 in Gustilo-Anderson grade III open tibia fractures treated with unreamed nails based on individual patient data from two previously published studies. Linear regression analysis was performed on raw data of 90 patients that were either treated by standard of care with soft tissue management and unreamed nailing (SOC group) (n=50) or with rhBMP-2 in addition to soft tissue management and unreamed nailing (rhBMP-2 group) (n=40). For all types of revision, a significant lower percentage of patients (27.5%) of the rhBMP-2 group had to be revised compared to 48% of the patients of the SOC group (p=0.04). When only invasive secondary interventions such as bone grafting and nail exchange were considered, there was also a statistically significant reduction in the rhBMP-2 group with a revision rate of 10.0% (4 of 40 patients) compared to the SOC group with a revision rate of 28.0% (14 of 50 patients) (p=0.01). Mean fracture healing time of 228 days in the rhBMP-2 compared to 266 days in the SOC group was not statistically significant (p=0.24). Health-economic analysis based on a societal perspective with calculation of overall treatment costs after initial surgery and including productivity losses revealed savings of €6,239 per patient for Germany and €4,752 for the UK in favour of rhBMP-2 which was mainly driven by reduction of productivity losses. In conclusion, rhBMP-2 reduces secondary interventions in patients with grade III open tibia fractures treated with an unreamed nail and its use leads to financial savings for Germany and the UK from a societal perspective. PMID:26374949

  16. Heterotopic ossification following single-level anterior cervical discectomy and fusion: results from the prospective, multicenter, historically controlled trial comparing allograft to an optimized dose of rhBMP-2.

    Science.gov (United States)

    Arnold, Paul M; Anderson, Karen K; Selim, Abdulhafez; Dryer, Randall F; Kenneth Burkus, J

    2016-09-01

    OBJECTIVE Heterotopic ossification (HO) has been reported following total hip, knee, cervical, and lumbar arthroplasty, as well as following posterolateral lumbar fusion using recombinant human bone morphogenetic protein-2 (rhBMP-2). Data regarding HO following anterior cervical discectomy and fusion (ACDF) with rhBMP-2 are sparse. A subanalysis was done of the prospective, multicenter, investigational device exemption trial that compared rhBMP-2 on an absorbable collagen sponge (ACS) versus allograft in ACDF for patients with symptomatic single-level cervical degenerative disc disease. METHODS To assess differences in types of HO observed in the treatment groups and effects of HO on functional and efficacy outcomes, clinical outcomes from previous disc replacement studies were compared between patients who received rhBMP-2/ACS versus allograft. Rate, location, grade, and size of ossifications were assessed preoperatively and at 24 months, and correlated with clinical outcomes. RESULTS Heterotopic ossification was primarily anterior in both groups. Preoperatively in both groups, and including osteophytes in the target regions, HO rates were high at 40.9% and 36.9% for the rhBMP-2/ACS and allograft groups, respectively (p = 0.350). At 24 months, the rate of HO in the rhBMP-2/ACS group was higher than in the allograft group (78.6% vs 59.2%, respectively; p disc spaces significantly reduced range of motion, more so in the rhBMP-2/ACS group. At 24 months, HO negatively affected Neck Disability Index scores (excluding neck/arm pain scores), neurological status, and overall success in patients in the rhBMP-2/ACS group, but not in patients in the allograft group. CONCLUSIONS Implantation of rhBMP-2/ACS at 1.5 mg/ml with polyetheretherketone spacer and titanium plate is effective in inducing fusion and improving pain and function in patients undergoing ACDF for symptomatic single-level cervical degenerative disc disease. At 24 months, the rate and dimensions (length and

  17. Generation of an rhBMP-2-loaded beta-tricalcium phosphate/hydrogel composite and evaluation of its efficacy on peri-implant bone formation

    International Nuclear Information System (INIS)

    Dental implant insertion on a site with low bone quality or bone defect should be preceded by a bone graft or artificial bone graft insertion to heal the defect. We generated a beta-tricalcium phosphate (β-TCP) and poloxamer 407-based hydrogel composite and penetration of the β-TCP/hydrogel composite into the peri-implant area of bone was evaluated by porous bone block experiments. The maximum penetration depth for porous bone blocks and dense bone blocks were 524 μm and 464 μm, respectively. We report the in-vivo performance of a composite of β-TCP/hydrogel composite as a carrier of recombinant human bone morphogenetic protein (rhBMP-2), implanted into a rabbit tibial defect model. Three holes drilled into each tibia of eight male rabbits were (1) grafted with dental implant fixtures; (2) filled with β-TCP/hydrogel composite (containing 5 μg of rhBMP-2), followed by grafting of the dental implant fixtures. Four weeks later, bone-implant contact ratio and peri-implant bone formation were analyzed by radiography, micro-CT and histology of undecalcified specimens. The micro-CT results showed a significantly higher level of trabecular thickness and new bone and peri-implant new bone formation in the experimental treatment compared to the control treatment. Histomorphometry revealed a significantly higher bone-implant contact ratio and peri-implant bone formation with the experimental treatment. The use of β-TCP/poloxamer 407 hydrogel composite as a carrier of rhBMP-2 significantly promoted new bone formation around the dental implant fixture and it also improved the quality of the new bone formed in the tibial marrow space. (paper)

  18. The multifaceted effects of agmatine on functional recovery after spinal cord injury through Modulations of BMP-2/4/7 expressions in neurons and glial cells.

    Directory of Open Access Journals (Sweden)

    Yu Mi Park

    Full Text Available Presently, few treatments for spinal cord injury (SCI are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm, a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm(2 weight for 1 min at thoracic vertebra (Th 9 segment. Mice that received an intraperitoneal (i.p. injection of Agm (100 mg/kg/day within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following

  19. CONSTRUCTION OF RECOMBINANT PLASMID pcDNA3.1/BMP-2 AND ITS INVOLVEMENT IN DIFFERENTIATION OF HUMAN DENTAL PULP-DERIVED CELLS INTO AN ODONTOBLASTIC LINEAGE

    Directory of Open Access Journals (Sweden)

    Boy M. Bachtiar

    2009-06-01

    Full Text Available Experimental studies have shown that dental pulp tissue has potential to regenerate dentine in response to adverse stimuli, such as caries and associated operative procedures. However, the potential of dental pulp regeneration seems to be limited by regenerative capacity of the cell involved. In this study, we report the effect of transfection of a recombinant plasmid containing human BMP-2 gene in proliferation and differentiation of dental pulp tissue in vitro. The regenerative capacity was analyzed by ALP production and calcium content. Results showed that the transfected dental pulp cell was able to differentiate into the odontoblast phenotype, indicating the presence of odontoblast progentitor cells in dental pulp tissue.

  20. The toxic effects of Tris-(2,3-dibromopropyl)isocyanurate(TBC) on genes expression of bmp2b and bmp4 of zebrafish embryos

    Institute of Scientific and Technical Information of China (English)

    JIA Wan-jun

    2016-01-01

    We exposed zebrafish embryos to Tris-(2,3-dibromopropyl)isocyanurate(TBC)at the concentration of 20ppb, 100ppb, 400ppb, 1000ppb for 120h and 0.1%DMSO was set as the control group. Bmp2b and bmp4 were chosen perform RT-PCR to determine their genes expression level. The results showed that, TBC influenced their genes expression level in some extent and it significantly raised the genes expression level at the concentration of 20ppb.

  1. 转染Ad-hBMP2的脂肪干细胞与壳聚糖/磷酸三钙复合物支架的相容性%Compatibility of Adipose-derived Stem Cells Transfected by Ad-hBMP2 Gene and CTCP Scaffold in vitro

    Institute of Scientific and Technical Information of China (English)

    方忠; 杨琴; 熊伟; 李光辉; 廖晖; 李锋; 肖骏

    2012-01-01

    Objective To observe the biological behavior of cultured adipose-derived stem cells(ADSCs)transfected by Ad-hBMP2 combined with chitosan/tricalcium phosphate(CTCP)scaffold and investigate the feasibility of the composite for cartilage tissue engineering. Methods The ADSCsQ X 106/mL) transfected with Ad-hBMP2 plasmid vector were co-cultured with the CTCP scaffold. The adhesion and proliferation of ADSCs, and the morphological changes were observed. RT-PCR, Western blot and immunohistochemistry were applied to detect the expression of Osteocalcin and collagen I in the scaffold. Results The poriferous CTCP scaffold has macro and micro poriferous structures,and the porous rate was 83%. The ADSCs transfected by Ad-hBMP2 have been successfully cultured in vitro. The induced cells adhered to the surface of the scaffold and proliferated well. The RT-PCR,Western blot and immunohistochemistry revealed that the expression of Osteocalcin and collagen I was detected after the co-culture. Conclusion The poriferous CTCP scaffold with excellent property should be a good "matrix" for ADSCs transfected by Ad-hBMP2, and could be used for bone tissue engineering.%目的 探讨转染腺病毒骨形态发生蛋白(Ad-hBMP2)基因的脂肪干细胞(ADSCs)与壳聚糖/磷酸三钙(CTCP)复合物支架的相容性,以期为ADSCs修复骨缺损寻找理想的组织工程骨支架材料.方法 将壳聚糖与磷酸三钙进行复合制成CTCP复合物材料,再将其与转染Ad-hBMP2的ADSCs(密度1×106/mL)复合培养,进行细胞复合物支架的一般与超微形态学观察,观察细胞粘附能力、增殖活力,RT-PCR及Western blot测定成骨细胞骨钙素和Ⅰ型胶原水平.结果 CTCP支架孔径200~350 μm,孔隙率83%;电镜显示转染Ad-hBMP2的ADSCs与CTCP复合物在体外培养期间支架无塌陷及形变,且其在支架上粘附、增殖良好,并能分泌细胞外基质如骨钙素和Ⅰ型胶原等;随着培养时间延长,骨的组织学特征

  2. Research of quaternized chitosan based hybrid scaffold as a delivery system for rhBMP-2%壳聚糖季铵盐基杂化支架的制备及其作为rhBMP-2载体的研究*

    Institute of Scientific and Technical Information of China (English)

    胡园园; 王靖; 刘昌胜

    2014-01-01

    Quaternized chitosan was synthesized by the reaction of chitosan and glycidyltrimethylammonium chloride (GTMAC)and named as N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC). A hydrogel system composed of HTCC andβ-glycerophosphate (β-GP)was obtained by intermolecular forces. Introduction ofβ-TCP granules improved the mechanical strength and controlled the swelling behavior and pro-longed the releasing period for rhBMP-2 of the hydrogel scaffold.Moreover,rhBMP-2 loaded scaffolds were as-sessed in ectopic bone formation.The results indicate that porous HTCC/β-GP/β-TCP/rhBMP-2 scaffolds are promising candidates for application in tissue engineering of bone.%采用环氧衍生物开环法制备壳聚糖季铵盐,通过其与甘油磷酸钠的分子间作用力交联成凝胶,冻干后得多孔支架,用于装载骨形态发生蛋白-2应用于骨修复领域;同时引入β-TCP 作为物理交联点,在提高支架力学强度的同时,更好地调控支架的溶胀行为,延长rhBMP-2的释放时间,并且在异位诱导成骨的动物实验中取得良好效果。

  3. 核心结合因子α1在BMP-2调控细胞外基质蛋白表达中的作用%Study of cbfα1 on the expression of extracellular matrix proteins in dental papilla cells induced by BMP-2 in vitro

    Institute of Scientific and Technical Information of China (English)

    余擎; 朱庆林; 孙汉堂; 田宇; 何文喜; 肖明振

    2008-01-01

    目的:探讨核心结合因子α1(cbfa1)在BMP-2调控体外培养的牙乳头细胞表达细胞外基质蛋白中的作用.方法:采用反义核酸技术,体外阻断培养的牙乳头细胞中cbfα1的表达,分别用RT-PCR、Western印迹等方法观察200ng/mL BMP-2作用6h后细胞中相关基质蛋白,碱性磷酸酶(ALP)、骨钙素(OC)、骨连蛋白(ON)、骨桥素(OPN)、骨涎蛋白(BSP)、牙本质基质蛋白1(DMP-1)以及牙本质涎磷蛋白(DSPP)的表达,采用SPSS 11.0软件包对数据进行方差分析.结果:外源性BMP-2能明显上调牙乳头细胞中ALP、OC含量以及OPN、BSP和ON的表达,当反义阻断cbfα1的表达时,ALP、OC、OPN和BSP的表达显著降低(P<0.01).结论:cbfα1参与了BMP-2调控体外培养的牙乳头细胞表达细胞外基质蛋白的信号转导过程.

  4. Hereditary hemochromatosis type 1 phenotype modifiers in Italian patients. The controversial role of variants in HAMP, BMP2, FTL and SLC40A1 genes.

    Science.gov (United States)

    Radio, Francesca Clementina; Majore, Silvia; Aurizi, Caterina; Sorge, Fiammetta; Biolcati, Gianfranco; Bernabini, Sara; Giotti, Irene; Torricelli, Francesca; Giannarelli, Diana; De Bernardo, Carmelilia; Grammatico, Paola

    2015-06-01

    Hereditary hemochromatosis (HH) is a heterogeneous disorder of iron metabolism. The most common form of the disease is Classic or type 1 HH, mainly caused by a biallelic missense p.Cys282Tyr (c.845G>A) mutation in the HFE gene. However, the penetrance of p.Cys282Tyr/p.Cys282Tyr genotype is incomplete in terms of both biochemical and clinical expressivity. Lack of penetrance is thought to be caused by several genetic and environmental factors. Recently, a lot of evidences on HH genetic modifiers were produced, often without conclusive results. We investigated 6 polymorphisms (rs10421768 in HAMP gene, rs235756 in BMP2 gene, rs2230267 in FTL gene, rs1439816 in SLC40A1 gene, rs41295942 in TFR2 gene and rs2111833 in TMPRSS6 gene) with uncertain function in order to further evaluate their role in an independent cohort of 109 HH type 1 patients. Our results make it likely the role of rs10421768, rs235756, rs2230267 and rs1439816 polymorphisms, respectively in HAMP, BMP2, FTL and SLC40A1 genes in HH expressivity. In addition, previous and our findings support a hypothetical multifactorial model of HH, characterized by a principal gene (HFE in HH type 1) and minor genetic and environmental factors that still have to be fully elucidated.

  5. Low-power GaAlAs laser irradiation promotes the proliferation and osteogenic differentiation of stem cells via IGF1 and BMP2.

    Directory of Open Access Journals (Sweden)

    Jyun-Yi Wu

    Full Text Available Low-power laser irradiation (LPLI has been found to induce various biological effects and cellular processes. Also, LPLI has been shown to promote fracture repair. Until now, it has been unclear how LPLI promotes bone formation and fracture healing. The aim of this study was to investigate the potential mechanism of LPLI-mediated enhancement of bone formation using mouse bone marrow mesenchymal stem cells (D1 cells. D1 cells were irradiated daily with a gallium-aluminum-arsenide (GaAlAs laser at dose of 0, 1, 2, or 4 J/cm(2. The lactate dehydrogenase (LDH assay showed no cytotoxic effects of LPLI on D1 cells, and instead, LPLI at 4 J/cm(2 significantly promoted D1 cell proliferation. LPLI also enhanced osteogenic differentiation in a dose-dependent manner and moderately increased expression of osteogenic markers. The neutralization experiments indicated that LPLI regulated insulin-like growth factor 1 (IGF1 and bone morphogenetic protein 2 (BMP2 signaling to promote cell proliferation and/or osteogenic differentiation. In conclusion, our study suggests that LPLI may induce IGF1 expression to promote both the proliferation and osteogenic differentiation of D1 cells, whereas it may induce BMP2 expression primarily to enhance osteogenic differentiation.

  6. 辽宁绒山羊BMP-2基因的克隆及序列比较分析%Cloning and Sequence Analyzing of B MP-2 Gene from Liaoning Cashmere Goats

    Institute of Scientific and Technical Information of China (English)

    薛冰; 郭丹; 王春艳; 郑旭; 高月; 张世伟

    2012-01-01

    根据GenBank上绵羊的BMP-2基因序列设计特异性引物,以辽宁绒山羊基因组DNA为模板,利用聚合酶链式反应,成功克隆了常年长绒型和季节长绒型辽宁绒山羊BMP-2部分基因片段,丰富了绒山羊BMP-2基因序列。经与绵羊、牛、鼠、猪和人的BMP-2基因进行的比对结果表明,季节与常年长绒型辽宁绒山羊的BMP-2基因同源片段的同源性达到99.7%,二者与绵羊同源性为98.2%和98.4%;与牛同源性为98.2%和97.9%;与鼠同源性为86.3%和86%;与人同源性为88.1%和88.1%。结果表明,辽宁绒山羊BMP-2基因部分核苷酸序列与其他哺乳动物同源性很高,与绵羊、牛的同源性高达97%以上,这与它们的种属关系相近一致。与人、鼠的同源性也在86%以上,说明BMP-2基因在不同物种之间具有较高的保守性。%According to the BMP-2 gene sequence of sheep on the GenBank, specific primers were designed and perennial and seasonal long-staple Cashmere goat BMP type-2 partial gene fragments were successfully cloned from the Liaoning cashmere goat genomic DNA, using the polymerase chain reaction. With the sheep, cat- tie, rats, pigs and human BMP-2 gene than on the results show that seasonal and perennial long-staple type of Liaoning cashmere goats homologous fragment of BMP-2 homology 99.7%, respeciively homology with the sheep was 98.2% and 98.4%; and bovine homology was 98.2% and 97.9%; and rat homolgy 86.3% and 86%; and human homology was 88.1% and 88.1%. The results showed that BMP-2 in Liaoning Cashmere goat gene partial nucleotide sequence homology with other mammals, which is similar to their relationship of species, and BMP-2 gene were conserved in different species.

  7. Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries

    Science.gov (United States)

    Hernandez-Hurtado, Adelina A.; Lara-Arias, Jorge; Romero-Diaz, Viktor J.; Abrego-Guerra, Adalberto; Vilchez-Cavazos, Jose F.; Elizondo-Riojas, Guillermo; Martinez-Rodriguez, Herminia G.; Espinoza-Juarez, Marcela A.; Mendoza Lemus, Oscar F.

    2016-01-01

    Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

  8. A new heterologous fibrin sealant as scaffold to recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins for the repair of tibial bone defects.

    Science.gov (United States)

    Machado, Eduardo Gomes; Issa, João Paulo Mardegan; Figueiredo, Fellipe Augusto Tocchini de; Santos, Geovane Ribeiro Dos; Galdeano, Ewerton Alexandre; Alves, Mariana Carla; Chacon, Erivelto Luis; Ferreira Junior, Rui Seabra; Barraviera, Benedito; Cunha, Marcelo Rodrigues da

    2015-04-01

    Tissue engineering has special interest in bone tissue aiming at future medical applications Studies have focused on recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins due to the osteogenic properties of rhBMP-2 and the angiogenic characteristic of fraction 1 protein (P-1) extracted from the rubber tree Hevea brasiliensis. Furthermore, heterologous fibrin sealant (FS) has been shown as a promising alternative in regenerative therapies. The aim of this study was to evaluate these substances for the repair of bone defects in rats. A bone defect measuring 3mm in diameter was created in the proximal metaphysis of the left tibia of 60 rats and was implanted with rhBMP-2 or P-1 in combination with a new heterologous FS derived from snake venom. The animals were divided into six groups: control (unfilled bone defect), rhBMP-2 (defect filled with 5μg rhBMP-2), P-1 (defect filled with 5μg P-1), FS (defect filled with 8μg FS), FS/rhBMP-2 (defect filled with 8μg FS and 5μg rhBMP-2), FS/P-1 (defect filled with 8μg FS and 5μg P-1). The animals were sacrificed 2 and 6 weeks after surgery. The newly formed bone projected from the margins of the original bone and exhibited trabecular morphology and a disorganized arrangement of osteocyte lacunae. Immunohistochemical analysis showed intense expression of osteocalcin in all groups. Histometric analysis revealed a significant difference in all groups after 2 weeks (p0.05). A statistically significant difference (p<0.05) was observed in all groups after 6 weeks in relation to the volume of newly formed bone in the surgical area. In conclusion, the new heterologous fibrin sealant was found to be biocompatible and the combination with rhBMP-2 showed the highest osteogenic and osteoconductive capacity for bone healing. These findings suggest a promising application of this combination in the regeneration surgery. PMID:25825118

  9. 丝素蛋白增强型磷酸钙复合rhBMP-2用于绵羊腰椎椎体间融合的实验研究%Experimental study on lumbar interbody fusion with silk fibroin enhanced calcium phosphate cement composite loaded with recombinant human bone morphogenetic protein-2 in sheep

    Institute of Scientific and Technical Information of China (English)

    陈亮; 顾勇; 陈晓庆; 干旻峰; 朱雪松; 杨惠林; 唐天驷

    2010-01-01

    Objective To evaluate the osteogenic characteristics of an injectable silk fibroin (SF) enhanced calcium phosphate cement (CPC) composite loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) on lumbar interbody fusion in sheep. Methods Twenty-four mature sheep were randomly divided into two groups. Each sheep underwent L1.2, L3.4 and L5.6 lumber interbody fusion, and the three disc spaces were randomly implanted with three of the following materials: SF/CPC, CPC/rhBMP-2, SF/CPC/rhBMP2 and autogenous iliac crest bone. One group was killed at 6 months and the other at 12 months. The fusion segments were observed and analyzed by manual palpation, CT scan, undestructive biomechanical testing, undecalcified histology, and histomorphology. Results The fusion rates of SF/CPC, CPC/rhBMP-2, SF/CPC/rhBMP-2 and autogenous bone assessed by manual palpation were 0, 33.33%, 55.56% and 77.78% respectively at 6 months. At 12 months, the fusion rates improved to 11.11%, 44.44%, 77.78% and 77.78%, respectively.The biomechanical results showed that fusion stiffness was significantly greater in autograft compared with SF/CPC/rhBMP-2, CPC/rhBMP-2, and SF/CPC in 4 degrees of freedom (flexion, extension, right bending, and left bending) at 6 months. The SF/CPC/rhBMP-2 composite showed similar stiffness as autograft, which was significantly greater than CPC/rhBMP-2 and SF/CPC at 12 nonths. Both CPC/rhBMP-2 and SF/CPC/rhBMP-2 showed significantly greater stiffness at 12 months compared with that of at 6 months. The results showed that bone volume was significantly greater in autograft compared with SF/CPC/rhBMP-2, CPC/rhBMP-2, and SF/CPC at 6 months. There was significant difference among ceramic residue among SF/CPC, CPC/rhBMP-2 and SF/CPC/rhBMP-2, with SF/CPC the greatest and SF/CPC/thBMP-2 the least. At 12 months, the bone volume of SF/CPC/rhBMP-2 composite was comparable with autograft, and greater than that of CPC/rhBMP-2 and SF/CPC. The bone volume of SF/CPC, CPC/rhBMP

  10. Delta-like 1/fetal antigen 1(DLK1/FA1) inhibits BMP2 induced osteoblast differentiation through modulation of NFκB signaling pathway

    DEFF Research Database (Denmark)

    Qiu, Weimin; Abdallah, Basem; Kassem, Moustapha

    as assessed by reduced Alp activity and osteogenic gene expression including Alp, Col1a1, Runx2 and Bglap. In addition, DLK1/FA1 inhibited BMP signaling as demonstrated by reduced gene expression of BMP-responsive genes: Junb and Id1, reduced BMP2 induced luciferase activity in C2C12 BMP luciferase reporter....... Besides, we observed that DLK1/FA1 induced strong NFκB activity evidenced by NFκB responsive luciferase reporter assay and real-time RT-PCR analysis of NFκB target genes. The inhibitory effect of NFκB signaling on BMP signaling was confirmed by luciferase assay in C2C12 BMP luciferase reporter cells...

  11. Construction and identification of human BMP2-IRES-HIF1αmu adenovirus expressing carrier and its expression in HEK293 cells%人BMP2-IRES-HIF1αmu腺病毒表达载体的构建及其在HEK293细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    李全营; 李谌; 郭威; 吴秀成; 王巍; 刘丹平

    2011-01-01

    目的 构建人BMP2-IRES-HIF1 αmu腺病毒表达载体,并转染HEK293细胞,为下一步转染骨髓基质细胞和体内实验打下基础.方法 PCR扩增HIF1αmu片段,用BstXⅠ和XbaⅠ双酶切回收目的片段.pIRES2-EGFP用BstXⅠ和Xba Ⅰ进行双酶切后回收大片段.将上述回收的目的基因与载体片段连接,然后转化感受态大肠杆菌DH5α扩增;PCR扩增BMP2片段,用Nhe Ⅰ和BamH Ⅰ双酶切后回收目的片段.把目的基因与载体片段连接,转化感受态大肠杆菌DH5α扩增重组腺病毒表达载体,通过酶切分析、PCR和测序进行鉴定.将构建好的质粒转染HEK293细胞,检测病毒液滴度.结果 构建了人BMP2-1RES-HIF1 αmu腺病毒表达载体,转染HEK293细胞见绿色荧光表达.结论 成功构建了人BMP2 -IRES-HIF1 αmu腺病毒表达载体,酶切分析及DNA测序证实质粒构建正确,质粒成功转染HEK293细胞,并见绿色荧光蛋白表达.%Objective To construct and identify human BMP2-IRES-HIFlotmu Adenovirus expressing carrier, trans-feet it in HEK293 cells, and determinate the virus droplet degrees. Methods PCR was used to amplify HIFlamu segments, BstX I and Xba I double enzyme cut pIRES2-EGFP and recycling purpose extract. Connecting the recovery target gene with carrier segment. Then introduced it into E. Coli for amplification. PCR was used to amplify BMP2 segments, Nhe I and BamH I double enzyme cut and recycling purpose extract. Connecting the recovery target gene with carrier segment. Then introduced it into E. Coli for amplification and restructuring adenovirus expressing carrier. Using the enzyme cut analysis, PCR for identification. The correct recombinant express plasmid was transfected into HEK293 cells, and detecting the virus droplet degrees. Results The adenovirus shuttle plasmid was constructed. Green fluorescent expression was seen in transfected HEK293 cells. Conclusion The adenovirus shuttle plasmid is constructed, it is successfully expressed in HEK

  12. Characteristics and Stimulation Potential with BMP-2 and BMP-7 of Tenocyte-Like Cells Isolated from the Rotator Cuff of Female Donors

    Science.gov (United States)

    Klatte-Schulz, Franka; Pauly, Stephan; Scheibel, Markus; Greiner, Stefan; Gerhardt, Christian; Hartwig, Jelka; Schmidmaier, Gerhard; Wildemann, Britt

    2013-01-01

    Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient’s age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml) in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and –III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff. PMID:23825642

  13. Characteristics and stimulation potential with BMP-2 and BMP-7 of tenocyte-like cells isolated from the rotator cuff of female donors.

    Directory of Open Access Journals (Sweden)

    Franka Klatte-Schulz

    Full Text Available Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient's age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and -III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff.

  14. Effects of administration rhBMP-2/rhVEGF165 on tendon-bone healing after anterior cruciate ligament reconstruction in rabbits%rhBMP-2联合rhVEGF165对兔前交叉韧带重建后腱-骨愈合的影响

    Institute of Scientific and Technical Information of China (English)

    周平; 赵其纯; 尚希福; 王姚斐; 李旭; 纪小枫; 凌晓冬; 朱亚林

    2013-01-01

    目的 探讨局部联合应用重组人骨形态发生蛋白(rhBMP)-2和重组人血管内皮生长因子165 (rhVEGF165) 对兔前叉韧带重建后腱-骨早期愈合的影响.方法 78只健康成年雄性新西兰大白兔,随机分为VB、B、V、F及正常组,前4组建立双膝自体半腱肌前交叉韧带重建模型,正常组未行手术.VB组腱-骨界面注入以纤维蛋白胶(FG)为载体的rhBMP-2/rhVEGF165混合物;B组注入rhBMP-2和FG混合物;V组注入rhVEGF165和FG混合物;F组单纯注入FG.术后第2、4、8周,每组随机抽取6只兔子,右膝标本行组织学观察,左膝标本行生物力学测试.结果 术后第2周:VB组腱-骨连接紧密,成纤维细胞大量增生;B组连接紧密,可见少量软骨细胞;V、F组连接疏松,主要为纤维血管组织构成.术后第4周:VB组腱-骨界面初步形成四层结构;V、B、F组则主要由成熟的成纤维细胞构成,腱-骨连接紧密,骨隧道壁出现一定量成骨反应;术后第8周:VB组四层结构更明显,可见潮线;B组骨隧道壁侧成骨反应进步加强;V、F组腱-骨界面宽度进步变窄,成纤维细胞数目明显减少.直至术后第4、8周,VB组平均最大载负荷及刚度才较V、B、F组大(P<0.05),均低于正常组;同时V、B组生物力学性能优于F组(P<0.05).结论 rhBMP-2联合rhVEGF165对兔前交叉韧带重建术后腱-骨早期愈合具有促进作用,且联合效应强于各自单独效应.%Objective To explore effects of local administration rhBMP-2/rhVEGF165 on tendon-bone ealy healing after anterior cruciate ligament reconsctruction in rabbits. Methods Seventy eight male New Zealand white rabbits were randomly divided into Group VB,B,V,F and normal group. Bilateral anterior cruciate ligaments of each animal were removed and reconstructed with autogenetic tendon of semitendinosus muscles while normal group did not receive any operation. The fibrin glue ( FG ) containing rhBMP-2/rhVEGF165 was injected into tendon-bone interface

  15. Effect of recombinant human bone morphogenetic protein 2/poly-lactide-co-glycolic acid (rhBMP-2/PLGA) with core decompression on repair of rabbit femoral head necrosis

    Institute of Scientific and Technical Information of China (English)

    Zhao-Xun Pan; Hong-Xin Zhang; Ye-Xin Wang; Long-Di Zhai; Wei Du

    2014-01-01

    Objective:To observe the effect of recombinant human bone morphogenetic protein 2/poly-lactide-co-glycolic acid (rhBMP-2/PLGA) with core decompression on repair of rabbit femoral head necrosis. Methods: Bilateral femoral head necrosis models of rabbit were established by steroid injection. A total of 48 rabbits (96 femoral head necrosis) were randomly divided into 4 groups: Group A, control group with12 rabbits, 24 femoral head necrosis;Group B, treated with rhBMP-2/PLGA implantation after core depression, with 12 rabbits, 24 femoral head necrosis;Group C, treated with rhBMP-2 implantation after core depression, with 12 rabbits, 24 femoral head necrosis;Group D treated with core depression group without implantation, with 12 rabbits, 24 femoral head necrosis. All animals were sacrificed after 12 weeks. The ability of repairing bone defect was evaluated by X-ray radiograph. Bone mineral density analysis of the defect regions were used to evaluate the level of ossification. The morphologic change and bone formation was assessed by HE staining. The angiogenesis was evaluated by VEGF immunohistochemistry. Results: The osteogenetic ability and quality of femoral head necrosis in group B were better than those of other groups after 12 weeks by X-ray radiograph and morphologic investigation. And the angiogenesis in group B was better than other groups. Group C had similar osteogenetic quality of femoral head necrosis and angiogenesis with group D. Conclusions:The treatment of rhBMP-2/PLGA implantation after core depression can promote the repair of rabbit femoral head necrosis. It is a promising and efficient synthetic bone material to treat the femoral head necrosis.

  16. Histological and radiographic evaluation of the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2 in a scaffold of inorganic bone and after stimulation with low-power laser light

    Directory of Open Access Journals (Sweden)

    Bengtson Antonio

    2010-01-01

    Full Text Available Objective: The present study histologically and radiologically evaluates the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2 in a natural inorganic bone mineral scaffold from a bull calf femur and irradiation with low-power light laser. Materials and Methods: The right and left hind limbs of 16 rats were shaved and an incision was made in the muscle on the face corresponding to the median portion of the tibia, into which rhBMP-2 in a scaffold of inorganic bone was implanted. Two groups of limbs were formed: control (G1 and laser irradiation (G2. G2 received diode laser light applied in the direction of the implant, at a dose of 8 J/cm2 for three minutes. On the 7th, 21st, 40th and 112th days after implantation, hind limbs of 4 animals were radiographed and their implants removed together with the surrounding tissue for study under the microscope. The histological results were graded as 0=absence, 1=slight presence, 2=representative and 3=very representative, with regard to the following events: formation of osteoid structure, acute inflammation, chronic inflammation, fibrin deposition, neovascularization, foreign-body granuloma and fibrosis. Results: There were no statistically significant differences in these events at each evaluation times, between the two groups (P > 0.05; Mann-Whitney test. Nevertheless, it could be concluded that the natural inorganic bone matrix with rhBMP-2, from the femur of a bull calf, is a biocompatible combination. Conclusions: Under these conditions, the inductive capacity of rhBMP-2 for cell differentiation was inhibited. There was a slight acceleration in tissue healing in the group that received irradiation with low-power laser light.

  17. Study on the Drug-loading Pattern of BMP2 Slow-release Microspheres%载骨形态发生蛋白2缓释微球载药形式研究

    Institute of Scientific and Technical Information of China (English)

    陈发明; 吴织芬; 王勤涛; 金岩; 王国芳; 杜岩

    2005-01-01

    目的:探讨右旋糖酐-甲基丙烯酸缩水甘油酯载骨形态发生蛋白 2(BMP2-DEX-GMA)凝胶微球的载药形式.方法:采用放射免疫分析法测定.结果: BMP2-DEX-GMA凝胶微球载药形式为内部包埋(DE)与表面吸附(DA)并存,且随着粒径增大, DA减少、 DE增加,但统计学检验结果显示粒径为 20μ m~ 40μ m的凝胶微球载药形式 DA、 DE间比较无显著性差异(P>0. 05).结论:粒径的改变可以影响 BMP2-DEX-GMA凝胶微球的载药形式,但不会改变其释药特征,尚需对载药材料进行改性才能实现对药物的控释.

  18. Parathyroid hormone stimulate osteoblast differentiation by up-regulation of BMP2 expression and function%骨形态发生蛋白2介导甲状旁腺素促进成骨细胞分化的实验研究

    Institute of Scientific and Technical Information of China (English)

    徐莹; 田野; 孟凌新

    2012-01-01

    Objective To study the important role of BMP2 in the PTH-induced osteoblast differentiation. Methods MC3T3-E1 cells were divided into 4 groups: 1) Controls; 2) PTH treatment; 3) Dorso-morphin treatment; 4) PTH + Dorsomorphin treatment. Gene and protein expression levels of BMP2, BMP2 downstream genes and osteoblastic genes and protein expressions were detected by Real-time PCR and Western blot respectively. Alkaline phosphatase (ALP) staining was performed to detect ALP activity. 12xSBE-OC luciferase activity was measured using dual luciferase reporter assays. Results The expression level of BMP2 and osteoblastic marker genes was higher in the PTH group than in controls. PTH also increases 12xSBE-OC luciferase activity significantly. In the other hand, the expression of BMP2, BMP2 downstream genes and osteoblastic genes was lower in Dorsomorphin and PTH + Dorsomorphin group, than in controls. However, the expression was similar in Dorsomorphin and PTH + Dorsomorphin groups. Conclusion PTH stimulates osteoblast differentiation by up-regulating BMP2 expression and function.%目的 探讨骨形态发生蛋白2(BMP2)在甲状旁腺素(PTH)促进成骨细胞分化过程中的重要介导作用.方法培养MC3T3-E1细胞,分为4组:1)盐水对照组;2)PTH组;3)6-[4-[2-(1-哌啶基)乙氧基]苯基]-3-(4-吡啶基)吡唑并[1,5-a]嘧啶 (Dorsomorphin) 组;4) PTH+Dorsomorphin组.Real-time PCR法和Westernblot方法检测细胞BMP2BMP2下游基因和成骨因子的表达,碱性磷酸酶(ALP)染色方法检测细胞ALP的活性;双荧光素酶报告基因检测方法检测12xSBE-OC荧光素酶的活性.结果:PTH组BMP-2、成骨因子的表达及其12xSBE-OC荧光素酶的活性,明显高于盐水对照组.Dorsomorphin组和PTH+Dorsomorphin组BMP-2BMP-2下游基因和成骨因子的表达,均明显低于盐水对照组;但其表达于两组间无明显差别.结论 BMP2介导PTH促进成骨细胞的分化,PTH可通过上调BMP2的表达,提高其功能,促进成骨细胞的成熟分化.

  19. 聚乳酸及其复合物修复下颌骨缺损的实验研究%THE EXPERIMENTAL STUDY OF REPAIRING THE MANDIBULAR BONE DEFECT USING PLA-rhBMP-2

    Institute of Scientific and Technical Information of China (English)

    黄相道; 刘境华; 赵明; 王会信

    2001-01-01

    Purpose A new synthetic material consists of Polylac tic acid[PLA] and recombinant human bone morphogenetic protein-2[PLA-rhBMP -2]. The capacity of repairing bone defect of biodegradable PLA and the effica cy of PLA as a carrier for bone morphogenetic protein [BMP] are determined wit h detecting the osteogenetic process. Materials and Methods Eighteen rabbits were picked up and divided i nto three groups randomly. The defects of 12mm×6mm size were made at bilateral mandibul ar edges. PLA-rhBMP-2, PLA were implanted in the defect respectively. The 18 r abbits were killed at 2,4,8 and 12 weeks after implanting. The sample was analyz ed by the methods of X-rays and histological examination. Results After 2 weeks, the new bone began to form in the defects. A fter 8 weeks, bulk bone could be obviously observed. After 12 weeks, most of the PLA in the defects were already biodegraded. The defects were restored with n ew bone. The results in PLA-rhBMP-2 group were better than those of other grou ps. Conclusions PLA could be a promising carrier for BMP. PLA-rhBM P-2 has also been considered a better biodegradable material for repairing mand ibular defect.%目的 聚乳酸与骨形成蛋白复合,研究该材料修复颌骨缺损的能力,探讨聚乳酸作为骨形成蛋白载体的有效性。方法 18只日本大耳白兔,随机分组,在双侧下颌骨体部形成12mm×6mm的缺损,分别植入聚乳酸-人骨形成蛋白-2复合物(PLA-rhBMP-2)、单纯聚乳酸(PLA),于2、4、8、12周分批处死。通过X线、组织学染色等方法进行观察。结果 PLA-rhBMP-2植入组于术后2周,缺损区部分新骨形成;术后8周,大片新骨形成并开始改建,术后12周,PLA大部分降解,由骨组织修复。结果优于对照组。结论 PLA可作为BMP的有效载体,PLA-rhBMP-2是良好的颌骨缺损修复材料。

  20. 糖尿病对大鼠牙槽骨缺损修复中骨形态发生蛋白-2表达影响的研究%Effects of diabetes on expression of BMP-2 during bone healing of alveolar defect in rats

    Institute of Scientific and Technical Information of China (English)

    聂莹; 张志宏; 袁晟; 鲍军燕

    2011-01-01

    Objective To investigate the effects of diabetes on the expression of bone morphogenetic protein - 2 (BMP-2) during the various bone healing periods of alveolar defect. Methods 48 male SD rats were randomly divided into diabetes group ( n = 24) and control group( n=24). Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) ,and alveolar defect was created through the diabetic duration time. 6 rats in each group were scarified respectively at the lth ,2th ,4th ,8th week ,then the alveolar bone were processed for histological examination. The expression of BMP-2 in different periods( 1,2,4,8 weeks)of alveolar defect healing of rats was investigated through immunohistochemistry method. The optical density (OD) of BMP-2 was analysed and compared between groups. Results Osteopenia in diabetes group were observed. The OD of BMP - 2 in the control group was statistically greater than that in the test group 1 and 2 weeks after alveolar defect. After 4 and 8 weeks, the expression of BMP-2 in the control group decreased,and no statistical difference was found in BMP -2 expression between these two groups. Conclusions Diabetes may affect the formation of BMP-2 ,leading to a reduction in bone healing. Diabetes is able to affect the differentiation of mesenchymal stem cells,leading to less osseointegration. Therefore, primary stability was decreased.%目的 观察糖尿病(diabetes mellitus,DM)对实验性大鼠牙槽骨缺损修复过程中不同时期骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)表达的影响.方法 将48 只雄性SD大鼠随机分为DM组和对照组,每组24 只,DM组大鼠经腹腔注射链脲佐菌素造成DM大鼠模型,建模成功后行大鼠牙槽骨骨缺损制备,2 组均分别于骨缺损制备后1、2、4、8 周各取6 只大鼠处死,取术区组织.苏木精-伊红染色(hematoxylin-eosin staining,HE染色)镜下观察缺损区内新生骨样组织形成情况;用免疫组化法检测术后1、2、4

  1. Autologous serum improves bone formation in a primary stable silica-embedded nanohydroxyapatite bone substitute in combination with mesenchymal stem cells and rhBMP-2 in the sheep model

    Directory of Open Access Journals (Sweden)

    Boos AM

    2014-11-01

    Full Text Available Anja M Boos,1,* Annika Weigand,1,* Gloria Deschler,1 Thomas Gerber,2 Andreas Arkudas,1 Ulrich Kneser,1 Raymund E Horch,1 Justus P Beier11Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg FAU, Erlangen, 2Institute of Physics, University of Rostock, Rostock, Germany *These authors contributed equally to this work Abstract: New therapeutic strategies are required for critical size bone defects, because the gold standard of transplanting autologous bone from an unharmed area of the body often leads to several severe side effects and disadvantages for the patient. For years, tissue engineering approaches have been seeking a stable, axially vascularized transplantable bone replacement suitable for transplantation into the recipient bed with pre-existing insufficient conditions. For this reason, the arteriovenous loop model was developed and various bone substitutes have been vascularized. However, it has not been possible thus far to engineer a primary stable and axially vascularized transplantable bone substitute. For that purpose, a primary stable silica-embedded nanohydroxyapatite (HA bone substitute in combination with blood, bone marrow, expanded, or directly retransplanted mesenchymal stem cells, recombinant human bone morphogenetic protein 2 (rhBMP-2, and different carrier materials (fibrin, cell culture medium, autologous serum was tested subcutaneously for 4 or 12 weeks in the sheep model. Autologous serum lead to an early matrix change during degradation of the bone substitute and formation of new bone tissue. The best results were achieved in the group combining mesenchymal stem cells expanded with 60 µg/mL rhBMP-2 in autologous serum. Better ingrowth of fibrovascular tissue could be detected in the autologous serum group compared with the control (fibrin. Osteoclastic activity indicating an active bone remodeling process was observed after 4 weeks, particularly

  2. Effects of TiO2 sandblasted and acid-etched titanium on expression of bone morphogenetic protein 2 in human osteoblasts%TiO2喷砂酸蚀处理对钛片表面人成骨细胞BMP-2表达水平的影响

    Institute of Scientific and Technical Information of China (English)

    陆斌; 李建武; 郭义; 杨艳

    2013-01-01

    目的 探讨钛片经过TiO2喷砂酸蚀处理后对人成骨细胞系MG63细胞骨形态发生蛋白2(bone morphogenetic protein,BMP-2)表达水平的影响.方法 将钛片分为3组进行处理:机械打磨组、喷砂组及喷砂酸蚀组,分别进行机械打磨、TiO2喷砂和喷砂酸蚀处理.将人成骨细胞系MG63细胞接种于钛片表面,采用实时定量聚合酶链反应(real-time polymerase chain reaction,RT-PCR)、Western blot检测BMP-2 mRNA及蛋白表达水平.结果 喷砂组及喷砂酸蚀组BMP-2 mRNA及蛋白水平增高,与机械打磨组相比差异有统计学意义(P<0.05),而喷砂组与喷砂酸蚀组之间差异无统计学意义(P>0.05).结论 使用经过TiO2喷砂及喷砂酸蚀处理的钛片进行人成骨细胞培养可促进BMP-2表达.%Objective To explore the effect of TiO 2 sandblasted and acid -etched titanium on the expression of bone morphogenetic pro -tein 2 (BMP-2) in human MG63 cells.Methods Titanium discs (15 mm diameter and 1 mm thickness ) were divided into 3 groups: machine polished group , sandblasted group , sandblasted and acid -etched group.Titanium discs were treated with mechanical polishing , TiO2 sandblasting, sandblasting and acid-etching in three groups , respectively.MG63 cells were cultured on the titanium.The mRNA and protein expression of BMP-2 in MG63 cells were analyzed by real-time polymerase chain reaction (RT-PCR) and Western blot.Results The mRNA and protein levels of BMP -2 were significantly higher in sandblasted group and sandblasted and acid -etched group than in machine polished group ( P 0.05 ).Conclusion After sandblasting and acid -etching, titanium could promote the expression of BMP-2 in human osteoblast.

  3. BMP-2联合温热化疗对SW480中GDF15和TFF3表达的影响%Effect of bone morphogenetic protein-2 combined with hyperthermic chemotherapy on GDF15 and TFF3 expression in SW480

    Institute of Scientific and Technical Information of China (English)

    毕德利; 王亚旭; 舒宁波; 谢凯

    2012-01-01

    目的:探讨骨形态发生蛋白-2(Bone morphogenetic protein-2,BMP-2)联合温热化疗对SW480中GDF15和TFF3表达的影响.方法:BMP-2作用于大肠癌SW480细胞系,置于43℃温热化疗30 min,培养6h.(1)流式细胞法检测SW480细胞的凋亡;(2) Western blot法检测GDF15和TFF3蛋白表达情况;(3) RT-PCR半定量检测GDF15和TFF3的mRNA表达.结果:BMP-2联合43℃温热化疗后SW480细胞凋亡增加,GDF15和TFF3蛋白表达水平下降,GDF15和TFF3的mRNA表达亦下调.结论:BMP-2联合温热化疗通过抑制大肠癌SW480的GDF15和TFF3蛋白及其相关基因表达,增强抑制SW480的增殖及转移复发的作用;温热疗法联合化疗对GDF15和TFF3表达抑制有协同作用.%Objective: To explore the effect of bone morphogenetic protein-2(BMP-2) combined with hyperthermic chemotherapy on GDF15 and TFF3 expression in SW480. Methods:BMP-2 was acted on SW480 colorectal cancer cell lines,placed in 43 t hyperthermic chemotherapy for 30 min and cultured for 6 h. (1 )Apoptosis of SW480 cells was detected by flow cytometry assay; (2)GDF15 and TFF3 protein expressions were detected by Western blot; (3)GDF15 and TFF3 mRNA expressions were detected by RT-PCR. Results: SW480 cells apoptosis was increased, GDF15 and TFF3 protein levels were decreased after BMP-2 combined with 43 t hyperthermic chemotherapy. GDF 15 and TFF3 mRNA expressions were also reduced. Conclusions: BMP-2 combined hyperthermic chemotherapy can inhibit proliferation and metastasis of SW480 by inhibiting GDF15 and TFF3 protein and mRNA expressions. Hyperthermia combined with chemotherapy has synergistic effect on the inhibition of GDF15 and TFF3 expression.

  4. Comparison of Peritoneal Low-Molecular-Weight-Protein-Removal in CCPD and CAPD Patients Based on C-Terminal Agrin Fragment Clearance

    Directory of Open Access Journals (Sweden)

    Dominik Steubl

    2016-03-01

    Full Text Available Background/Aims: This study compares the peritoneal elimination of the low-molecular-weight-protein (LMWP C-terminal agrin fragment (tCAF, size 22 kDa, a promising biomarker for kidney function, in continuous cycling peritoneal dialysis (CCPD and continuous ambulatory peritoneal dialysis (CAPD. Methods: 103 sets of serum, 24h-urine and dialysate samples were obtained in 15 CCPD (63 sets and 11 CAPD (40 sets patients. Total, renal and peritoneal substrate removals/clearances were measured/calculated for tCAF, creatinine, blood-urea-nitrogen (BUN, cystatin C and albumin and correlated with the peritoneal transport type. Results: Serum und urine concentrations of all biomarkers did not differ between both groups, urinary substrate removal was higher in CAPD patients for all biomarkers due to better residual renal function. Peritoneal substrate removal of tCAF and albumin were significantly higher in CAPD (tCAF: 35.3 vs. 19.3 µg/d, p2 body-surface-area, pConclusions: CAPD was superior to CCPD concerning peritoneal tCAF removal. This finding was pronounced in high-transporters and CAPD patients using icodextrin twice daily.

  5. 活血化瘀补肾壮骨中药促进BMP基因克隆转染犬牙髓细胞增殖的实验研究%THE EXPERIMENTAL STUDY ON THE PROMOTIVE EFFECT OF CHINESE TRADITION MEDICINE ON PROLIFERATION OF DOG DENTAL PULP CELLS TRANSFECTED BY BMP2 GENE

    Institute of Scientific and Technical Information of China (English)

    刁志虹; 高毅; 李威

    2012-01-01

    Objective To observe the effect of Chinese tradition medicine serum on the proliferation of dog dental pulp cells( DDPCs )transfected with pEGFP - Nl - BMP2 eukaryotic expression plasmid in vitro. To investigate the mechanism of Chinese tradition medicine on formation of dentin. Methods BMP2 - DDPCs were cultivated respectively in Chinese tradition medicine serum group( group A),non - medicine serum group( group B ) and fetal bovine serum group( group C ). The proliferation activity of BMP2 - DDPCs was detected respectively by the 3 -(4,5 - dimethythiazol -2-yl)-2,5-diphenyl tetrazolium bromide( MTT )after 24 hours ,48 hours and 72hours. Results After BMP2 - DDPCs were cultivated in three kinds of serum, there were no differences in the cells morphology, but there were differences in the number of the cells. With the prolongation of culture time, the number of the cells gradually increased in the 3 groups, among which medicine serum group increased more prominently. There were significant differences between Chinese traditional medicine serum group and the other two groups( P <0. 05 ). Conclusion The Chinese tradition medicine could promote proliferation of BMP2 - DDPCs and obviously increase the quantity of seed cells in dentin tissue engineering.%目的 观察活血化瘀补肾壮骨含药血清对骨形成蛋白2(bone morphogenetic proteins 2,BMP2)绿色荧光融合蛋白(green-fluorescent protein,GFP)真核表达质粒(pEGFP-N1-BMP2)在体外转染的犬牙髓细胞(dog dental pulp cells,DDPCs)增殖情况的影响,探讨活血化瘀补肾壮骨中药在牙本质改建形成中的作用机制.方法 将 BMP2-DDPCs分别于活血化瘀补肾壮骨含药血清组(A组)、无药血清组(B组)和胎牛血清组(C组)中培养.分别于24、48、72h用溴化-3-(4,5-二甲基噻唑基-2)-2,5二苯基四氮唑[3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide,MTT]法检测BMP2-DDPCs增殖活性.结果 BMP2-DDPCs分别在A、B、C 3组中

  6. In vivo experimental study of hollow porous femoral prosthesis filled with rhBMP-2%中空多孔股骨柄假体骨长入方式的在体实验研究

    Institute of Scientific and Technical Information of China (English)

    蔡谞; 王达文; 樊宇平; 赵斌; 王岩

    2008-01-01

    Objective To study hone formation and osteointegration at the interface and inside the hollow porous femoral porsthesis filled with recombinant human bone morphogentic protein-2 (rhBMP-2). Methods 18 health adult hybrid dogs were divided into 3 groups randomly. Each animal received total hip arthroplasty at right hip joint. The femoral stems implanted were solid center prosthesis (group A), hollow porous prosthesis filled with cancellous bone taken from the femur head and neck (group B), and hollow porous prosthesis filled with gelfoam and 5 mg rhBMP-2(group C), respectively. All prosthesis were made of CoCrMo alloy with hydroxyapatite coating at the proximal half. The animals were sacrificed at 2,4,8 months post operation and X-ray of the femur samples were taken. The histological changes, interface osseointega- tion rate and bone ingrowth rate round and inside prosthesis were evaluated. Results The bone formation of group C was faster than group A and B histologically. The interface osteointegration rate of group C was sig- nificantly higher than that of group A and B (P< 0.05) at 2, 4 months, but there was no significantly differ- ence between them at 8 months (P 0.05). Also the bone ingrowth rate of group C was higher than that of group B(P<0.05), but no significant difference at 8 months(P 0.05). There was nosignificant difference be- tween goup A and B at interface osteointegration rate. Conclusion The hollow porous femoral prosthesis filled with rhBMP-2 can enhance interface osteointegration and bone ingrowth effectively, which can make the prosthesis fast fixed in the bone bed right at the early stage. And this may provide reliable experimental evidence for developing a kind of hollow porous femoral prosthesis for human.%目的 观察多孔中空人工股骨柄假体周围骨床骨质经界面长入金属空腔及其在腔室内成骨的特征,比较腔内复合骨诱导因子(rhBMP-2)与自身骨长入的成骨差异.方法 健康成年杂交犬18

  7. 重组人BMP-2诱导C3H10T1/2间质干细胞定向成骨分化早期基因表达谱分析%Identification and analysis of gene expression profiles for early osteoblastic differentiation on C3H10T1/2 mesenchymal stem cells induced by rhBMP-2

    Institute of Scientific and Technical Information of China (English)

    左长清; 汪宗桂; 钟月春; 卢旱云; 戴忠; 吴铁; 崔燎

    2014-01-01

    目的 研究间质干细胞早期定向成骨分化基因表达谱,为研究基因对早期成骨定向分化调控机制提供实验基础.方法 分别提取重组人骨形成蛋白2(rhBMP-2)诱导组和对照组C3H10T1/2细胞总RNA,进行扩增标记后,与ArraySTAR小鼠基因芯片杂交,应用生物信息学软件GeneSpring和GATHER对基因芯片数据进行分析.应用STRING在线软件对差异表达基因构建蛋白互作网络并进行网络分析.结果 C3H10T1/2早期成骨分化中,主要富集发育、器官形成等分子功能本体以及细胞因子-细胞因子受体作用信号通路.成骨分化1d和4d均上调表达基因42个,下调表达基因45个.网络分析研究表明:Egfr、Cxcl 12等信号分子参与调控rhBMP-2诱导成骨分化.结论 筛选的差异表达基因和信号分子对早期成骨分化调控具有重要作用,为进一步全面解析早期成骨定向分化提供实验基础.

  8. Subcutaneous ectopic osteogenesis induced by porous calcium phosphate cement and gelatin sponge as the carrier of recombinant bone morphogenetic protein-2 in rats:A comparative study%两种材料复合rhBMP-2诱导大鼠皮下异位成骨的比较研究

    Institute of Scientific and Technical Information of China (English)

    李想; 董纪元; 彭江; 汪爱媛; 睢翔; 赵斌; 刘道宏

    2011-01-01

    Objective To analyze the difference in subcutaneous ectopic osteogenesis induced by porous calcium phosphate cement (CPC) and gelatin sponge as a carrier of recombinant bone morphogenetic protein-2 (rhBMP-2). Methods Thirty Sprague Dawley rats with an average body weight of 200g were divided into groups A-D. CPC+rhBMP-2, CPC, gelatin sponge+rhBMP-2, and gelatin sponge were implanted into the rats after anesthesia. Ten rats were killed 2, 4 and 8 weeks after they were fed under sterile environment. Bone tissue samples were collected from the implantation sites. Tissue mineral density (TMD) and trabecular thickness were detected with micro-CT scanner and analyzed with SPSS 1 OX) statistical software. Bone tissue was fixed in 4% paraformaldehyde for 2 days, embedded in paraffin, and cut into sections. The sections were stained with H&E to observe their histological change. Results The tissue mineral density and trabecular thickness of the samples with rhBMP-2 were higher in two experimental groups 2,4 and 8 weeks after implantation, which increased with the prolongation of time (P<0.05). Conclusion Porous CPC can be used as a carrier of rhBMP-2 for osteogenesis.%目的 分析多孔自固化磷酸钙骨水泥(Calcium Phosphate Cement,CPC)和明胶海绵复合重组人骨形态发生蛋白(Recombinantion Humen Bone Morphogenetic Protein-2,rhBMP-2)诱导大鼠皮下异位成骨的区别.方法 平均质量200g SD大鼠30只,麻醉后分别植入A:多孔CPC复合rhBMP-2(2μg);B:多孔CPC;C:明胶海绵复合rhBMP-2(2μg);D:空白明胶海绵,无菌喂养后分别于2、4、8周各处死10只.对植入部位组织取材,分别进行micro-CT扫描,并使用Micview V2.1三维重建处理软件扫及ABA骨形态分析软件检测,记录组织骨密度(Tissue Mineral Density,TMD)及骨小梁厚度(Trabecular Thickness,Tb.Th).运用SPSS10.0统计软件进行统计学分析.后行甲醛固定2周,石蜡包埋切片,HE染色进行组织学观察.结果 在2、4、8周时,加入rhBMP

  9. Allogeneic Platelet Releasate Preparations Derived via a Novel Rapid Thrombin Activation Process Promote Rapid Growth and Increased BMP-2 and BMP-4 Expression in Human Adipose-Derived Stem Cells.

    Science.gov (United States)

    McLaughlin, Michael; Gagnet, Paul; Cunningham, Elizabeth; Yeager, Randi; D'Amico, Michael; Guski, Katie; Scarpone, Michael; Kuebler, Daniel

    2016-01-01

    The administration of human adipose-derived stem cells (ASCs) represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR). ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase) and BMP-2 (4.7 ± 1.3-fold increase) and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease) and FGF-2 (33 ± 9.0-fold decrease). No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications.

  10. Allogeneic Platelet Releasate Preparations Derived via a Novel Rapid Thrombin Activation Process Promote Rapid Growth and Increased BMP-2 and BMP-4 Expression in Human Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Michael McLaughlin

    2016-01-01

    Full Text Available The administration of human adipose-derived stem cells (ASCs represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR. ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase and BMP-2 (4.7 ± 1.3-fold increase and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease and FGF-2 (33 ± 9.0-fold decrease. No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications.

  11. Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

    Science.gov (United States)

    Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

    2015-11-01

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells.

  12. Role of bone morphogenetic protein 2 in early acetabulum development and dysplastic acetabulum remodeling%BMP-2在髋臼软骨发育早期及发育不良髋臼软骨可逆性恢复过程中的作用研究

    Institute of Scientific and Technical Information of China (English)

    莫越强; 裴新红; 马瑞雪

    2015-01-01

    目的 研究BMP-2在髋臼软骨发育早期及发育不良髋臼软骨可逆性恢复过程中的作用.方法 通过伸髋内收、模拟襁褓体位固定新生大鼠双后肢,建立发育不良髋臼软骨模型.将髋臼标本经HE染色后观察比较正常及发育不良髋臼软骨组织形态学变化特点,同时用ELISA方法和PCR方法分别检测BMP-2、BMP-4、BMP-6、BMP-7的分泌及基因表达情况.将捆绑不同时间后的大鼠松绑,其中部分当场处死,其余大鼠继续喂养,最终至30日龄,建立发育不良髋臼软骨可逆性恢复模型.研究其髋臼软骨组织形态学恢复及BMP-2分泌变化情况.结果 正常大鼠髋臼软骨呈半圆形、容积大、表面光滑.发育不良髋臼软骨髋臼上缘肥厚,软骨发生变性,与周围组织分界不清.髋臼软骨BMP-2的分泌在正常大鼠7日龄和9日龄时出现高峰,分别为(13.7±0.29) ng/ml和(13.9±0.38) ng/ml.而在发育不良髋臼软骨中这一分泌高峰消失.在发育不良髋臼软骨可逆性恢复组,捆绑4d和6d的大鼠,BMP-2的分泌高峰出现延迟,都在15日龄时出现;而在捆绑8d及以上的大鼠,在松绑后继续喂养至30日龄,髋臼软骨组织形态无法恢复正常,并且BMP-2的分泌高峰未出现.结论 BMP-2的分泌可能是髋臼软骨早期发育情况的生物学标记之一.%Objective To explore the early-stage acetabulum development in normal and dysplastic acetabula and elucidate the function of bone morphogenetic protein 2 (BMP-2) in early acetabulum development and dysplastic acetabulum remodeling.Methods The rat model of dysplastic acetabulum was established by maintaining hips in a swaddling position.By analyzing the cartilage histologic characteristics,early-stage acetabulum developments were examined in normal and dysplastic acetabulum animals.Meantime,the mRNA expression and chondrocyte secretion of functional BMP-2,bone morphogenetic protein 4 (BMP-4),bone morphogenetic protein 6 (BMP-6) and bone

  13. Detection of growth factor binding to gelatin and heparin using a photonic crystal optical biosensor

    International Nuclear Information System (INIS)

    Drug-carrier interactions are important to protein controlled release systems to protect the protein from denaturation and ensure properly timed release. A novel photonic crystal biosensor was used to investigate a gelatin-protein controlled release system to determine the amount of protein bound to the carrier at physiological conditions. The Biomolecular Interaction Detection (BIND) system reflects a narrow band of wavelengths when white light is shone incident to the grating. As mass is deposited onto the surface, the peak wavelength value is shifted due to changes in the optical density of the biosensor. The BIND system was used to detect the binding of growth factors onto acidic gelatin, basic gelatin, and heparin on the sensor surface. Through a series of experiments, including functionalizing the sensor, adjusting the ionic strength of the solution, adjusting the substrate concentration, and minimizing non-specific signal, the adsorption of the gelatins and heparin on the sensor was enhanced. The binding interaction of recombinant human transforming growth factor (rhTGF)-β1 and bone morphogenetic protein (rhBMP)-2 with the two types of gelatin and heparin were investigated. The strength of the interaction between rhTGF-β1 and the substrates is in the following order: heparin > acidic gelatin > basic gelatin. RhBMP-2 bound to the substrates but with less intensity than TGF-β1: heparin > basic gelatin > acidic gelatin. This work provides support for the controlled release mechanism through degradation of the gelatin carrier.

  14. The effects of BMP-2 gene medication on the reconstruction of osteolytic bone defect around implant%假体周围骨溶解性骨缺损的转骨形态发生蛋白-2基因治疗

    Institute of Scientific and Technical Information of China (English)

    严孟宁; 戴尅戎; 汤亭亭

    2008-01-01

    目的 模拟假体周围骨溶解环境,观察骨形态发生蛋白-2(BMP-2)基因治疗假体周围骨溶解性骨缺损的效果.方法 成年雄性Beagle犬6条,于股骨外髁造成假体周围3mm骨缺损区.1条动物的左侧缺损区植入1ml平均直径1μm的钛合金颗粒混悬液,右侧植入1ml磷酸盐缓冲液(PBS),观察造模结果;其他5条动物双侧植入1ml钛合金颗粒混悬液,于术后2个月取出假体,植入转BMP-2基因冻干骨或单纯冻干骨,二次术后3个月取材,行组织学、组织形态计量学观察植骨愈合替代及界面骨整合情况.结果 颗粒造模术后2个月可见典型的骨溶解界膜组织形成.翻修术后3个月,冻干骨组见较多植骨残余,假体-骨界面基本为软组织界膜,假体骨接触率(BIC)为(1.38±1.22)%;基因治疗组见少量植骨残余,假体-骨界面有点状骨接触,BIC为(12.96±1.61)%,两组差异有统计学意义(P<0.01).结论 采用BMP-2基因治疗可提高假体周围骨溶解性骨缺损的界面骨整合.%Objective With the osteolystic model,the simulating revision was done to investigate the effects of BMP-2 gene therapy on the reconstruction of periprosthetic bone defect.Methods A 3 mm bone defect around Ti alloy implant was created in both femoral lateral condyla of 6 adult Beagle dogs.One animal was left as a model to evaluate the effects of particles on the defect,with 1 ml Ti alloy particles averaged diameter of 1μm implanted on the left bone defect and 1 ml PBS on the right.Ten defects of the other 5 animals were implanted 1 ml Ti alloy particle and the revisions were done 2 months postoperatively.With the impaction grafting technique,two defects of each animal were reconstructed with freeze-dried allograft (FDB group),freeze-dried allograft loading autogenous bone marrow stromal cells transfected by Adv-BMP2 gene (gene group) respectively.The allograft healing and osseointegration of bone-implant interface were evaluated by histological

  15. LRP4 Is Critical for Neuromuscular Junction Maintenance

    OpenAIRE

    Barik, Arnab; Lu, Yisheng; Sathyamurthy, Anupama; Bowman, Andrew; Shen, Chengyong; Li, Lei(Beijing Institute of Petrochemical Technology, Beijing, 102617, People's Republic of China); Xiong, Wen-Cheng; Mei, Lin

    2014-01-01

    The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critic...

  16. Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B

    DEFF Research Database (Denmark)

    Olsen, Oddrun Elise; Wader, Karin Fahl; Hella, Hanne;

    2015-01-01

    BACKGROUND: Activins are members of the TGF-β family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling...... by activin A through canonical ALK4-ACVR2 receptor complexes activates the transcription factors SMAD2 and SMAD3. Activin A has a strong affinity to type 2 receptors, a feature that they share with some of the bone morphogenetic proteins (BMPs). Activin A is also elevated in myeloma patients with advanced...... disease and is involved in myeloma bone disease. RESULTS: In this study we investigated effects of activin A binding to receptors that are shared with BMPs using myeloma cell lines with well-characterized BMP-receptor expression and responses. Activin A antagonized BMP-6 and BMP-9, but not BMP-2 and BMP-4...

  17. Restoration of segmental bone defects by using chitosan-coated pressed calcium sulfate pellet com-bined with rhBMP-2%壳聚糖包衣加压硫酸钙片复合重组人骨形态发生蛋白-2修复兔节段性骨缺损

    Institute of Scientific and Technical Information of China (English)

    崔旭; 张伯勋

    2009-01-01

    Objective To compare the effect of calcium sulfate pellets made by different methods in repair of segmental radial defect of rabbits. Methods Eighty white New Zealand rabbits were sub-jected to defects of middle part of the left radial bone and divided into four groups according to repair ma-terials: control group (Group A, implanted with no artificial bone substitute), uncoated pressed calcium sulfate pellets (Group B), coated pressed calcium sulfate pellets (Group C) and coated pressed calcium sulfate pellets combined with rhBMP-2 (Group D). Histologic examination and biological test were done at 4, 8 and 12 weeks after operation. The data were processed with mono-factor variance analysis. Re-sults New bone formation was found on the defected bone in Group D and Group C, with better in Group D. The bone strength test showed that the anti-bending strength was (39.6±1.7) % in Group C and (47.5±2.1) % in Group D, which were higher than (21.3±2.7) % in Group A and (23.6±3.3) % in Group B, with higher anti-bending strength in Group D than that in Group C (F = 125.3 ,P <0.01). Conclusions For restoration of segmental bone defects, chitosan-coated pressed calcium sulfate pellet shows relatively high density and slightly slow resorption, which closely coincides with the growth rate of new bone. The coated pellet combined with rhBMP-2 can enhance its osteogeneais in restoring segmental Done defects.%目的 比较不同方法 制备的硫酸钙片修复兔桡骨节段性骨缺损的效果. 方法 新西兰大白兔80只随机数字表法分为A、B、C、D组,造成左桡骨中段骨缺损,采用三种经不同方法 制备的硫酸钙片修复.A组:空白对照组;B组:加压方法 制备的硫酸钙组;C组:壳聚糖包衣的加压硫酸钙组;D组:壳聚糖包衣的复合重组人骨形态发生蛋白-2(rhBMP-2)加压硫酸钙组.术后4,8,12周进行组织学检查和生物力学测试,实验数据采用单因素方差分析. 结果 D组、C组骨缺损愈合,而

  18. Binding Procurement

    Science.gov (United States)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  19. Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

    Science.gov (United States)

    Picke, Ann-Kristin; Salbach-Hirsch, Juliane; Hintze, Vera; Rother, Sandra; Rauner, Martina; Kascholke, Christian; Möller, Stephanie; Bernhardt, Ricardo; Rammelt, Stefan; Pisabarro, M Teresa; Ruiz-Gómez, Gloria; Schnabelrauch, Matthias; Schulz-Siegmund, Michaela; Hacker, Michael C; Scharnweber, Dieter; Hofbauer, Christine; Hofbauer, Lorenz C

    2016-07-01

    Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function. PMID:27131598

  20. 复合骨形态蛋白-2的聚乳酸-乙醇酸共聚物/磷酸三钙人工骨结合带血供组织修复羊大段骨缺损的试验研究%An experimental research of composite of BMP-2 and PLGA/tricalcium phosphate with vascularized ulna or vascularized periosteum in repairing large segmental defect of sheeps' radius

    Institute of Scientific and Technical Information of China (English)

    徐建强; 张树明; 周密; 李长庚

    2012-01-01

    Objective To study the effects of the composite of PLGA-TCP-BMP-2 artificial bone with vascularized ulna or vascularized periosteum in repairing the large segmental defect of sheep's radius. Methods Thirty millimeter defects were made in middle segments of sheep's radius and ulna. In group A, artificial bone with vascularized ulna were implanted into the radial defects. Artificial bone with vascularized periosteum were implanted into the radial defects in group B. Artificial bone were implanted into the radial defects in group C. No material was implanted in group D. Plates were used to fix the bone. X ray was used to evaluate the repairing effects. Animals were sacrificed 24 weeks after operation to make CT analysis and histological evaluation. Radiological and scoring histological systems were used to evaluate the plains. Results Twenty four weeks after surgery, radiological scoring systems revealed that group A was better than other groups. Evaluation of the morphologic features of the regenerating bone showed that active osteogenesis occurred at the site of bone defect and complete bony union was confirmed in animals of group A. In group B, corporation between new bone and fracture site could be seen. However, the lamellar bone was rather slim. In group C, new lamellar bone and lacuna were found in deranged alignment. In group D, deranged collagenous fibrous tissue, instead of osseointegration was found at the site of bone defect. Within the porous structure in animals of group A, B, and C, no residual artificial materials or muscle fibers were observed. According to histological scoring systems revealed that group A was better than other groups. Scores of group D was lower than other groups. Conclusion The composite of BMP-2 and PLGA/tricalcium phosphate with vascularized ulna or vascularized periosteum could repair large segmental defect of sheeps' radius satisfactorily.%目的 研究复合骨形态蛋白-2的聚乳酸-乙醇酸共聚物/磷酸三钙(PLGA-TCP-BMP

  1. Insulin-like growth factor binding protein-3 affects osteogenic efficacy on dental implants in rat mandible

    International Nuclear Information System (INIS)

    Insulin like growth factor binding protein-3 (IGFBP-3) in bone cells and its utilization in dental implants have not been well studied. The aim of this study was to determine the osteogenic efficacy of chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 coated titanium (Ti) implants. Ch-GNPs were conjugated with IGFBP-3 plasmid DNA through a coacervation process. Conjugation was cast over Ti surfaces, and cells were seeded on coated surfaces. For in vitro analysis the expression of different proteins was analyzed by immunoblotting. For in vivo analysis, Ch-GNP/IGFBP-3 coated implants were installed in rat mandibles. Four weeks post-implantation, mandibles were examined by microcomputed tomography (μCT), immunohistochemistry, hematoxylin & eosin and tartrate resistance acid phosphatase staining. In vitro overexpressed Ch-GNP/IGFBP-3 coated Ti surfaces was associated with activation of extracellular signal related kinase (ERK), inhibition of the stress activated protein c-Jun N-terminal kinase (JNK) and enhanced bone morphogenetic protein (BMP)-2 and 7 compared to control. Further, in vivo, Ch-GNP/IGFBP-3 coated implants were associated with inhibition of implant induced osteoclastogenesis molecules, receptor activator of nuclear factor kappa-B ligand (RANKL) and enhanced expression of osteogenic molecules including BMP2/7 and osteopontin (OPN). The μCT analysis demonstrated that IGFBP-3 increased the volume of newly formed bone surrounding the implants compared to control (n = 5; p < 0.05). These results support the view that IGFBP-3 overexpression diminishes osteoclastogenesis and enhances osteogenesis of Ti implants, and can serve as a potent molecule for the development of good implantation. - Highlights: • Chitosan gold nanoparticles were conjugated with IGFBP-3 and coated onto surface of the titanium implants for gene delivery to bone. • Implants were inserted in rat mandible for 4 weeks. • Parameters studied: histopathology and radiology.

  2. Insulin-like growth factor binding protein-3 affects osteogenic efficacy on dental implants in rat mandible

    Energy Technology Data Exchange (ETDEWEB)

    Bhattarai, Govinda; Lee, Young-Hee [Department of Oral Biochemistry, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Lee, Min-Ho [Department of Dental Materials, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Park, Il-Song [Division of Advanced Materials Engineering, Research Center for Advanced Materials, Development and Institute of Biodegradable Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Yi, Ho-Keun, E-mail: yihokn@chonbuk.ac.kr [Department of Oral Biochemistry, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of)

    2015-10-01

    Insulin like growth factor binding protein-3 (IGFBP-3) in bone cells and its utilization in dental implants have not been well studied. The aim of this study was to determine the osteogenic efficacy of chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 coated titanium (Ti) implants. Ch-GNPs were conjugated with IGFBP-3 plasmid DNA through a coacervation process. Conjugation was cast over Ti surfaces, and cells were seeded on coated surfaces. For in vitro analysis the expression of different proteins was analyzed by immunoblotting. For in vivo analysis, Ch-GNP/IGFBP-3 coated implants were installed in rat mandibles. Four weeks post-implantation, mandibles were examined by microcomputed tomography (μCT), immunohistochemistry, hematoxylin & eosin and tartrate resistance acid phosphatase staining. In vitro overexpressed Ch-GNP/IGFBP-3 coated Ti surfaces was associated with activation of extracellular signal related kinase (ERK), inhibition of the stress activated protein c-Jun N-terminal kinase (JNK) and enhanced bone morphogenetic protein (BMP)-2 and 7 compared to control. Further, in vivo, Ch-GNP/IGFBP-3 coated implants were associated with inhibition of implant induced osteoclastogenesis molecules, receptor activator of nuclear factor kappa-B ligand (RANKL) and enhanced expression of osteogenic molecules including BMP2/7 and osteopontin (OPN). The μCT analysis demonstrated that IGFBP-3 increased the volume of newly formed bone surrounding the implants compared to control (n = 5; p < 0.05). These results support the view that IGFBP-3 overexpression diminishes osteoclastogenesis and enhances osteogenesis of Ti implants, and can serve as a potent molecule for the development of good implantation. - Highlights: • Chitosan gold nanoparticles were conjugated with IGFBP-3 and coated onto surface of the titanium implants for gene delivery to bone. • Implants were inserted in rat mandible for 4 weeks. • Parameters studied: histopathology and radiology.

  3. Control of growth factor binding and release in bisphosphonate functionalized hydrogels guides rapid differentiation of precursor cells in vitro.

    Science.gov (United States)

    Kootala, Sujit; Zhang, Yu; Ghalib, Sara; Tolmachev, Vladimir; Hilborn, Jöns; Ossipov, Dmitri A

    2016-02-01

    An in situ cross-linkable hyaluronan hydrogel functionalized with bisphosphonate (BP) groups allows tunable release of bone morphogenetic protein-2 (BMP-2) determined by the amount of BP groups. The high affinity of matrix-anchored BP groups towards BMP-2 permits guided differentiation of entrapped progenitor cells in 3-D cultures. PMID:26610690

  4. Control of growth factor binding and release in bisphosphonate functionalized hydrogels guides rapid diff erentiation of precursor cells in vitro

    OpenAIRE

    Kootala, Sujit; Zhang, Yu; Ghalib, Sara; Tolmachev, Vladmir; Hilborn, Jöns; Ossipov, Dmitri

    2016-01-01

    An in situ cross-linkable hyaluronan hydrogel functionalized with bisphosphonate (BP) groups allows tunable release of bone morphogenetic protein-2 (BMP-2) determined by the amount of BP groups. The high affinity of matrix-anchored BP groups towards BMP-2 permits guided differentiation of entrapped progenitor cells in 3-D cultures.

  5. Analyzing binding data.

    Science.gov (United States)

    Motulsky, Harvey J; Neubig, Richard R

    2010-07-01

    Measuring the rate and extent of radioligand binding provides information on the number of binding sites, and their affinity and accessibility of these binding sites for various drugs. This unit explains how to design and analyze such experiments.

  6. Total iron binding capacity

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003489.htm Total iron binding capacity To use the sharing features on this page, please enable JavaScript. Total iron binding capacity (TIBC) is a blood test to ...

  7. Plant Hormone Binding Sites

    OpenAIRE

    Napier, Richard

    2004-01-01

    • Aims Receptors for plant hormones are becoming identified with increasing rapidity, although a frustrating number remain unknown. There have also been many more hormone‐binding proteins described than receptors. This Botanical Briefing summarizes what has been discovered about hormone binding sites, their discovery and descriptions, and will not dwell on receptor functions or activities except where these are relevant to understand binding.

  8. Analysis of binding heterogeneity.

    NARCIS (Netherlands)

    Nederlof, M.M.

    1992-01-01

    Binding heterogeneity, due to different functional groups on a reactive surface, plays an important role in the binding of small molecules or ions to many adsorbents, both in industrial processes and in natural environments. The binding heterogeneity is described by a distribution of affinity consta

  9. Analyzing radioligand binding data.

    Science.gov (United States)

    Motulsky, Harvey; Neubig, Richard

    2002-08-01

    Radioligand binding experiments are easy to perform, and provide useful data in many fields. They can be used to study receptor regulation, discover new drugs by screening for compounds that compete with high affinity for radioligand binding to a particular receptor, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling, via measurements of agonist binding and its regulation by ions, nucleotides, and other allosteric modulators. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.

  10. Python bindings for libcloudph++

    OpenAIRE

    Jarecka, Dorota; Arabas, Sylwester; Del Vento, Davide

    2015-01-01

    This technical note introduces the Python bindings for libcloudph++. The libcloudph++ is a C++ library of algorithms for representing atmospheric cloud microphysics in numerical models. The bindings expose the complete functionality of the library to the Python users. The bindings are implemented using the Boost.Python C++ library and use NumPy arrays. This note includes listings with Python scripts exemplifying the use of selected library components. An example solution for using the Python ...

  11. DNS & Bind Cookbook

    CERN Document Server

    Liu, Cricket

    2011-01-01

    The DNS & BIND Cookbook presents solutions to the many problems faced by network administrators responsible for a name server. Following O'Reilly's popular problem-and-solution cookbook format, this title is an indispensable companion to DNS & BIND, 4th Edition, the definitive guide to the critical task of name server administration. The cookbook contains dozens of code recipes showing solutions to everyday problems, ranging from simple questions, like, "How do I get BIND?" to more advanced topics like providing name service for IPv6 addresses. It's full of BIND configuration files that yo

  12. Python bindings for libcloudph++

    CERN Document Server

    Jarecka, Dorota; Del Vento, Davide

    2015-01-01

    This technical note introduces the Python bindings for libcloudph++. The libcloudph++ is a C++ library of algorithms for representing atmospheric cloud microphysics in numerical models. The bindings expose the complete functionality of the library to the Python users. The bindings are implemented using the Boost.Python C++ library and use NumPy arrays. This note includes listings with Python scripts exemplifying the use of selected library components. An example solution for using the Python bindings to access libcloudph++ from Fortran is presented.

  13. Melanin-binding radiopharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Packer, S; Fairchild, R G; Watts, K P; Greenberg, D; Hannon, S J

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed. (PSB)

  14. DNS BIND Server Configuration

    Directory of Open Access Journals (Sweden)

    Radu MARSANU

    2011-01-01

    Full Text Available After a brief presentation of the DNS and BIND standard for Unix platforms, the paper presents an application which has a principal objective, the configuring of the DNS BIND 9 server. The general objectives of the application are presented, follow by the description of the details of designing the program.

  15. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues.

    Science.gov (United States)

    Myers, Jennifer S; von Lersner, Ariana K; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  16. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues

    Science.gov (United States)

    Myers, Jennifer S.; von Lersner, Ariana K.; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  17. How are ion pumps and agrin signaling integrated?

    DEFF Research Database (Denmark)

    Tidow, Henning; Aperia, Anita; Nissen, Poul

    2010-01-01

    Na(+),K(+)-ATPase (NKA) has a fundamental role in ion transport across the plasma membrane of animal cells and uses approximately 50% of brain energy consumption. Recent work has uncovered additional roles for NKA in signal transduction. How might such different functions of the sodium-potassium ......Na(+),K(+)-ATPase (NKA) has a fundamental role in ion transport across the plasma membrane of animal cells and uses approximately 50% of brain energy consumption. Recent work has uncovered additional roles for NKA in signal transduction. How might such different functions of the sodium...

  18. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... as: Testosterone-estrogen Binding Globulin; TeBG Formal name: Sex Hormone Binding Globulin Related tests: Testosterone , Free Testosterone, ... I should know? How is it used? The sex hormone binding globulin (SHBG) test may be used ...

  19. Cellulose binding domain proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL); Shpiegl, Itai (Rehovot, IL); Goldstein, Marc (Davis, CA); Doi, Roy (Davis, CA)

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  20. Sequential memory: Binding dynamics

    Science.gov (United States)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

  1. Lectin binding in meningiomas.

    Science.gov (United States)

    Kleinert, R; Radner, H

    1987-01-01

    Forty-two meningiomas of different morphological sub-type were examined to determine their pattern of binding to 11 different lectins which characterize cell surface components such as carbohydrate residues. Histiocytic and xanthoma cells within meningiomas could be demonstrated with six different lectins: wheat germ agglutinin (WGA), peanut agglutinin (PNA) Bauhinia purpurea agglutinin (BPA), Helix pomatia agglutinin (HPA), Vicia fava agglutinin (VFA) and Soyabean agglutinin (SBA). Vascular elements including endothelial cells and intimal cells, bound Ulex europaeus agglutinin type 1 (UEA 1), WGA and HPA. The fibrous stroma in fibrous and fibroblastic meningiomas bound PNA, Laburnum alpinum agglutinin (LAA) and SBA. Tumour cells in meningotheliomatous meningiomas and some areas of anaplastic meningiomas bound Concanavalin A, PNA, LAA and VFA whereas tumour cells in fibrous and fibroblastic meningiomas bound BPA, LAA and VFA. Lectin binding has proved to be of value in detecting histiocytic and xanthoma cells together with vascular elements within meningiomas. In addition, the different lectin binding patterns allow different histological sub-types of meningioma to be distinguished although the biological significance of the binding patterns is unclear. PMID:3658105

  2. Bone marrow mesenchymal stem cells, collagen scaffold and BMP-2 for rat spinal fusio

    OpenAIRE

    Arrabal, Pilar M.; de Visser, R; Cifuentes, Manuel; Becerra Ratia, José; Jiménez-Enjuto, E.

    2013-01-01

    The use of autograft for posterolateral spinal fusion, continue being considered the gold standard for the treatment of spine pathologies. However, due to complications such as donor site morbidity, increased operating time, and limited supply, the use of allograft has become an acceptable practice especially in multisegment arthrodesis or in patients with previous graft harvests. Since their use involves the risk of immune response or disease transmission and fusion rates are not as good as ...

  3. Vessel formation is induced prior to the appearance of cartilage in BMP-2-mediated heterotopic ossification

    Science.gov (United States)

    Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. ...

  4. Stimulation of porcine bone marrow stromal cells by hyaluronan, dexamethasone and rhBMP-2

    DEFF Research Database (Denmark)

    Zou, Xuenong; Li, Haisheng; Chen, Li;

    2004-01-01

    In the interest of optimizing osteogenesis in in vitro, the present study sought to determine how porcine bone marrow stromal cell (BMSc) would respond to different concentrations of hyaluronan (HY) and its different combinations with dexamethasone (Dex) and recombinant human bone morphogenic pro...

  5. Derivation of a novel undifferentiated human foetal phenotype in serum-free cultures with BMP-2

    OpenAIRE

    Mirmalek-Sani, Sayed-Hadi; Stokes, Paula J; Tare, Rahul S; Ralph, Esther J; Inglis, Stefanie; Hanley, Neil A.; Franchesca D. Houghton; Oreffo, Richard OC

    2009-01-01

    Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduce...

  6. Iron transferrin regulates hepcidin synthesis in primary hepatocyte culture through hemojuvelin and BMP2/4

    OpenAIRE

    Lin, Lan; Valore, Erika V.; Nemeth, Elizabeta; Goodnough, Julia B; Gabayan, Victoria; Ganz, Tomas

    2007-01-01

    The peptide hormone hepcidin is the principal regulator of systemic iron homeostasis. We examined the pathway by which iron stimulates the production of hepcidin. In humans who ingested 65 mg of iron, the increase in transferrin saturation preceded by hours the increase in urinary hepcidin excretion. Increases in urinary hepcidin concentrations were proportional to the increment in transferrin saturation. Paradoxically, in previous studies in primary hepatocytes and cell lines, hepcidin respo...

  7. Sustained Release of BMP-2 in Bioprinted Alginate for Osteogenicity in Mice and Rats

    NARCIS (Netherlands)

    Poldervaart, M.T.; Wang, H.; Stok, J. van der; Weinans, H.; Leeuwenburgh, S.C.G.; Oner, F.C.; Dhert, W.J.; Alblas, J.

    2013-01-01

    The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the s

  8. Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats

    NARCIS (Netherlands)

    Poldervaart, M.T.; Wang, H.; Van der Stok, J.; Weinans, H.H.; Leeuwenburgh, S.C.G.; Cumhur Öner, F.; Dhert, W.J.A.; Alblas, J.

    2013-01-01

    The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the s

  9. Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration

    NARCIS (Netherlands)

    Kempen, Diederik H. R.; Lu, Lichun; Heijink, Andras; Hefferan, Theresa E.; Creemers, Laura B.; Maran, Avudaiappan; Yaszemski, Michael J.; Dhert, Wouter J. A.

    2009-01-01

    Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone form

  10. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan;

    2005-01-01

    Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known...... to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...

  11. Carboplatin binding to histidine

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M. [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom); Diederichs, Kay [University of Konstanz, D-78457 Konstanz (Germany); Kroon-Batenburg, Loes M. J. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Levy, Colin [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Schreurs, Antoine M. M. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  12. Analytic QCD Binding Potentials

    CERN Document Server

    Fried, H M; Grandou, T; Sheu, Y -M

    2011-01-01

    This paper applies the analytic forms of a recent non-perturbative, manifestly gauge- and Lorentz-invariant description (of the exchange of all possible virtual gluons between quarks ($Q$) and/or anti-quarks ($\\bar{Q}$) in a quenched, eikonal approximation) to extract analytic forms for the binding potentials generating a model $Q$-$\\bar{Q}$ "pion", and a model $QQQ$ "nucleon". Other, more complicated $Q$, $\\bar{Q}$ contributions to such color-singlet states may also be identified analytically. An elementary minimization technique, relevant to the ground states of such bound systems, is adopted to approximate the solutions to a more proper, but far more complicated Schroedinger/Dirac equation; the existence of possible contributions to the pion and nucleon masses due to spin, angular momentum, and "deformation" degrees of freedom is noted but not pursued. Neglecting electromagnetic and weak interactions, this analysis illustrates how the one new parameter making its appearance in this exact, realistic formali...

  13. Quarkonium Binding and Entropic Force

    CERN Document Server

    Satz, Helmut

    2015-01-01

    A Q-Qbar bound state represents a balance between repulsive kinetic and attractive potential energy. In a hot quark-gluon plasma, the interaction potential experiences medium effects. Color screening modifies the attractive binding force between the quarks, while the increase of entropy with Q-Qbar separation gives rise to a growing repulsion. We study the role of these phenomena for in-medium Q-Qbar binding and dissociation. It is found that the relevant potential for Q-Qbar binding is the free energy F; with increasing Q-Qbar separation, further binding through the internal energy U is compensated by repulsive entropic effects.

  14. Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities

    OpenAIRE

    Ader, Lauren; Jones, David N. M.; Lin, Hai

    2010-01-01

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as cr...

  15. Binding Energy and Enzymatic Catalysis.

    Science.gov (United States)

    Hansen, David E.; Raines, Ronald T.

    1990-01-01

    Discussed is the fundamental role that the favorable free energy of binding of the rate-determining transition state plays in catalysis. The principle that all of the catalytic factors discussed are realized by the use of this binding energy is reviewed. (CW)

  16. 基于同位素标记相对和绝对定量技术核因子-KB必需分子结合的小分子多肽改善肿瘤坏死因子-α抑制成骨细胞分化的蛋白质组学研究%Isobaric tags for relative and absolute quantitation-based quantitative protein expression profiling of NEMO-binding domain peptide promotes osteoblast differentiation impaired by tumor necrosis factor alpha

    Institute of Scientific and Technical Information of China (English)

    戚芮榛; 许长鹏; 周一林; 侯毅龙; 冯冬阳; 江艺; 余斌

    2015-01-01

    目的 应用同位素标记相对和绝对定量(iTRAQ)技术观察核因子-κB(NF-κB)必需分子结合的小分子多肽(NBD)干预肿瘤坏死因子-α(TNF-α)刺激下成骨细胞分化过程中蛋白质表达组的变化.方法 肌原C2C12细胞接种于骨形态发生蛋白-2(BMP-2)诱导分化体系中诱导作为分化细胞模型,实验分为3组:对照组(B组)即分化细胞模型未加其他刺激,实验组(BT组)即分化细胞模型添加TNF-α,干预组(BTP组)即分化细胞模型添加TNF-α及NBD多肽,共同孵育分化7d后提取蛋白,以iTRAQ试剂标记后进行质谱检测并以软件分析差异表达的蛋白质.结果 TNF-α明显抑制成骨细胞分化,而NBD多肽可部分改善TNF-α对成骨细胞分化的抑制.iTRAQ试剂标记的B组与B+T组细胞蛋白质表达谱分析筛选出明显差异表达蛋白质点为76个,表达上调的蛋白质点59个,下调的蛋白质点17个;BT组与BTP组细胞蛋白质表达谱分析筛选出明显差异表达蛋白质点为43个,表达上调的蛋白质点25个,下调的蛋白质点18个.其中3组间成骨细胞特异因子(Postn)、ATP酶钙离子运输因子(Atp2a3)、SR依赖蛋白CTD关联因子-1(Scafl)、肌动蛋白-F交联蛋白(Actn2)、ATP合成酶,氢离子转运,线粒体复合体-F1,Delta亚基(Atp5d)、延伸体乙酰转移酶复合体亚基-2(Elp2)、Atp5]、C1型尼曼-匹克疾病(Npc1)等8个蛋白表达出现动态变化,提示上述蛋白可能与炎症刺激成骨分化机制有关,NBD多肽之外尚有其他药物靶点干预炎症对成骨分化的抑制.结论 iTRAQ技术是研究细胞分子蛋白改变的有效的蛋白质组学方法.Postn、Atp2a3、Scaf1、Actn2、Atp5d、Elp2、Atp5l及Npc1可能作为炎症刺激成骨分化机制研究的候选靶标.%Objective To screen for the differentially expressed proteins of NEMO-binding domain peptide promotes osteoblast differentiation impaired by tumor necrosis factor alpha.Methods The myoblast C2C12 cells as

  17. Cooperative binding: a multiple personality.

    Science.gov (United States)

    Martini, Johannes W R; Diambra, Luis; Habeck, Michael

    2016-06-01

    Cooperative binding has been described in many publications and has been related to or defined by several different properties of the binding behavior of the ligand to the target molecule. In addition to the commonly used Hill coefficient, other characteristics such as a sigmoidal shape of the overall titration curve in a linear plot, a change of ligand affinity of the other binding sites when a site of the target molecule becomes occupied, or complex roots of the binding polynomial have been used to define or to quantify cooperative binding. In this work, we analyze how the different properties are related in the most general model for binding curves based on the grand canonical partition function and present several examples which highlight differences between the cooperativity characterizing properties which are discussed. Our results mainly show that among the presented definitions there are not two which fully coincide. Moreover, this work poses the question whether it can make sense to distinguish between positive and negative cooperativity based on the macroscopic binding isotherm only. This article shall emphasize that scientists who investigate cooperative effects in biological systems could help avoiding misunderstandings by stating clearly which kind of cooperativity they discuss.

  18. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    High (17 nM) and low (603 nM) affinity binding sites for [3]tetrahydrotrazodone ([3] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [3]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [3] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [3]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  19. Structural studies of sugar binding proteins

    OpenAIRE

    Sooriyaarachchi, Sanjeewani

    2010-01-01

    Binding proteins, which are themselves non-enzymatic, play an important role in enzymatic reactions as well as non-enzymatic processes by providing a binding platform for the specific recognition of particular molecules. For example, periplasmic binding proteins play a vital role in nutrient uptake in Gram-negative bacteria. In the present study, three sugar binding proteins, including two periplasmic binding proteins and a β-glucan binding protein, are described. The glucose/galactose bindin...

  20. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen;

    2012-01-01

    profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides...... around the phosphorylated residue are important for the binding affinity of ILKAP. We conclude that solid-phase affinity pull-down of proteins from complex mixtures can be applied in phosphoproteomics and systems biology.......Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...

  1. Water binding in legume seeds

    Science.gov (United States)

    Vertucci, C. W.; Leopold, A. C.

    1987-01-01

    The physical status of water in seeds has a pivotal role in determining the physiological reactions that can take place in the dry state. Using water sorption isotherms from cotyledon and axis tissue of five leguminous seeds, the strength of water binding and the numbers of binding sites have been estimated using van't Hoff analyses and the D'Arcy/Watt equation. These parameters of water sorption are calculated for each of the three regions of water binding and for a range of temperatures. Water sorption characteristics are reflective of the chemical composition of the biological materials as well as the temperature at which hydration takes place. Changes in the sorption characteristics with temperature and hydration level may suggest hydration-induced structural changes in cellular components.

  2. Computational Prediction of RNA-Binding Proteins and Binding Sites.

    Science.gov (United States)

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  3. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  4. Skyrmions with low binding energies

    Directory of Open Access Journals (Sweden)

    Mike Gillard

    2015-06-01

    Full Text Available Nuclear binding energies are investigated in two variants of the Skyrme model: the first replaces the usual Skyrme term with a term that is sixth order in derivatives, and the second includes a potential that is quartic in the pion fields. Solitons in the first model are shown to deviate significantly from ansätze previously assumed in the literature. The binding energies obtained in both models are lower than those obtained from the standard Skyrme model, and those obtained in the second model are close to the experimental values.

  5. Skyrmions with low binding energies

    Energy Technology Data Exchange (ETDEWEB)

    Gillard, Mike, E-mail: m.n.gillard@leeds.ac.uk; Harland, Derek, E-mail: d.g.harland@leeds.ac.uk; Speight, Martin, E-mail: speight@maths.leeds.ac.uk

    2015-06-15

    Nuclear binding energies are investigated in two variants of the Skyrme model: the first replaces the usual Skyrme term with a term that is sixth order in derivatives, and the second includes a potential that is quartic in the pion fields. Solitons in the first model are shown to deviate significantly from ansätze previously assumed in the literature. The binding energies obtained in both models are lower than those obtained from the standard Skyrme model, and those obtained in the second model are close to the experimental values.

  6. Synthetic heparin-binding growth factor analogs

    Science.gov (United States)

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  7. Sex hormone binding globulin phenotypes

    DEFF Research Database (Denmark)

    Cornelisse, M M; Bennett, Patrick; Christiansen, M;

    1994-01-01

    Human sex hormone binding globulin (SHBG) is encoded by a normal and a variant allele. The resulting SHBG phenotypes (the homozygous normal SHBG, the heterozygous SHBG and the homozygous variant SHBG phenotype) can be distinguished by their electrophoretic patterns. We developed a novel detection...

  8. BINDING ISOTHERMS SURFACTANT-PROTEINS

    OpenAIRE

    Elena Irina Moater; Cristiana Radulescu; Ionica Ionita

    2011-01-01

    The interactions between surfactants and proteins shows some similarities with interactions between surfactants and polymers, but the hydrophobic amphoteric nature of proteins and their secondary and tertiary structure components make them different from conventional polymer systems. Many studies from the past about surfactant - proteins bonding used the dialysis techniques. Other techniques used to determine the binding isotherm, included ultrafiltration, ultracentrifugation, potentiometry, ...

  9. Cellulose binding domain fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL); Shpiegl, Itai (Rehovot, IL); Goldstein, Marc A. (Davis, CA); Doi, Roy H. (Davis, CA)

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  10. Protein binding assay for hyaluronate

    Energy Technology Data Exchange (ETDEWEB)

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  11. Antimicrobial Peptide-Lipid Binding Interactions and Binding Selectivity

    OpenAIRE

    Lad, Mitaben D.; Birembaut, Fabrice; Clifton, Luke A.; Frazier, Richard A.; Webster, John R. P.; Green, Rebecca J.

    2007-01-01

    Surface pressure measurements, external reflection-Fourier transform infrared spectroscopy, and neutron reflectivity have been used to investigate the lipid-binding behavior of three antimicrobial peptides: melittin, magainin II, and cecropin P1. As expected, all three cationic peptides were shown to interact more strongly with the anionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-(phosphor-rac-(1-glycerol)) (DPPG), compared to the zwitterionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-phosphocho...

  12. Characteristics of human erythrocyte insulin binding sites.

    OpenAIRE

    Okada, Yoshio

    1981-01-01

    Insulin and human erythrocyte cell membrane interactions were studied with respect to binding and dissociation. The per cent of specific binding of 125I-labeled insulin to erythrocytes was directly proportional to the cell concentration. The optimum pH for binding was 8.1. The initial binding rate was directly proportional to, and the steady state insulin binding was reversely proportional to, the incubation temperature. The per cent of specific binding of 125I-labeled insulin was 12.10 +/- 1...

  13. Dissection of the Critical Binding Determinants of Cellular Retinoic Acid Binding Protein II by Mutagenesis and Fluorescence Binding Assay

    OpenAIRE

    Vasileiou, Chrysoula; Lee, Kin Sing Stephen; Crist, Rachael M.; Vaezeslami, Soheila; Goins, Sarah M.; Geiger, James H.; Borhan, Babak

    2009-01-01

    The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by ...

  14. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B;

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...

  15. Binding effects and nuclear shadowing

    OpenAIRE

    Indumathi, D.; Wei ZHU

    1996-01-01

    The effects of nuclear binding on nuclear structure functions have so far been studied mainly at fixed target experiments, and there is currently much interest in obtaining a clearer understanding of this phenomenon. We use an existing dynamical model of nuclear structure functions, that gives good agreement with current data, to study this effect in a kinematical regime (low $x$, high $Q^2$) that can possibly be probed by an upgrade of {\\sc hera} at {\\sc desy} into a nuclear accelerator.

  16. Calcium binding by dietary fibre

    International Nuclear Information System (INIS)

    Dietary fibre from plants low in phytate bound calcium in proportion to its uronic-acid content. This binding by the non-cellulosic fraction of fibre reduces the availability of calcium for small-intestinal absorption, but the colonic microbial digestion of uronic acids liberates the calcium. Thus the ability to maintain calcium balance on high-fibre diets may depend on the adaptive capacity on the colon for calcium. (author)

  17. Material Binding Peptides for Nanotechnology

    Directory of Open Access Journals (Sweden)

    Urartu Ozgur Safak Seker

    2011-02-01

    Full Text Available Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.

  18. Anion binding in biological systems

    Science.gov (United States)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  19. Anion binding in biological systems

    Energy Technology Data Exchange (ETDEWEB)

    Feiters, Martin C [Department of Organic Chemistry, Institute for Molecules and Materials, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Meyer-Klaucke, Wolfram [EMBL Hamburg Outstation at DESY, Notkestrasse 85, D-22607 Hamburg (Germany); Kostenko, Alexander V; Soldatov, Alexander V [Faculty of Physics, Southern Federal University, Sorge 5, Rostov-na-Donu, 344090 (Russian Federation); Leblanc, Catherine; Michel, Gurvan; Potin, Philippe [Centre National de la Recherche Scientifique and Universite Pierre et Marie Curie Paris-VI, Station Biologique de Roscoff, Place Georges Teissier, BP 74, F-29682 Roscoff cedex, Bretagne (France); Kuepper, Frithjof C [Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA37 1QA, Scotland (United Kingdom); Hollenstein, Kaspar; Locher, Kaspar P [Institute of Molecular Biology and Biophysics, ETH Zuerich, Schafmattstrasse 20, Zuerich, 8093 (Switzerland); Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R, E-mail: m.feiters@science.ru.n [Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (Netherlands)

    2009-11-15

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L{sub 3} (2p{sub 3/2}) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  20. Solute-vacancy binding in aluminum

    International Nuclear Information System (INIS)

    Previous efforts to understand solute-vacancy binding in aluminum alloys have been hampered by a scarcity of reliable, quantitative experimental measurements. Here, we report a large database of solute-vacancy binding energies determined from first-principles density functional calculations. The calculated binding energies agree well with accurate measurements where available, and provide an accurate predictor of solute-vacancy binding in other systems. We find: (i) some common solutes in commercial Al alloys (e.g., Cu and Mg) possess either very weak (Cu), or even repulsive (Mg), binding energies. Hence, we assert that some previously reported large binding energies for these solutes are erroneous. (ii) Large binding energies are found for Sn, Cd and In, confirming the proposed mechanism for the reduced natural aging in Al-Cu alloys containing microalloying additions of these solutes. (iii) In addition, we predict that similar reduction in natural aging should occur with additions of Si, Ge and Au. (iv) Even larger binding energies are found for other solutes (e.g., Pb, Bi, Sr, Ba), but these solutes possess essentially no solubility in Al. (v) We have explored the physical effects controlling solute-vacancy binding in Al. We find that there is a strong correlation between binding energy and solute size, with larger solute atoms possessing a stronger binding with vacancies. (vi) Most transition-metal 3d solutes do not bind strongly with vacancies, and some are even energetically strongly repelled from vacancies, particularly for the early 3d solutes, Ti and V

  1. Erythropoietin binding protein from mammalian serum

    Energy Technology Data Exchange (ETDEWEB)

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  2. Erythropoietin binding protein from mammalian serum

    Energy Technology Data Exchange (ETDEWEB)

    Clemons, Gisela K. (Berkeley, CA)

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  3. Synthetic heparin-binding factor analogs

    Science.gov (United States)

    Pena, Louis A.; Zamora, Paul O.; Lin, Xinhua; Glass, John D.

    2010-04-20

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain, and preferably two peptide chains branched from a dipeptide branch moiety composed of two trifunctional amino acid residues, which peptide chain or chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a linker, which may be a hydrophobic linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  4. Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

    Science.gov (United States)

    Lehrberg, Jeffrey; Gardiner, David M

    2015-01-01

    We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response. PMID:25923915

  5. E. coli-Produced BMP-2 as a Chemopreventive Strategy for Colon Cancer : A Proof-of-Concept Study

    NARCIS (Netherlands)

    Yuvaraj, Saravanan; Al-Lahham, Sa'ad H.; Somasundaram, Rajesh; Figaroa, Patrick A.; Peppelenbosch, Maikel P.; Bos, Nicolaas A.

    2012-01-01

    Colon cancer is a serious health problem, and novel preventive and therapeutical avenues are urgently called for. Delivery of proteins with anticancer activity through genetically modified bacteria provides an interesting, potentially specific, economic and effective approach here. Interestingly, bo

  6. BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1α stability and MGMT expression

    OpenAIRE

    Persano, L; Pistollato, F; Rampazzo, E; Della Puppa, A; Abbadi, S; Frasson, C; Volpin, F; S. Indraccolo; Scienza, R; G. Basso

    2012-01-01

    Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in...

  7. Radiographic Assessment of Bone Formation Using rhBMP2 at Maxillary Periapical Surgical Defects: A Case Series.

    Science.gov (United States)

    Kumar, M Siva; Kumar, M Hari; Vishalakshi, K; Sabitha, H

    2016-04-01

    Periapical cysts are the most common inflammatory odontogenic cysts arising from untreated dental caries with pulp necrosis and periapical infection. The choice of treatment is often influenced by various factors like size, extension of the lesion, proximity to vital structures, systemic condition and compliance of the patient too. The treatment protocol for management of periapical cysts is still under discussion and options vary from conservative treatment by means of endodontic technique to surgical treatment like decompression or a marsupialisation or even to enucleation. Large bony defect secondary to periapical surgery compromising the tooth integrity often requires bone graft to enhance bone formation and thus restoring function at the earliest. The present case series included 10 patients who had established periapical pathology secondary to history of trauma on upper anterior teeth as well patients with history of carious teeth with an apparent failure in root canal therapy. All ten patients were treated with cyst enucleation and apiceotomy along with 1.4cc Recombinant Human Bone Morphogenetic Protein-2 soaked Absorbable Collagen Sponge implantation at surgical defect. Radiographs and clinical examinations were done upto 3 months to evaluate healing. Radiographic and clinical assessments revealed bone regeneration and restoration of the maxillary surgical defects in all 10 patients. No evidence of graft failure was noted. The Recombinant Human Bone Morphogenetic Protein-2 soaked Absorbable Collagen Sponge carrier is thus proved to be a viable option for the treatment of maxillary periapical surgical defects. PMID:27190972

  8. Radiographic Assessment of Bone Formation Using rhBMP2 at Maxillary Periapical Surgical Defects: A Case Series

    Science.gov (United States)

    Kumar, M. Hari; Vishalakshi, K.; Sabitha, H.

    2016-01-01

    Periapical cysts are the most common inflammatory odontogenic cysts arising from untreated dental caries with pulp necrosis and periapical infection. The choice of treatment is often influenced by various factors like size, extension of the lesion, proximity to vital structures, systemic condition and compliance of the patient too. The treatment protocol for management of periapical cysts is still under discussion and options vary from conservative treatment by means of endodontic technique to surgical treatment like decompression or a marsupialisation or even to enucleation. Large bony defect secondary to periapical surgery compromising the tooth integrity often requires bone graft to enhance bone formation and thus restoring function at the earliest. The present case series included 10 patients who had established periapical pathology secondary to history of trauma on upper anterior teeth as well patients with history of carious teeth with an apparent failure in root canal therapy. All ten patients were treated with cyst enucleation and apiceotomy along with 1.4cc Recombinant Human Bone Morphogenetic Protein-2 soaked Absorbable Collagen Sponge implantation at surgical defect. Radiographs and clinical examinations were done upto 3 months to evaluate healing. Radiographic and clinical assessments revealed bone regeneration and restoration of the maxillary surgical defects in all 10 patients. No evidence of graft failure was noted. The Recombinant Human Bone Morphogenetic Protein-2 soaked Absorbable Collagen Sponge carrier is thus proved to be a viable option for the treatment of maxillary periapical surgical defects. PMID:27190972

  9. Evaluation of collagen/heparin coated TCP/HA granules for long-term delivery of BMP-2

    NARCIS (Netherlands)

    Hannink, G.J.; Geutjes, P.J.; Daamen, W.F.; Buma, P.

    2013-01-01

    Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. However, a delivery system is essential to take advantage of the osteoinductive effect of BMPs. The purpose of this study was to develop a sustained delivery system for recombinant human bone morphogenetic protein-

  10. Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

    Science.gov (United States)

    Lehrberg, Jeffrey; Gardiner, David M

    2015-01-01

    We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

  11. Regulation of Axolotl (Ambystoma mexicanum Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

    Directory of Open Access Journals (Sweden)

    Jeffrey Lehrberg

    Full Text Available We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

  12. Gelatin Tight-Coated Poly(lactide-co-glycolide) Scaffold Incorporating rhBMP-2 for Bone Tissue Engineering

    OpenAIRE

    Juan Wang; Dongsong Li; Tianyi Li; Jianxun Ding; Jianguo Liu; Baosheng Li; Xuesi Chen

    2015-01-01

    Surface coating is the simplest surface modification. However, bioactive molecules can not spread well on the commonly used polylactone-type skeletons; thus, the surface coatings of biomolecules are typically unstable due to the weak interaction between the polymer and the bioactive molecules. In this study, a special type of poly(lactide-co-glycolide) (PLGA)-based scaffold with a loosened skeleton was fabricated by phase separation, which allowed gelatin molecules to more readily diffuse th...

  13. Drug binding properties of neonatal albumin

    DEFF Research Database (Denmark)

    Brodersen, R; Honoré, B

    1989-01-01

    Neonatal and adult albumin was isolated by gel chromatography on Sephacryl S-300, from adult and umbilical cord serum, respectively. Binding of monoacetyl-diamino-diphenyl sulfone, warfarin, sulfamethizole, and diazepam was studied by means of equilibrium dialysis and the binding data were analyzed...... by the method of several acceptable fitted curves. It was found that the binding affinity to neonatal albumin is less than to adult albumin for monoacetyl-diamino-diphenyl sulfone and warfarin. Sulfamethizole binding to the neonatal protein is similarly reduced when more than one molecule of the drug...... is bound per albumin molecule, and binding of the first sulfamethizole molecule is possibly reduced as well. Diazepam binds with equal affinity to the fetal and adult proteins. Among the two main albumin drug-binding functions, for warfarin and diazepam, the former is thus compromised in the newborn...

  14. Infinite sets and double binds.

    Science.gov (United States)

    Arden, M

    1984-01-01

    There have been many attempts to bring psychoanalytical theory up to date. This paper approaches the problem by discussing the work of Gregory Bateson and Ignacio Matte-Blanco, with particular reference to the use made by these authors of Russell's theory of logical types. Bateson's theory of the double bind and Matte-Blanco's bilogic are both based on concepts of logical typing. It is argued that the two theories can be linked by the idea that neurotic symptoms are based on category errors in thinking. Clinical material is presented from the analysis of a middle-aged woman. The intention is to demonstrate that the process of making interpretations can be thought of as revealing errors in thinking. Changes in the patient's inner world are then seen to be the result of clarifying childhood experiences based on category errors. Matte-Blanco's theory of bilogic and infinite experiences is a re-evaluation of the place of the primary process in mental life. It is suggested that a combination of bilogic and double bind theory provides a possibility of reformulating psychoanalytical theory.

  15. Erythropoietin binding sites in human foetal tissues

    Energy Technology Data Exchange (ETDEWEB)

    Pekonen, F.; Rosenloef, K.; Rutanen, E.-M.

    1987-01-01

    Using /sup 125/I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has a equilibrium association constant of 4.1-6.2 x 10/sup 9/l/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4-16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37 deg. C in 1 h and at 4 deg. C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked /sup 125/I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively.

  16. Binding characteristics of swine erythrocyte insulin receptors

    International Nuclear Information System (INIS)

    Crossbred gilts had 8.8 +/- 1.1% maximum binding of [125I]insulin to insulin receptors on erythrocytes. The number of insulin-binding sites per cell was 137 +/- 19, with a binding affinity ranging from 7.4 X 10(7)M-1 to 11.2 X 10(7)M-1 and mean of 8.8 X 10(7)M-1. Pregnant sows had a significant increase in maximum binding due to an increase in number of receptor sites per cell. Lactating sows fed a high-fiber diet and a low-fiber diet did not develop a significant difference in maximum binding of insulin. Sows fed the low-fiber diet had a significantly higher number of binding sites and a significantly lower binding affinity than did sows fed a high-fiber diet. Receptor-binding affinity was lower in the low-fiber diet group than in cycling gilts, whereas data from sows fed the high-fiber diet did not differ from data for cycling gilts. Data from this study indicated that insulin receptors of swine erythrocytes have binding characteristics similar to those in other species. Pregnancy and diet will alter insulin receptor binding in swine

  17. Erythropoietin binding sites in human foetal tissues

    International Nuclear Information System (INIS)

    Using 125I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has a equilibrium association constant of 4.1-6.2 x 109l/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4-16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37 deg. C in 1 h and at 4 deg. C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked 125I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively. (author)

  18. Adaptive evolution of transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  19. Synthetic LPS-Binding Polymer Nanoparticles

    Science.gov (United States)

    Jiang, Tian

    Lipopolysaccharide (LPS), one of the principal components of most gram-negative bacteria's outer membrane, is a type of contaminant that can be frequently found in recombinant DNA products. Because of its strong and even lethal biological effects, selective LPS removal from bioproducts solution is of particular importance in the pharmaceutical and health care industries. In this thesis, for the first time, a proof-of-concept study on preparing LPS-binding hydrogel-like NPs through facile one-step free-radical polymerization was presented. With the incorporation of various hydrophobic (TBAm), cationic (APM, GUA) monomers and cross-linkers (BIS, PEG), a small library of NPs was constructed. Their FITC-LPS binding behaviors were investigated and compared with those of commercially available LPS-binding products. Moreover, the LPS binding selectivity of the NPs was also explored by studying the NPs-BSA interactions. The results showed that all NPs obtained generally presented higher FITC-LPS binding capacity in lower ionic strength buffer than higher ionic strength. However, unlike commercial poly-lysine cellulose and polymyxin B agarose beads' nearly linear increase of FITC-LPS binding with particle concentration, NPs exhibited serious aggregation and the binding quickly saturated or even decreased at high particle concentration. Among various types of NPs, higher FITC-LPS binding capacity was observed for those containing more hydrophobic monomers (TBAm). However, surprisingly, more cationic NPs with higher content of APM exhibited decreased FITC-LPS binding in high ionic strength conditions. Additionally, when new cationic monomer and cross-linker, GUA and PEG, were applied to replace APM and BIS, the obtained NPs showed improved FITC-LPS binding capacity at low NP concentration. But compared with APM- and BIS-containing NPs, the FITC-LPS binding capacity of GUA- and PEG-containing NPs saturated earlier. To investigate the NPs' binding to proteins, we tested the NPs

  20. Why tight-binding theory?

    Science.gov (United States)

    Harrison, Walter A.

    2002-12-01

    In the context of computational physics other methods are more accurate, but tight-binding theory allows very direct physical interpretation and is simple enough to allow much more realistic treatments beyond the local density approximation. We address several important questions of this last category: How does the gap enhancement from Coulomb correlations vary from material to material? Should the enhanced gap be used for calculating the dielectric constant? For calculating the effective mass in k-dot-p theory? How valid is the scissors approximation? How does one line up bands at an interface? How should we match the envelope function at interfaces in effective-mass theory? Why can the resulting quantum-well states seem to violate the uncertainty principle? How should f-shell electrons be treated when they are intermediate between band-like and core-like? The answers to all of these questions are given and discussed.

  1. DNA binding hydroxyl radical probes

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Vicky J.; Konigsfeld, Katie M.; Aguilera, Joe A. [Department of Radiology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0610 (United States); Milligan, Jamie R., E-mail: jmilligan@ucsd.edu [Department of Radiology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0610 (United States)

    2012-01-15

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores, which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA. - Highlights: > Examined four aromatic groups as a means to detect hydroxyl radicals by fluorescence. > Coumarin system suffers from the fewest disadvantages. > Characterized its reactivity when linked to a hexa-arginine peptide.

  2. Gamma Oscillations and Visual Binding

    Science.gov (United States)

    Robinson, Peter A.; Kim, Jong Won

    2006-03-01

    At the root of visual perception is the mechanism the brain uses to analyze features in a scene and bind related ones together. Experiments show this process is linked to oscillations of brain activity in the 30-100 Hz gamma band. Oscillations at different sites have correlation functions (CFs) that often peak at zero lag, implying simultaneous firing, even when conduction delays are large. CFs are strongest between cells stimulated by related features. Gamma oscillations are studied here by modeling mm-scale patchy interconnections in the visual cortex. Resulting predictions for gamma responses to stimuli account for numerous experimental findings, including why oscillations and zero-lag synchrony are associated, observed connections with feature preferences, the shape of the zero-lag peak, and variations of CFs with attention. Gamma waves are found to obey the Schroedinger equation, opening the possibility of cortical analogs of quantum phenomena. Gamma instabilities are tied to observations of gamma activity linked to seizures and hallucinations.

  3. The helical structure of DNA facilitates binding

    Science.gov (United States)

    Berg, Otto G.; Mahmutovic, Anel; Marklund, Emil; Elf, Johan

    2016-09-01

    The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

  4. Methods for Improving Aptamer Binding Affinity

    OpenAIRE

    Hijiri Hasegawa; Nasa Savory; Koichi Abe; Kazunori Ikebukuro

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of a...

  5. Collective bargaining under non-binding contracts

    OpenAIRE

    Dobbelaere, S.; Luttens, R.I.

    2011-01-01

    We introduce collective bargaining in a static framework where the firm and its risk-neutral employees negotiate over wages in a non-binding contract setting. Our main result is the equivalence between the non-binding collective equilibrium wage-employment contract and the equilibrium contract under binding risk-neutral efficient bargaining. We also demonstrate that our non-cooperative equilibrium wages and profits coincide with the Owen values of the corresponding cooperative game with the c...

  6. A computational model for feature binding

    Institute of Scientific and Technical Information of China (English)

    SHI ZhiWei; SHI ZhongZhi; LIU Xi; SHI ZhiPing

    2008-01-01

    The "Binding Problem" is an important problem across many disciplines, including psychology, neuroscience, computational modeling, and even philosophy. In this work, we proposed a novel computational model, Bayesian Linking Field Model, for feature binding in visual perception, by combining the idea of noisy neuron model, Bayesian method, Linking Field Network and competitive mechanism.Simulation Experiments demonstrated that our model perfectly fulfilled the task of feature binding in visual perception and provided us some enlightening idea for future research.

  7. A computational model for feature binding

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The "Binding Problem" is an important problem across many disciplines, including psychology, neuroscience, computational modeling, and even philosophy. In this work, we proposed a novel computational model, Bayesian Linking Field Model, for feature binding in visual perception, by combining the idea of noisy neuron model, Bayesian method, Linking Field Network and competitive mechanism. Simulation Experiments demonstrated that our model perfectly fulfilled the task of feature binding in visual perception and provided us some enlightening idea for future research.

  8. RNA Binding Specificity of Drosophila Muscleblind†

    OpenAIRE

    Goers, Emily S.; Voelker, Rodger B.; Gates, Devika P.; Berglund, J. Andrew

    2008-01-01

    Members of the muscleblind family of RNA binding proteins found in Drosophila and mammals are key players in both the human disease myotonic dystrophy and the regulation of alternative splicing. Recently, the mammalian muscleblind-like protein, MBNL1, has been shown to have interesting RNA binding properties with both endogenous and disease-related RNA targets. Here we report the characterization of RNA binding properties of the Drosophila muscleblind protein Mbl. Mutagenesis of double-strand...

  9. Copper(II) binding properties of hepcidin

    OpenAIRE

    Kulprachakarn, Kanokwan; Chen, Yu-Lin; Kong, Xiaole; Arno, Maria Chiara; Hider, Robert Charles; Srichairatanakool, Somdet; Bansal, Sukhvinder

    2016-01-01

    Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding CuII and NiII through the amino terminal copper–nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of CuII with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidi...

  10. Advances on Plant Pathogenic Mycotoxin Binding Proteins

    Institute of Scientific and Technical Information of China (English)

    WANG Chao-hua; DONG Jin-gao

    2002-01-01

    Toxin-binding protein is one of the key subjects in plant pathogenic mycotoxin research. In this paper, new advances in toxin-binding proteins of 10 kinds of plant pathogenic mycotoxins belonging to Helminthosporium ,Alternaria ,Fusicoccum ,Verticillium were reviewed, especially the techniques and methods of toxin-binding proteins of HS-toxin, HV-toxin, HMT-toxin, HC-toxin. It was proposed that the isotope-labeling technique and immunological chemistry technique should be combined together in research of toxin-binding protein, which will be significant to study the molecular recognition mechanism between host and pathogenic fungus.

  11. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  12. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B;

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  13. Biodiscovery of aluminum binding peptides

    Science.gov (United States)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  14. CTCF Binding Polarity Determines Chromatin Looping

    NARCIS (Netherlands)

    de Wit, Elzo; Vos, Erica S M; Holwerda, Sjoerd J B; Valdes-Quezada, Christian; Verstegen, Marjon J A M; Teunissen, Hans; Splinter, Erik; Wijchers, Patrick J; Krijger, Peter H L; de Laat, Wouter

    2015-01-01

    CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that ch

  15. Localization-enhanced biexciton binding in semiconductors

    DEFF Research Database (Denmark)

    Langbein, Wolfgang Werner; Hvam, Jørn Märcher

    1999-01-01

    The influence of excitonic localization on the binding energy of biexcitons is investigated for quasi-three-dimensional and quasi-two-dimensional AlxGa1-xAs structures. An increase of the biexciton binding energy is observed for localization energies comparable to or larger than the free biexcito...

  16. Gravitational Binding Energy in Charged Cylindrical Symmetry

    CERN Document Server

    Sharif, M

    2014-01-01

    We consider static cylindrically symmetric charged gravitating object with perfect fluid and investigate the gravitational binding energy. It is found that only the localized part of the mass function provides the gravitational binding energy, whereas the non-localized part generated by the electric coupling does not contribute for such energy.

  17. Triazatriangulene as binding group for molecular electronics

    DEFF Research Database (Denmark)

    Wei, Zhongming; Wang, Xintai; Borges, Anders;

    2014-01-01

    The triazatriangulene (TATA) ring system was investigated as a binding group for tunnel junctions of molecular wires on gold surfaces. Self-assembled monolayers (SAMs) of TATA platforms with three different lengths of phenylene wires were fabricated, and their electrical conductance was recorded ...... with its high stability and directionality make this binding group very attractive for molecular electronic measurements and devices. (Figure Presented)....

  18. Binding of Quasi-Two-Dimensional Biexcitons

    DEFF Research Database (Denmark)

    Birkedal, Dan; Singh, Jai; Vadim, Lyssenko;

    1996-01-01

    Biexciton binding in GaAs quantum wells has been investigated for a range of well thicknesses (80-160 Angstrom) with spectrally resolved photoluminescence and transient degenerate four-wave mixing. Both light and heavy hole biexcitons are observed. The ratio of the binding energy of the heavy hole...

  19. (TH) diazepam binding to human granulocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bond, P.A.; Cundall, R.L.; Rolfe, B.

    1985-07-08

    (TH)-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These (TH)-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14 M). Binding of (TH) diazepam at 0 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table.

  20. [3H] diazepam binding to human granulocytes

    International Nuclear Information System (INIS)

    [3H]-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These [3H]-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14μM). Binding of [3H] diazepam at 00 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table

  1. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    Science.gov (United States)

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  2. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  3. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  4. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    Energy Technology Data Exchange (ETDEWEB)

    Serck-Hanssen, G.; Soevik, O.

    1987-12-28

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of /sup 125/I-insulin was carried out at 15/sup 0/C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table.

  5. Calmodulin Binding Proteins and Alzheimer's Disease.

    Science.gov (United States)

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  6. DNA Triplexes That Bind Several Cofactor Molecules.

    Science.gov (United States)

    Vollmer, Sven; Richert, Clemens

    2015-12-14

    Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA. PMID:26561335

  7. Copper(II) binding properties of hepcidin.

    Science.gov (United States)

    Kulprachakarn, Kanokwan; Chen, Yu-Lin; Kong, Xiaole; Arno, Maria C; Hider, Robert C; Srichairatanakool, Somdet; Bansal, Sukhvinder S

    2016-06-01

    Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding Cu(II) and Ni(II) through the amino terminal copper-nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of Cu(II) with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for Cu(II) than that of native hepcidin. The log K 1 value of hepcidin for Cu(II) was determined as 7.7. Cu(II) binds to albumin more tightly than hepcidin (log K 1 = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile Cu(II) present in blood will be bound to albumin. It is estimated that the concentration of Cu(II)-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin. PMID:26883683

  8. [Binding to chicken liver lactatedehydrogenase (author's transl)].

    Science.gov (United States)

    Lluís, C; Bozal, J

    1976-06-01

    Some information about the lactate dehydrogenase NAD binding site has been obtained by working with coenzymes analogs of incomplete molecules. 5'AMP, 5'-ADP, ATP, 5'-c-AMP and 3'(2)-AMP inhibit chicken liver LDH activity competitively with NADH. 5"-AMP and 5'-ADP show a stronger inhibition power than ATP, suggesting that the presence of one or two phosphate groups at the 5' position of adenosine, is essential for the binding of the coenzyme analogs at the enzyme binding site. Ribose and ribose-5'-P do not appear to inhibit the LDH activity, proving that purine base lacking mononucleotides do not bind to the enzyme. 5"-ADPG inhibits LDH activity in the exactly as 5'-ADP, showing that ribose moiety may be replaced by glucose, without considerable effects on the coenzyme analog binding. 2'-desoxidenosin-5'-phosphate proves to be a poorer inhibitor of the LDH activity than 5'-AMP, indicating that an interaction between the--OH groups and the amino-acids of the LDH active center takes place. Nicotinamide does not produce any inhibition effect, while NMN and CMP induce a much weaker inhibition than the adenine analogues, thus indicating a lesser binding capacity to the enzyme. Therefore, the LDH binding site seems to show some definite specificity towards the adenina groups of the coenzyme.

  9. Glycolipid binding preferences of Shiga toxin variants.

    Directory of Open Access Journals (Sweden)

    Sayali S Karve

    Full Text Available The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx, an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3 and globotetraosylceramide (Gb4 in the presence of cell membrane components such as phosphatidylcholine (PC, cholesterol (Ch and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.

  10. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  11. Estradiol Binds to Insulin and Insulin Receptor Decreasing Insulin Binding in vitro

    Directory of Open Access Journals (Sweden)

    Robert eRoot-Bernstein

    2014-07-01

    Full Text Available Rationale: Insulin resistance associated with hyperestrogenemias occurs in gestational diabetes mellitus, polycystic ovary syndrome, ovarian hyperstimulation syndrome, estrogen therapies, metabolic syndrome and obesity. The mechanism by which insulin and estrogen interact is unknown. We hypothesize that estrogen binds directly to insulin and the insulin receptor producing insulin resistance.Objectives: To determine the binding constants of steroid hormones to insulin, the insulin receptor, and insulin-like peptides derived from the insulin receptor; and to investigate the effect of estrogens on the binding of insulin to its receptor.Methods: Ultraviolet spectroscopy, capillary electrophoresis and NMR demonstrated estrogen binding to insulin and its receptor. Horse-radish peroxidase-linked insulin was used in an ELISA-like procedure to measure the effect of estradiol on binding of insulin to its receptor. Measurements: Binding constants for estrogens to insulin and the insulin receptor were determined by concentration-dependent spectral shifts. The effect of estradiol on insulin-HRP binding to its receptor was determined by shifts in the insulin binding curve. Main Results: Estradiol bound to insulin with a Kd of 12 x 10-9 M and to the insulin receptor with a Kd of 24 x 10-9 M, while other hormones had significantly less affinity. 200 nM estradiol shifted the binding curve of insulin to its receptor 0.8 log units to the right. Conclusions: Estradiol concentrations in many hyperestrogenemic syndromes are sufficient to interfere with insulin binding to its receptor producing significant insulin resistance.

  12. Acyl-coenzyme A binding protein (ACBP)

    DEFF Research Database (Denmark)

    Kragelund, B B; Knudsen, J; Poulsen, F M

    1999-01-01

    Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein...... and of the complex with palmitoyl-CoA using NMR spectroscopy. In the four alpha-helix bundle structure, a set of 21 highly conserved residues present in more that 90% of all known sequences of acyl-coenzyme A binding proteins constitutes three separate mini-cores. These residues are predominantly located...... at the helix-helix interfaces. From studies of a large set of mutant proteins the role of the conserved residues has been related to structure, function, folding and stability....

  13. Acyl-coenzyme A binding protein, ACBP

    DEFF Research Database (Denmark)

    Kragelund, Birthe Brandt; Knudsen, J.; Poulsen, Flemming

    1999-01-01

    Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein...... and of the complex with palmitoyl-CoA using NMR spectroscopy. In the four a-helix bundle structure, a set of 21 highly conserved residues present in more that 90% of all known sequences of acyl-coenzyme A binding proteins constitutes three separate mini-cores. These residues are predominantly located at the helix......-helix interfaces. From studies of a large set of mutant proteins the role of the conserved residues has been related to structure, function, folding and stability....

  14. Tau Induces Cooperative Taxol Binding to Microtubules

    Science.gov (United States)

    Ross, Jennifer; Santangelo, Christian; Victoria, Makrides; Fygenson, Deborah

    2004-03-01

    Taxol and tau are two ligands which stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds β tubulin in the MT interior. Tau is a MT-associated protein that binds both α and β tubulin on the MT exterior. Both taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts as a buttress to bundle, stiffen, and space MTs. A structural study recently suggested that taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau and yields a measure of taxol cooperativity when tau is present.

  15. Adjustment of legally binding local plans

    DEFF Research Database (Denmark)

    Hvingel, Line Træholt; Aunsborg, Christian; Christensen, Finn Kjær;

    2012-01-01

    Traditionally, and by law, new urban areas in Denmark are regulated and planned through legally binding local plans. Recently a tendency has occurred: The municipalities make the legally binding local plans quite open for future adjustment, and they are using a substantial amount of ‘empowerment......, which seem to be beyond the scope of the Danish Planning Act. This paper deals with this problem through case studies and a legal analysis of present law. If the combination of the legally binding local plan and subsequent added requirements is misused, it will weaken the legal rights of the citizens...... the considerations of legal rights, the extend of the legal use of empowerment provisions and the combination of the use of legal binding local plans and other legal instruments such as easements and sales agreements....

  16. Hardware device binding and mutual authentication

    Science.gov (United States)

    Hamlet, Jason R; Pierson, Lyndon G

    2014-03-04

    Detection and deterrence of device tampering and subversion by substitution may be achieved by including a cryptographic unit within a computing device for binding multiple hardware devices and mutually authenticating the devices. The cryptographic unit includes a physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generates a binding PUF value. The cryptographic unit uses the binding PUF value during an enrollment phase and subsequent authentication phases. During a subsequent authentication phase, the cryptographic unit uses the binding PUF values of the multiple hardware devices to generate a challenge to send to the other device, and to verify a challenge received from the other device to mutually authenticate the hardware devices.

  17. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.;

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  18. System Support for Managing Invalid Bindings

    CERN Document Server

    Das, Lachhman; Shah, Azhar; Khoumbati, Khalil; 10.5121/iju.2011.2303

    2011-01-01

    Context-aware adaptation is a central aspect of pervasive computing applications, enabling them to adapt and perform tasks based on contextual information. One of the aspects of context-aware adaptation is reconfiguration in which bindings are created between application component and remote services in order to realize new behaviour in response to contextual information. Various research efforts provide reconfiguration support and allow the development of adaptive context-aware applications from high-level specifications, but don't consider failure conditions that might arise during execution of such applications, making bindings between application and remote services invalid. To this end, we propose and implement our design approach to reconfiguration to manage invalid bindings. The development and modification of adaptive context-aware applications is a complex task, and an issue of an invalidity of bindings further complicates development efforts. To reduce the development efforts, our approach provides ...

  19. Genetics Home Reference: mannose-binding lectin deficiency

    Science.gov (United States)

    ... Health Conditions mannose-binding lectin deficiency mannose-binding lectin deficiency Enable Javascript to view the expand/collapse ... PDF Open All Close All Description Mannose-binding lectin deficiency is a condition that affects the immune ...

  20. DNA-Aptamers Binding Aminoglycoside Antibiotics

    OpenAIRE

    Nadia Nikolaus; Beate Strehlitz

    2014-01-01

    Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminog...

  1. Liver Fatty Acid Binding Protein and Obesity

    OpenAIRE

    Atshaves, B.P.; Martin, G G; Hostetler, H.A.; McIntosh, A.L.; Kier, A B; Schroeder, F.

    2010-01-01

    While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them f...

  2. Binding capacity: cooperativity and buffering in biopolymers.

    Science.gov (United States)

    Di Cera, E; Gill, S J; Wyman, J

    1988-01-01

    The group of linkage potentials resulting from the energy of a physicochemical system expressed per mol of a reference component, say a polyfunctional macromolecule, leads to the concept of binding capacity. This concept applies equally to both chemical and physical ligands and opens the way to consideration of higher-order linkage relationships. It provides a means of exploring the consequences of thermodynamic stability on generalized binding phenomena in biopolymers. PMID:3422436

  3. Penicillin-Binding Protein Imaging Probes

    OpenAIRE

    Kocaoglu, Ozden; Carlson, Erin E.

    2013-01-01

    Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, the apparent functional redundancy of the ~5–15 proteins that most bacteria possess makes determination of their individual roles difficult. Existing techniques to st...

  4. Photonic Binding in Silicon-Colloid Microcavities

    OpenAIRE

    Xifré-Pérez, E.; García de Abajo, Francisco Javier; Fenollosa Esteve, Roberto; Meseguer, Francisco

    2009-01-01

    Photonic binding between two identical silicon-colloid-based microcavities is studied by using a generalized multipolar expansion. In contrast with previous works, we focus on low-order cavity modes that resemble low-energy electronic orbitals. For conservative light intensities, the interaction between cavity modes with moderate Q factors produces extremely large particle acceleration values. Optical forces dominate over vanderWaals, gravity, and Brownian motion, and they show a binding-anti...

  5. The readiness potential reflects intentional binding

    Directory of Open Access Journals (Sweden)

    Han-Gue eJo

    2014-06-01

    Full Text Available When a voluntary action is causally linked with a sensory outcome, the action and its consequent effect are perceived as being closer together in time. This effect is called intentional binding. Although many experiments were conducted on this phenomenon, the underlying neural mechanisms are not well understood. While intentional binding is specific to voluntary action, we presumed that preconscious brain activity (the readiness potential, RP, which occurs before an action is made, might play an important role in this binding effect. In this study, the brain dynamics were recorded with electroencephalography (EEG and analyzed in single-trials in order to estimate whether intentional binding is correlated with the early neural processes. Moreover, we were interested in different behavioral performance between meditators and non-meditators since meditators are expected to be able to keep attention more consistently on a task. Thus, we performed the intentional binding paradigm with twenty mindfulness meditators and compared them to matched controls. Although, we did not observe a group effect on either behavioral data or EEG recordings, we found that self-initiated movements following ongoing negative deflections of slow cortical potentials (SCPs result in a stronger binding effect compared to positive potentials, especially regarding the perceived time of the consequent effect. Our results provide the first direct evidence that the early neural activity within the range of SCPs affects perceived time of a sensory outcome that is caused by intentional action.

  6. The readiness potential reflects intentional binding.

    Science.gov (United States)

    Jo, Han-Gue; Wittmann, Marc; Hinterberger, Thilo; Schmidt, Stefan

    2014-01-01

    When a voluntary action is causally linked with a sensory outcome, the action and its consequent effect are perceived as being closer together in time. This effect is called intentional binding. Although many experiments were conducted on this phenomenon, the underlying neural mechanisms are not well understood. While intentional binding is specific to voluntary action, we presumed that preconscious brain activity (the readiness potential, RP), which occurs before an action is made, might play an important role in this binding effect. In this study, the brain dynamics were recorded with electroencephalography (EEG) and analyzed in single-trials in order to estimate whether intentional binding is correlated with the early neural processes. Moreover, we were interested in different behavioral performance between meditators and non-meditators since meditators are expected to be able to keep attention more consistently on a task. Thus, we performed the intentional binding paradigm with 20 mindfulness meditators and compared them to matched controls. Although, we did not observe a group effect on either behavioral data or EEG recordings, we found that self-initiated movements following ongoing negative deflections of slow cortical potentials (SCPs) result in a stronger binding effect compared to positive potentials, especially regarding the perceived time of the consequent effect. Our results provide the first direct evidence that the early neural activity within the range of SCPs affects perceived time of a sensory outcome that is caused by intentional action.

  7. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  8. Electrostatically biased binding of kinesin to microtubules.

    Directory of Open Access Journals (Sweden)

    Barry J Grant

    2011-11-01

    Full Text Available The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules.

  9. Theoretical studies of binding of mannose-binding protein to monosaccharides

    Science.gov (United States)

    Aida-Hyugaji, Sachiko; Takano, Keiko; Takada, Toshikazu; Hosoya, Haruo; Kojima, Naoya; Mizuochi, Tsuguo; Inoue, Yasushi

    2004-11-01

    Binding properties of mannose-binding protein (MBP) to monosaccharides are discussed based on ab initio molecular orbital calculations for cluster models constructed. The calculated binding energies indicate that MBP has an affinity for N-acetyl- D-glucosamine, D-mannose, L-fucose, and D-glucose rather than D-galactose and N-acetyl- D-galactosamine, which is consistent with the biochemical experimental results. Electrostatic potential surfaces at the binding site of four monosaccharides having binding properties matched well with that of MBP. A vacant frontier orbital was found to be localized around the binding site of MBP, suggesting that MBP-monosaccharide interaction may occur through electrostatic and orbital interactions.

  10. To Bind or not to Bind: It’s in the Contract

    DEFF Research Database (Denmark)

    Tvarnø, Christina D.

    2016-01-01

    discusses, in a theoretical perspective, the legal reasoning behind the different partnering approaches, both from a historical and contract law perspective, and furthermore applies a game theoretical approach in evaluating binding versus non-binding partnering contracts. The analysis focuses on private......This article discusses the formalization of collaboration through partnering contracts in the construction industry in the USA, Great Britain and Denmark. The article compares the different types of collaborative partnering contracts in the three countries, and provides a conclusion on whether...... the collaborative partnering contract should be binding or non-binding, based on the three empirical contracts analyzed in this article. The partnering contracts in Great Britain and Denmark are legally binding, while in the USA the partnering agreements are non-binding charters or letters of intent. This article...

  11. Effect of solid surface charge on the binding behaviour of a metal-binding peptide

    OpenAIRE

    Donatan, Senem; Sarikaya, Mehmet; TAMERLER, Candan; Urgen, Mustafa

    2012-01-01

    Over the last decade, solid-binding peptides have been increasingly used as molecular building blocks coupling bio- and nanotechnology. Despite considerable research being invested in this field, the effects of many surface-related parameters that define the binding of peptide to solids are still unknown. In the quest to control biological molecules at solid interfaces and, thereby, tailoring the binding characteristics of the peptides, the use of surface charge of the solid surface may proba...

  12. Fucose-binding Lotus tetragonolobus lectin binds to human polymorphonuclear leukocytes and induces a chemotactic response.

    Science.gov (United States)

    VanEpps, D E; Tung, K S

    1977-09-01

    Fucose-binding L. tetragonolobus lectin to the surface of human polymorphonuclear leukocytes (PMN) and induces a chemotactic response. Both surface binding and chemotaxis are inhibited by free fucose but not by fructose, mannose, or galactose. The lectin-binding sites on PMN are unrelated to the A, B, or O blood group antigen. Utilization of this lectin should be a useful tool in isolating PMN membrane components and in analyzing the mechanism of neutrophil chemotaxis. PMID:330752

  13. Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

    OpenAIRE

    Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

    2012-01-01

    Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the p...

  14. Binding site turnover produces pervasive quantitative changes in transcription factor binding between closely related Drosophila species.

    Directory of Open Access Journals (Sweden)

    Robert K Bradley

    2010-03-01

    Full Text Available Changes in gene expression play an important role in evolution, yet the molecular mechanisms underlying regulatory evolution are poorly understood. Here we compare genome-wide binding of the six transcription factors that initiate segmentation along the anterior-posterior axis in embryos of two closely related species: Drosophila melanogaster and Drosophila yakuba. Where we observe binding by a factor in one species, we almost always observe binding by that factor to the orthologous sequence in the other species. Levels of binding, however, vary considerably. The magnitude and direction of the interspecies differences in binding levels of all six factors are strongly correlated, suggesting a role for chromatin or other factor-independent forces in mediating the divergence of transcription factor binding. Nonetheless, factor-specific quantitative variation in binding is common, and we show that it is driven to a large extent by the gain and loss of cognate recognition sequences for the given factor. We find only a weak correlation between binding variation and regulatory function. These data provide the first genome-wide picture of how modest levels of sequence divergence between highly morphologically similar species affect a system of coordinately acting transcription factors during animal development, and highlight the dominant role of quantitative variation in transcription factor binding over short evolutionary distances.

  15. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  16. Bone induction at physiological doses of BMP through localization by clay nanoparticle gels.

    Science.gov (United States)

    Gibbs, D M R; Black, C R M; Hulsart-Billstrom, G; Shi, P; Scarpa, E; Oreffo, R O C; Dawson, J I

    2016-08-01

    Bone Morphogenic Protein 2 (BMP2) can induce ectopic bone. This ability, which first motivated the widespread application of BMP2 in fracture healing and spinal arthrodesis has, more recently, been indicated as one of several serious adverse effects associated with the supra-physiological doses of BMP2 relied upon for clinical efficacy. Key to harnessing BMPs and other agents safely and effectively will be the ability to localize activity at a target site at substantially reduced doses. Clay (Laponite) nanoparticles can self assemble into gels under physiological conditions and bind growth factors for enhanced and localized efficacy. Here we show the ability to localize and enhance the activity of BMP2 to achieve ectopic bone formation at doses within the sub-microgram per ml range of concentrations sufficient to induce differentiation of responsive cell populations in vitro and at approximately 3000 fold lower than those employed in clinical practice. PMID:27209259

  17. LRP4 is critical for neuromuscular junction maintenance.

    Science.gov (United States)

    Barik, Arnab; Lu, Yisheng; Sathyamurthy, Anupama; Bowman, Andrew; Shen, Chengyong; Li, Lei; Xiong, Wen-cheng; Mei, Lin

    2014-10-15

    The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms. PMID:25319686

  18. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    Science.gov (United States)

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  19. Haptenation: Chemical Reactivity and Protein Binding

    Directory of Open Access Journals (Sweden)

    Itai Chipinda

    2011-01-01

    Full Text Available Low molecular weight chemical (LMW allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

  20. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  1. Endocytosis of integrin-binding human picornaviruses.

    Science.gov (United States)

    Merilahti, Pirjo; Koskinen, Satu; Heikkilä, Outi; Karelehto, Eveliina; Susi, Petri

    2012-01-01

    Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  2. Conformational heterogeneity of the calmodulin binding interface

    Science.gov (United States)

    Shukla, Diwakar; Peck, Ariana; Pande, Vijay S.

    2016-04-01

    Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.

  3. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins.

    Science.gov (United States)

    Clifton, Ben E; Jackson, Colin J

    2016-02-18

    The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity.

  4. Estradiol Binds to Insulin and Insulin Receptor Decreasing Insulin Binding in vitro

    OpenAIRE

    RobertRoot-Bernstein

    2014-01-01

    Rationale: Insulin resistance associated with hyperestrogenemias occurs in gestational diabetes mellitus, polycystic ovary syndrome, ovarian hyperstimulation syndrome, estrogen therapies, metabolic syndrome and obesity. The mechanism by which insulin and estrogen interact is unknown. We hypothesize that estrogen binds directly to insulin and the insulin receptor producing insulin resistance. Objectives: To determine the binding constants of steroid hormones to insulin, the insulin recepto...

  5. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    Science.gov (United States)

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  6. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    Science.gov (United States)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  7. The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins.

    Science.gov (United States)

    Frankel, G; Lider, O; Hershkoviz, R; Mould, A P; Kachalsky, S G; Candy, D C; Cahalon, L; Humphries, M J; Dougan, G

    1996-08-23

    Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin. PMID:8702771

  8. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.;

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology...

  9. Receptor binding studies of the living heart

    International Nuclear Information System (INIS)

    Receptors form a class of intrinsic membrane proteins (or glycoproteins) defined by the high affinity and specificity with which they bind ligands. Many receptors are associated directly or indirectly with membrane ion channels that open or close after a conformational change of the receptor induced by the binding of the neurotransmitter. Changes in number and/or affinity of cardiac neurotransmitter receptors have been associated with myocardial ischemia and infarction, congestive heart failure, and cardiomyopathy as well as diabetes or thyroid-induced heart muscle disease. These alterations of cardiac receptors have been demonstrated in vitro on membrane homogenates from samples collected mainly during surgery or postmortem. The disadvantage of these in vitro binding techniques is that receptors lose their natural environment and their relationships with the other components of the tissue

  10. Binding of calcium and carbonate to polyacrylates.

    Science.gov (United States)

    Tribello, Gareth A; Liew, CheeChin; Parrinello, Michele

    2009-05-21

    Polyacrylate molecules can be used to slow the growth of calcium carbonate. However, little is known about the mechanism by which the molecules impede the growth rate. A recent computational study (Bulo et al. Macromolecules 2007, 40, 3437) used metadynamics to investigate the binding of calcium to polyacrylate chains and has thrown some light on the coiling and precipitation of these polymers. We extend these simulations to examine the binding of calcium and carbonate to polyacrylate chains. We show that calcium complexed with both carbonate and polyacrylate is a very stable species. The free energies of calcium-carbonate-polyacrylate complexes, with different polymer configurations, are calculated, and differences in the free energy of the binding of carbonate are shown to be due to differences in the amount of steric hindrance about the calcium, which prevents the approach of the carbonate ion. PMID:19400592

  11. Predicting binding free energies in solution

    CERN Document Server

    Jensen, Jan H

    2015-01-01

    Recent predictions of absolute binding free energies of host-guest complexes in aqueous solution using electronic structure theory have been encouraging for some systems, while other systems remain problematic for others. In paper I summarize some of the many factors that could easily contribute 1-3 kcal/mol errors at 298 K: three-body dispersion effects, molecular symmetry, anharmonicity, spurious imaginary frequencies, insufficient conformational sampling, wrong or changing ionization states, errors in the solvation free energy of ions, and explicit solvent (and ion) effects that are not well-represented by continuum models. While the paper is primarily a synthesis of previously published work there are two new results: the adaptation of Legendre transformed free energies to electronic structure theory and a use of water clusters that maximizes error cancellation in binding free energies computed using explicit solvent molecules. While I focus on binding free energies in aqueous solution the approach also a...

  12. Binding in short-term visual memory.

    Science.gov (United States)

    Wheeler, Mary E; Treisman, Anne M

    2002-03-01

    The integration of complex information in working memory, and its effect on capacity, shape the limits of conscious cognition. The literature conflicts on whether short-term visual memory represents information as integrated objects. A change-detection paradigm using objects defined by color with location or shape was used to investigate binding in short-term visual memory. Results showed that features from the same dimension compete for capacity, whereas features from different dimensions can be stored in parallel. Binding between these features can occur, but focused attention is required to create and maintain the binding over time, and this integrated format is vulnerable to interference. In the proposed model, working memory capacity is limited both by the independent capacity of simple feature stores and by demands on attention networks that integrate this distributed information into complex but unified thought objects. PMID:11900102

  13. [Water binding of adsorptive immobilized lipases].

    Science.gov (United States)

    Loose, S; Meusel, D; Muschter, A; Ruthe, B

    1990-01-01

    It is supposed that not only the total water content of lipase preparations but more their state of water binding is of technological importance in enzymatic interesterification reactions in systems nearly free from water. The isotherms at 65 degrees C of two microbial lipases immobilized on various adsorbents as well as different adsorbents themselves are shown. The water binding capacity in the range of water content of technological interest decreases from the anion exchange resin Amberlyst A 21 via nonpolar adsorbent Amberlite XAD-2 to kieselguhr Celite 545. It is demonstrated that water binding by lipases is depending on temperature but is also affected by adsorptive immobilization. Adsorptive immobilized lipases show hysteresis, which is very important for preparing a definite water content of the enzyme preparations. PMID:2325750

  14. Dendrimers bind antioxidant polyphenols and cisplatin drug.

    Directory of Open Access Journals (Sweden)

    Amine Abderrezak

    Full Text Available Synthetic polymers of a specific shape and size play major role in drug delivery systems. Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape with potential applications in gene and drug delivery. We examine the interaction of several dendrimers of different compositions mPEG-PAMAM (G3, mPEG-PAMAM (G4 and PAMAM (G4 with hydrophilic and hydrophobic drugs cisplatin, resveratrol, genistein and curcumin at physiological conditions. FTIR and UV-visible spectroscopic methods as well as molecular modeling were used to analyse drug binding mode, the binding constant and the effects of drug complexation on dendrimer stability and conformation. Structural analysis showed that cisplatin binds dendrimers in hydrophilic mode via Pt cation and polymer terminal NH(2 groups, while curcumin, genistein and resveratrol are located mainly in the cavities binding through both hydrophobic and hydrophilic contacts. The overall binding constants of durg-dendrimers are ranging from 10(2 M(-1 to 10(3 M(-1. The affinity of dendrimer binding was PAMAM-G4>mPEG-PAMAM-G4>mPEG-PAMAM-G3, while the order of drug-polymer stability was curcumin>cisplatin>genistein>resveratrol. Molecular modeling showed larger stability for genisten-PAMAM-G4 (ΔG = -4.75 kcal/mol than curcumin-PAMAM-G4 ((ΔG = -4.53 kcal/mol and resveratrol-PAMAM-G4 ((ΔG = -4.39 kcal/mol. Dendrimers might act as carriers to transport hydrophobic and hydrophilic drugs.

  15. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.;

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article...... discusses the current knowledge on the structure and potential function of this protein. Several putative binding partners of ALG-2 have been identified hinting to functions of ALG-2 in apoptosis and possibly also in proliferation, endocytosis and transcriptional regulation during development. Gene deletion...

  16. Ice-Binding Proteins and Their Function.

    Science.gov (United States)

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-01

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities. PMID:27145844

  17. Binding energies of hypernuclei and hypernuclear interactions

    Energy Technology Data Exchange (ETDEWEB)

    Bodmer, A.R. [Argonne National Lab., IL (United States)]|[Univ. of Illinois, Chicago, IL (United States). Dept. of Physics; Murali, S.; Usmani, Q.N. [Jamia Millia Islamia, New Delhi (India). Dept. of Physics

    1996-05-01

    In part 1 the effect of nuclear core dynamics on the binding energies of {Lambda} hypernuclei is discussed in the framework of variational correlated wave functions. In particular, the authors discuss a new rearrangement energy contribution and its effect on the core polarization. In part 2 they consider the interpretation of the {Lambda} single-particle energy in terms of basic {Lambda}-nuclear interactions using a local density approximation based on a Fermi hypernetted chain calculation of the A binding to nuclear matter. To account for the data strongly repulsive 3-body {Lambda}NN forces are required. Also in this framework they discuss core polarization for medium and heavier hypernuclei.

  18. On Feature Binding in Space and Time

    OpenAIRE

    Chennu, Srivas

    2008-01-01

    When presented with a yellow Volkswagen and a red Ferrari, how does the brain �gure out which color goes with which car? The binding problem refers to how the visual system pre-consciously combines visual features of objects in the physical world to create coherent mental equivalents in our consciousness. I discuss why feature binding is a problem for our brains despite its seemingly e�ortless resolution in every-day life. Drawing from experimental cognitive psychology, I demonstrate how i...

  19. Studies on folate binding and a radioassay for serum and whole blood folate using goat milk as binding agent

    International Nuclear Information System (INIS)

    Preparations of cow, goat, buffalo, and human milk in addition to pig plasma were tested for folate binding properties. Of these, only pig plasma and goat milk showed sufficient binding to enable use as binding agents in a radioassay for serum and whole blood folate. The binding of folate by cow mild preparations in particular was found to be very poor. (orig.)

  20. Oxytocin binding sites in bovine mammary tissue

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xin.

    1989-01-01

    Oxytocin binding sites were identified and characterized in bovine mammary tissue. ({sup 3}H)-oxytocin binding reached equilibrium by 50 min at 20{degree}C and by 8 hr at 4{degree}C. The half-time of displacement at 20{degree}C was approximately 1 hr. Thyrotropin releasing hormone, adrenocorticotropin, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive. In the presence of 10 nM LiCl, addition of oxytocin to dispersed bovine mammary cells, in which phosphatidylinositol was pre-labelled, caused a time and dose-dependent increase in radioactive inositiol monophosphate incorporation. The possibility that there are distinct vasopressin receptors in bovine mammary tissue was investigated. ({sup 3}H)-vasopressin binding reached equilibrium by 40 min at 20{degree}. The half-time of displacement at 20{degree}C was approximately 1 hr. The ability of the peptides to inhibit ({sup 3}H)-vasopressin binding was: (Thr{sup 4},Gly{sup 7})-oxytocin > Arg{sup 8}-vasopressin > (lys{sup 8})-vasopressin > (Deamino{sup 1},D-arg{sup 8})-vasopressin > oxytocin > d (CH{sub 2}){sub 5}Tyr(Me)AVP.

  1. Binding of cationic surfactants to humic substances

    NARCIS (Netherlands)

    Ishiguro, M.; Tan, W.; Koopal, L.K.

    2007-01-01

    Commercial surfactants are introduced into the environment either through waste products or site-specific contamination. The amphiphilic nature of both surfactants and humic substances (HS) leads to their mutual attraction especially when surfactant and HS are oppositely charged. Binding of the cati

  2. Information flow through calcium binding proteins

    Science.gov (United States)

    Bak, Ji Hyun; Bialek, William

    2013-03-01

    Calcium signaling is a ubiquitous mode of biological communication, which regulates a great variety of vital processes in living systems. Such a signal typically begins with an elementary event, in which calcium ions bind to a protein, inducing a change in the protein's structure. Information can only be lost, from what was conveyed through this initial event, as the signal is further transduced through the downstream networks. In the present work we analyze and optimize the information flow in the calcium binding process. We explicitly calculate the mutual information between the calcium concentration and the states of the protein, using a simple model for allosteric regulation in a dimeric protein. The optimal solution depends on the dynamic range of the input as well as on the timescale of signal integration. According to our result, the optimizing strategy involves allowing the calcium-binding protein to be ``activated'' by a partial occupation of its sites, and tuning independently the strengths of cooperative interactions in the binding and unbinding processes.

  3. Lipid Binding Proteins from Parasitic Platyhelmithes

    Directory of Open Access Journals (Sweden)

    Gabriela eAlvite

    2012-09-01

    Full Text Available Two main families of lipid binding proteins have been identified in parasitic Platyhelminthes: hydrophobic ligand binding proteins (HLBPs and fatty acid binding proteins (FABPs. Members of the former family of proteins are specific to the Cestoda class, while FABPs are conserved across a wide range of animal species. Because Platyhelminthes are unable to synthesise their own lipids, these lipid-binding proteins are important molecules in these organisms.HLBPs are a high molecular mass complex of proteins and lipids. They are composed of subunits of low molecular mass proteins and a wide array of lipid molecules ranging from CoA esters to cholesterol. These proteins are excretory-secretory molecules and are key serological tools for diagnosis of diseases caused by cestodes. FABPs are mainly intracellular proteins of low molecular weight. They are also vaccine candidates.Despite that the knowledge of their function is scarce, the differences in their molecular organisation, ligand preferences, intra/extracellular localisation, evolution, and phylogenetic distribution, suggest that platyhelminths HLBPs and FABPs should play different functions. FABPs might be involved in the removal of fatty acids from the inner surface of the cell membrane and in their subsequent targeting to specific cellular destinations. In contrast, HLBPs might be involved in fatty acid uptake from the host environment.

  4. Alkali binding in hydrated Portland cement paste

    NARCIS (Netherlands)

    Chen, W.; Brouwers, H.J.H.

    2010-01-01

    The alkali-binding capacity of C–S–H in hydrated Portland cement pastes is addressed in this study. The amount of bound alkalis in C–S–H is computed based on the alkali partition theories firstly proposed by Taylor (1987) and later further developed by Brouwers and Van Eijk (2003). Experimental data

  5. Nucleic acids encoding a cellulose binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL); Shpiegl, Itai (Rehovot, IL); Goldstein, Marc A. (Davis, CA); Doi, Roy H. (Davis, CA)

    1996-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  6. Tension-induced binding of semiflexible biopolymers

    CERN Document Server

    Benetatos, Panayotis; Zippelius, Annette

    2014-01-01

    We investigate theoretically the effect of polymer tension on the collective behavior of reversibly binding cross-links. For this purpose, we employ a model of two weakly bending wormlike chains aligned in parallel by a tensile force, with a sequence of inter-chain binding sites regularly spaced along the contours. Reversible cross-links attach and detach at the sites with an affinity controlled by a chemical potential. In a mean-field approach, we calculate the free energy of the system and find the emergence of a free-energy barrier which controls the reversible (un)binding. The tension affects the conformational entropy of the chains which competes with the binding energy of the cross-links. This competition gives rise to a sudden increase in the fraction of bound sites as the tension increases. We show that this transition is related to the cross-over between weak and strong localization of a directed polymer in a pinning potential. The cross-over to the strongly bound state can be interpreted as a mechan...

  7. Stabilized sulfur binding using activated fillers

    Energy Technology Data Exchange (ETDEWEB)

    Kalb, Paul D.; Vagin, Vyacheslav P.; Vagin, Sergey P.

    2015-07-21

    A method of making a stable, sulfur binding composite comprising impregnating a solid aggregate with an organic modifier comprising unsaturated hydrocarbons with at least one double or triple covalent bond between adjacent carbon atoms to create a modifier-impregnated aggregate; heating and drying the modifier-impregnated aggregate to activate the surface of the modifier-impregnated aggregate for reaction with sulfur.

  8. tPA-binding RNA Aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils

    2015-01-01

    -density lipoprotein receptor Related Protein-1 (LRP-1). Here, we describe the selection and characterisation of structured RNA ligands (“RNA aptamers”) to tPA, K18 and K32. Both aptamers were truncated to minimal 32-nucleotide constructs (v2) with improved or unchanged activities, and were shown to bind tPA with low...

  9. The Double Bind: The next Generation

    Science.gov (United States)

    Malcom, Lindsey E.; Malcom, Shirley M.

    2011-01-01

    In this foreword, Shirley Malcom and Lindsey Malcom speak to the history and current status of women of color in science, technology, engineering, and mathematics (STEM) fields. As the author of the seminal report "The Double Bind: The Price of Being a Minority Woman in Science", Shirley Malcom is uniquely poised to give us an insightful…

  10. The Case against Binding Interest Arbitration.

    Science.gov (United States)

    Ecker, Charles I.

    1984-01-01

    The author contends that districts should reject binding interest arbitration as a means of resolving an impasse in contract negotiations, charging that it hampers good faith bargaining, adversely affects fiscal and operational management of the school system, and diminishes the governing role of the board of education. (MJL)

  11. CD36 binds oxidized low density lipoprotein (LDL) in a mechanism dependent upon fatty acid binding.

    Science.gov (United States)

    Jay, Anthony G; Chen, Alexander N; Paz, Miguel A; Hung, Justin P; Hamilton, James A

    2015-02-20

    The association of unesterified fatty acid (FA) with the scavenger receptor CD36 has been actively researched, with focuses on FA and oxidized low density lipoprotein (oxLDL) uptake. CD36 has been shown to bind FA, but this interaction has been poorly characterized to date. To gain new insights into the physiological relevance of binding of FA to CD36, we characterized FA binding to the ectodomain of CD36 by the biophysical method surface plasmon resonance. Five structurally distinct FAs (saturated, monounsaturated (cis and trans), polyunsaturated, and oxidized) were pulsed across surface plasmon resonance channels, generating association and dissociation binding curves. Except for the oxidized FA HODE, all FAs bound to CD36, with rapid association and dissociation kinetics similar to HSA. Next, to elucidate the role that each FA might play in CD36-mediated oxLDL uptake, we used a fluorescent oxLDL (Dii-oxLDL) live cell assay with confocal microscopy imaging. CD36-mediated uptake in serum-free medium was very low but greatly increased when serum was present. The addition of exogenous FA in serum-free medium increased oxLDL binding and uptake to levels found with serum and affected CD36 plasma membrane distribution. Binding/uptake of oxLDL was dependent upon the FA dose, except for docosahexaenoic acid, which exhibited binding to CD36 but did not activate the uptake of oxLDL. HODE also did not affect oxLDL uptake. High affinity FA binding to CD36 and the effects of each FA on oxLDL uptake have important implications for protein conformation, binding of other ligands, functional properties of CD36, and high plasma FA levels in obesity and type 2 diabetes.

  12. Perturbation Approaches for Exploring Protein Binding Site Flexibility to Predict Transient Binding Pockets.

    Science.gov (United States)

    Kokh, Daria B; Czodrowski, Paul; Rippmann, Friedrich; Wade, Rebecca C

    2016-08-01

    Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design. PMID:27399277

  13. STARD4 Membrane Interactions and Sterol Binding.

    Science.gov (United States)

    Iaea, David B; Dikiy, Igor; Kiburu, Irene; Eliezer, David; Maxfield, Frederick R

    2015-08-01

    The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/β helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix.

  14. Automatic Binding Time Analysis for a Typed Lambda-Calculus

    DEFF Research Database (Denmark)

    Nielson, Hanne Riis; Nielson, Flemming

    1988-01-01

    A binding time analysis imposes a distinction between the computations to be performed early (e.g. at compile-time) and those to be performed late (e.g. at run-time). For the lambda-calculus this distinction is formalized by a two-level lambda-calculus. The authors present an algorithm for static...... analysis of the binding times of a typed lambda-calculus with products, sums, lists and general recursive types. Given partial information about the binding times of some of the subexpressions it will complete that information such that (i) early bindings may be turned into late bindings but not vice versa......, (ii) the resulting two-level lambda-expression reflects our intuition about binding times, e.g. that early bindings are performed before late bindings, and (iii) as few changes as possible have been made compared with the initial binding information. The results can be applied in the implementation...

  15. Metal binding stoichiometry and isotherm choice in biosorption

    Energy Technology Data Exchange (ETDEWEB)

    Schiewer, S.; Wong, M.H.

    1999-11-01

    Seaweeds that possess a high metal binding capacity may be used as biosorbents for the removal of toxic heavy metals from wastewater. The binding of Cu and Ni by three brown algae (Sargassum, Colpomenia, Petalonia) and one green alga (Ulva) was investigated at pH 4.0 and pH 3.0. The greater binding strength of Cu is reflected in a binding constant that is about 10 times as high as that of Ni. The extent of metal binding followed the order Petalonia {approximately} Sargassum > Colpomenia > Ulva. This was caused by a decreasing number of binding sites and by much lower metal binding constants for Ulva as compared to the brown algae. Three different stoichiometric assumptions are compared for describing the metal binding, which assume either that each metal ion M binds to one binding site B forming a BM complex or that a divalent metal ion M binds to two monovalent sites B forming BM{sub 0.5} or B{sub 2}M complexes, respectively. Stoichiometry plots are proposed as tools to discern the relevant binding stoichiometry. The pH effect in metal binding and the change in proton binding were well predicted for the B{sub 2}M or BM{sub 0.5} stoichiometries with the former being better for Cu and the latter preferable for Ni. Overall, the BM{sub 0.5} model is recommended because it avoids iterations.

  16. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B;

    1999-01-01

    We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence...... spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two...... and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains...

  17. A new zinc binding fold underlines the versatility of zinc binding modules in protein evolution.

    Science.gov (United States)

    Sharpe, Belinda K; Matthews, Jacqueline M; Kwan, Ann H Y; Newton, Anthea; Gell, David A; Crossley, Merlin; Mackay, Joel P

    2002-05-01

    Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.

  18. Oligomerization of mannan-binding lectin dictates binding properties and complement activation

    DEFF Research Database (Denmark)

    Kjaer, Troels R; Jensen, Lisbeth; Hansen, Annette;

    2016-01-01

    altered self-surfaces. Associated with the collagen-like stems of these PRMs are three mannan-binding lectin (MBL)-associated serine proteases (MASPs) and two MBL-associated proteins (MAps). The most studied of the PRMs, MBL, is present in serum mainly as trimeric and tetrameric oligomers of the...... structural subunit. We hypothesized that oligomerization of MBL may influence both the potential to bind to microorganisms and the interaction with the MASPs and MAps, thus influencing the ability to initiate complement activation. When testing binding at 37°C, we found higher binding of tetrameric MBL to...... Staphylococcus aureus (S. aureus) than trimeric and dimeric MBL. In serum, we found that tetrameric MBL was the main oligomeric form present in complexes with the MASPs and MAp44. Such preference was confirmed using purified forms of recombinant MBL (rMBL) oligomers, where tetrameric rMBL interacted stronger...

  19. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T;

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...... by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were...... bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range. Oleic acid was bound with the highest affinity and a Kd of 0.26 microM. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay...

  20. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel M; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless...... potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area...... around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro...

  1. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  2. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  3. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    Science.gov (United States)

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  4. Evidence for an intrinsic binding force between dodecaborate dianions and receptors with hydrophobic binding pockets.

    Science.gov (United States)

    Warneke, Jonas; Jenne, Carsten; Bernarding, Johannes; Azov, Vladimir A; Plaumann, Markus

    2016-05-01

    A gas phase binding study revealed strong intrinsic intermolecular interactions between dianionic halogenated closo-dodecaborates [B12X12](2-) and several neutral organic receptors. Oxidation of a tetrathiafulvalene host allowed switching between two host-guest binding modes in a supramolecular complex. Complexes of β-cyclodextrin with [B12F12](2-) show remarkable stability in the gas phase and were successfully tested as carriers for the delivery of boron clusters into cancer cells. PMID:27087168

  5. Factor XII binding to endothelial cells depends on caveolae

    DEFF Research Database (Denmark)

    Schousboe, Inger; Thomsen, Peter; van Deurs, Bo

    2004-01-01

    It is now generally accepted that factor XII (FXII) binds to cellular surfaces in the vascular system. One of the suggested receptors of this binding is the glycosylphosphatidylinositol-anchored urokinase-like plasminogen activator (u-PAR) harbored in caveolae/lipid rafts. However, binding of FXII...... lipid rafts. Accordingly, cholesterol-depleted cells were found to bind significantly reduced amounts of FXII. These observations, combined with the presence of a minority of u-PAR in caveolae concomitant with FXII binding, indicate that FXII binding to u-PAR may be secondary and depends upon...

  6. Is there a link between selectivity and binding thermodynamics profiles?

    Science.gov (United States)

    Tarcsay, Ákos; Keserű, György M

    2015-01-01

    Thermodynamics of ligand binding is influenced by the interplay between enthalpy and entropy contributions of the binding event. The impact of these binding free energy components, however, is not limited to the primary target only. Here, we investigate the relationship between binding thermodynamics and selectivity profiles by combining publicly available data from broad off-target assay profiling and the corresponding thermodynamics measurements. Our analysis indicates that compounds binding their primary targets with higher entropy contributions tend to hit more off-targets compared with those ligands that demonstrated enthalpy-driven binding.

  7. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

    1989-01-01

    Cobalamin and its binding protein, haptocorrin, are present in rat milk throughout the lactation period. The concentration of cobalamin is approximately 0.3-times the concentration of the unsaturated binding protein. The concentration of the unsaturated cobalamin-binding protein varies between 18...... nmol l-1 and 16 nmol l-1. The binding protein has a Stokes radius of 2.49 nm when saturated with cobalamin and 2.61 nm when unsaturated. It binds cobalamin over a broad range of pH and is able to bind cobinamide also. With immunohistochemistry, we find haptocorrin immunoreactivity in the mammary glands...

  8. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  9. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  10. Simultaneous optimal experimental design for in vitro binding parameter estimation.

    Science.gov (United States)

    Ernest, C Steven; Karlsson, Mats O; Hooker, Andrew C

    2013-10-01

    Simultaneous optimization of in vitro ligand binding studies using an optimal design software package that can incorporate multiple design variables through non-linear mixed effect models and provide a general optimized design regardless of the binding site capacity and relative binding rates for a two binding system. Experimental design optimization was employed with D- and ED-optimality using PopED 2.8 including commonly encountered factors during experimentation (residual error, between experiment variability and non-specific binding) for in vitro ligand binding experiments: association, dissociation, equilibrium and non-specific binding experiments. Moreover, a method for optimizing several design parameters (ligand concentrations, measurement times and total number of samples) was examined. With changes in relative binding site density and relative binding rates, different measurement times and ligand concentrations were needed to provide precise estimation of binding parameters. However, using optimized design variables, significant reductions in number of samples provided as good or better precision of the parameter estimates compared to the original extensive sampling design. Employing ED-optimality led to a general experimental design regardless of the relative binding site density and relative binding rates. Precision of the parameter estimates were as good as the extensive sampling design for most parameters and better for the poorly estimated parameters. Optimized designs for in vitro ligand binding studies provided robust parameter estimation while allowing more efficient and cost effective experimentation by reducing the measurement times and separate ligand concentrations required and in some cases, the total number of samples. PMID:23943088

  11. Causal binding of actions to their effects.

    Science.gov (United States)

    Buehner, Marc J; Humphreys, Gruffydd R

    2009-10-01

    According to widely held views in cognitive science harking back to David Hume, causality cannot be perceived directly, but instead is inferred from patterns of sensory experience, and the quality of these inferences is determined by perceivable quantities such as contingency and contiguity. We report results that suggest a reversal of Hume's conjecture: People's sense of time is warped by the experience of causality. In a stimulus-anticipation task, participants' response behavior reflected a shortened experience of time in the case of target stimuli participants themselves had generated, relative to equidistant, equally predictable stimuli they had not caused. These findings suggest that causality in the mind leads to temporal binding of cause and effect, and extend and generalize beyond earlier claims of intentional binding between action and outcome.

  12. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  13. Autologous antibodies that bind neuroblastoma cells.

    Science.gov (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  14. Lectin binding in normal donkey eyeball

    Directory of Open Access Journals (Sweden)

    Khaled Aly

    2013-10-01

    Full Text Available In the present study, the distribution of various sugar residues in the eyeball tissues of sexually mature donkey was examined by employing fluorescein isothiocyanate-conjugated lectins. Our results revealed the presence of mannose (labeled by lectins ConA, galactose (labeled by PNA, GSAI, ECA, GalNAc (labeled by SBA, VVA, and GlcNAc (labeled by WGA residues in the donkey ocular tissues. The epithelium and stroma of the ocular tissues were labeled with mannose (ConA and GlcNAc (WGA binding lectins. Binding sites for WGA and PNA to the rod and cone cells of the retina were evident. The lectins Con A, WGA and GSAI are bound strongly to the endothelium of blood vessels and to smooth muscle cells of the iris. In conclusion, the findings of the present study clearly indicate that the donkey eyeball contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosly residues, and it lacks fucosyl residues.

  15. Insulin-induced lipid binding to hemoglobin

    Directory of Open Access Journals (Sweden)

    VESNA NIKETIC

    2003-01-01

    Full Text Available Under hypoglycemic conditions, concomitant hyperinsulinism causes an apparent modification of hemoglobin (Hb which is manifested by its aggregation (Niketi} et al., Clin. Chim. Acta 197 (1991 47. In the present work the causes and mechanisms underlying this Hb modification were studied. Hemoglobin isolated from normal erythrocytes incubated with insulin was analyzed by applying 31P-spectrometry and lipid extraction and analysis. To study the dynamics of the plasma membrane during hyperinsulinism, a fluorescent lipid-analog was applied. In the presence of insulin, phosphatidylserine (PS, phosphatidylethanolamine (PE and cholesterol were found to bind to Hb. Lipid binding resulted in Hb aggregation, a condition that can be reproduced when phospholipids are incubated with Hb in vitro. Using a fluorescent lipid-analog, it was also shown that exposing erythrocytes to supraphysiological concentrations of insulin in vitro resulted in the internalization of lipids. The results presented in this work may have relevance to cases of diabetes mellitus and hypoglycemia.

  16. Tight-binding treatment of conjugated polymers

    DEFF Research Database (Denmark)

    Lynge, Thomas Bastholm

    This PhD thesis concerns conjugated polymers which constitute a constantly growing research area. Today, among other things, conjugated polymers play a role in plastic based solar cells, photodetectors and light emitting diodes, and even today such plastic-based components constitute an alternative...... of tomorrow. This thesis specifically treats the three conjugated polymers trans-polyacetylene (tPA), poly(para-phenylene) (PPP) and poly(para-phe\\-nylene vinylene) (PPV). The present results, which are derived within the tight-binding model, are divided into two parts. In one part, analytic results...... are derived for the optical properties of the polymers expressed in terms of the optical susceptibility both in the presence and in the absence of a static electric field. In the other part, the cumputationally efficient Density Functional-based Tight-Binding (DFTB) model is applied to the description...

  17. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    OpenAIRE

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesi...

  18. Binding of Actinobacillus pleuropneumoniae to Phosphatidylethanolamine

    OpenAIRE

    Jeannotte, Marie-Eve; Abul-Milh, Maan; Dubreuil, J. Daniel; Jacques, Mario

    2003-01-01

    The gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, a disease that causes important economic losses to the swine industry worldwide. In general, the initial step of bacterial colonization is attachment to host cells. The purpose of the present study was to evaluate the binding of A. pleuropneumoniae serotype 1 to phospholipids, which are the major constituents of biological membranes. Phospholipids serve...

  19. Copper Binding in the Prion Protein†

    OpenAIRE

    Millhauser, Glenn L.

    2004-01-01

    A conformational change of the prion protein is responsible for a class of neurodegenerative diseases called the transmissible spongiform encephalopathies that include mad cow disease and the human afflictions kuru and Creutzfeldt–Jakob disease. Despite the attention given to these diseases, the normal function of the prion protein in healthy tissue is unknown. Research over the past few years, however, demonstrates that the prion protein is a copper binding protein with high selectivity for ...

  20. Brain hyaluronan binding protein inhibits tumor growth

    Institute of Scientific and Technical Information of China (English)

    高锋; 曹曼林; 王蕾

    2004-01-01

    Background Great efforts have been made to search for the angiogenic inhibitors in avascular tissues. Several proteins isolated from cartilage have been proved to have anti-angiogenic or anti-tumour effects. Because cartilage contains a great amount of hyaluronic acid (HA) oligosaccharides and abundant HA binding proteins (HABP), therefore, we speculated that HABP might be one of the factors regulating vascularization in cartilage or anti-angiogenesis in tumours. The purpose of this research was to evaluale the effects of hyaluronan binding protein on inhibiting tumour growth both in vivo and vitro. Methods A unique protein termed human brain hyaluronan (HA) binding protein (b-HABP) was cloned from human brain cDNA library. MDA-435 human breast cancer cell line was chosen as a transfectant. The in vitro underlying mechanisms were investigated by determining the possibilities of MDA-435/b-HABP colony formation on soft agar, the effects of the transfectant on the proliferation of endothelial cells and the expression levels of caspase 3 and FasL from MDA-435/b-HABP. The in vivo study included tumour growth on the chorioallantoic membrane (CAM) of chicken embryos and nude mice. Results Colony formation assay revealed that the colonies formed by MDA-435/b-HABP were greatly reduced compared to mock transfectants. The conditioned media from MDA-435/b-HABP inhibited the growth of endothelial cells in culture. Caspase 3 and FasL expressions were induced by MDA-435/b-HABP. The size of tumours of MDA-435/b-HABP in both CAM and nude mice was much smaller than that of MDA-435 alone. Conclusions Human brain hyaluronan binding protein (b-HABP) may represent a new kind of naturally existing anti-tumour substance. This brain-derived glycoprotein may block tumour growth by inducing apoptosis of cancer cells or by decreasing angiogenesis in tumour tissue via inhibiting proliferation of endothelial cells.

  1. Tight Binding Models in Cold Atoms Physics

    Science.gov (United States)

    Zakrzewski, J.

    2007-05-01

    Cold atomic gases placed in optical lattice potentials offer a unique tool to study simple tight binding models. Both the standard cases known from the condensed matter theory as well as novel situations may be addressed. Cold atoms setting allows for a precise control of parameters of the systems discussed, stimulating new questions and problems. The attempts to treat disorder in a controlled fashion are addressed in detail.

  2. Alternative polyadenylation and RNA-binding proteins.

    Science.gov (United States)

    Erson-Bensan, Ayse Elif

    2016-08-01

    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  3. Binding Energy and Equilibrium of Compact Objects

    Directory of Open Access Journals (Sweden)

    Germano M.

    2014-04-01

    Full Text Available The theoretical analysis of the existence of a limit mass for compact astronomic ob- jects requires the solution of the Einstein’s equations of g eneral relativity together with an appropriate equation of state. Analytical solutions exi st in some special cases like the spherically symmetric static object without energy sou rces that is here considered. Solutions, i.e. the spacetime metrics, can have a singular m athematical form (the so called Schwarzschild metric due to Hilbert or a nonsingula r form (original work of Schwarzschild. The former predicts a limit mass and, conse quently, the existence of black holes above this limit. Here it is shown that, the origi nal Schwarzschild met- ric permits compact objects, without mass limit, having rea sonable values for central density and pressure. The lack of a limit mass is also demonst rated analytically just imposing reasonable conditions on the energy-matter densi ty, of positivity and decreas- ing with radius. Finally the ratio between proper mass and to tal mass tends to 2 for high values of mass so that the binding energy reaches the lim it m (total mass seen by a distant observer. As it is known the negative binding energ y reduces the gravitational mass of the object; the limit of m for the binding energy provides a mechanism for stable equilibrium of any amount of mass to contrast the gravitatio nal collapse.

  4. STUDY OF ESTROGEN BINDING SITE ON HUMAN EJACULATED SPERMATOZOA

    Institute of Scientific and Technical Information of China (English)

    CHUJin-Shong; WANGYi-Fei

    1989-01-01

    The specific estrogen binding site for 17β-estradiol has been investigated on human spermatozoa by electron microscopec autoradiography. The results show that the binding sites were distributed over the surface of human spermatozoa: acrosomal cap, equatorial

  5. Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

    Science.gov (United States)

    Pleass, R J; Areschoug, T; Lindahl, G; Woof, J M

    2001-03-16

    Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function. PMID:11096107

  6. AB-Bind: Antibody binding mutational database for computational affinity predictions.

    Science.gov (United States)

    Sirin, Sarah; Apgar, James R; Bennett, Eric M; Keating, Amy E

    2016-02-01

    Antibodies (Abs) are a crucial component of the immune system and are often used as diagnostic and therapeutic agents. The need for high-affinity and high-specificity antibodies in research and medicine is driving the development of computational tools for accelerating antibody design and discovery. We report a diverse set of antibody binding data with accompanying structures that can be used to evaluate methods for modeling antibody interactions. Our Antibody-Bind (AB-Bind) database includes 1101 mutants with experimentally determined changes in binding free energies (ΔΔG) across 32 complexes. Using the AB-Bind data set, we evaluated the performance of protein scoring potentials in their ability to predict changes in binding free energies upon mutagenesis. Numerical correlations between computed and observed ΔΔG values were low (r = 0.16-0.45), but the potentials exhibited predictive power for classifying variants as improved vs weakened binders. Performance was evaluated using the area under the curve (AUC) for receiver operator characteristic (ROC) curves; the highest AUC values for 527 mutants with |ΔΔG| > 1.0 kcal/mol were 0.81, 0.87, and 0.88 using STATIUM, FoldX, and Discovery Studio scoring potentials, respectively. Some methods could also enrich for variants with improved binding affinity; FoldX and Discovery Studio were able to correctly rank 42% and 30%, respectively, of the 80 most improved binders (those with ΔΔG < -1.0 kcal/mol) in the top 5% of the database. This modest predictive performance has value but demonstrates the continuing need to develop and improve protein energy functions for affinity prediction. PMID:26473627

  7. Biophysical characterization of DNA binding from single molecule force measurements

    OpenAIRE

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-01-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as hig...

  8. Quantitative analysis of pheromone-binding protein specificity

    OpenAIRE

    Katti, S.; Lokhande, N.; D González; Cassill, A.; Renthal, R

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-p...

  9. The hepcidin-binding site on ferroportin is evolutionarily conserved

    OpenAIRE

    De Domenico, Ivana; Nemeth, Elizabeta; Nelson, Jenifer M.; Phillips, John D.; Ajioka, Richard S.; Kay, Michael S.; Kushner, James P.; Ganz, Tomas; Ward, Diane M.; Kaplan, Jerry

    2008-01-01

    Mammalian iron homeostasis is regulated by the interaction of the liver-produced peptide hepcidin and its receptor, the iron transporter ferroportin. Hepcidin binds to ferroportin resulting in degradation of ferroportin and decreased cellular iron export. We identify the hepcidin-binding domain (HBD) on ferroportin and show that a synthetic 19 amino acid peptide corresponding to the HBD recapitulates the characteristics and specificity of hepcidin binding to cell surface ferroportin. The bind...

  10. Specific receptor binding of staphylococcal enterotoxins by murine splenic lymphocytes.

    OpenAIRE

    Buxser, S; Bonventre, P F; Archer, D L

    1981-01-01

    We describe a reliable assay to measure the specific binding of 125I-labeled staphylococcal enterotoxin A (SEA) by murine spleen cells. Toxin binding by lymphocytes was specific in that it was inhibited by unlabeled SEA but not by unrelated proteins. The biological activity of SEA (T-lymphocyte mitogenesis) correlated with toxin binding to splenic lymphocytes. In the presence of high concentrations of [125I]SEA, specific binding increased rapidly and approached saturation after 2 h. Toxin bin...

  11. Extraembryonic signals under the control of MGA, Max, and Smad4 are required for dorsoventral patterning.

    Science.gov (United States)

    Sun, Yuhua; Tseng, Wei-Chia; Fan, Xiang; Ball, Rebecca; Dougan, Scott T

    2014-02-10

    In vertebrates, extraembryonic tissues can act as signaling centers that impose a reproducible pattern of cell types upon the embryo. Here, we show that the zebrafish yolk syncytial layer (YSL) secretes a ventralizing signal during gastrulation. This activity is mediated by Bmp2b/Swirl (Swr) expressed under the control of Max's giant associated protein (MGA) and its binding partners, Max and Smad4. MGA coimmunoprecipitates with both Max and Smad4 in embryo extracts, and the three proteins form a complex in vitro. Furthermore, all three proteins bind to a DNA fragment upstream of the bmp2b transcription start site. Targeted depletion of MGA, its binding partners, or Bmp2b/Swr from the YSL reduces BMP signaling throughout the embryo, resulting in a mildly dorsalized phenotype. We conclude that MGA, Max, and Smad4 act in the extraembryonic YSL to initiate a positive feedback loop of Bmp signaling within the embryo. PMID:24525188

  12. Landscape of protein-small ligand binding modes.

    Science.gov (United States)

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.

  13. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert; Rydström, Jan

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  14. Landscape of protein-small ligand binding modes.

    Science.gov (United States)

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  15. Biomimetic supramolecular metallohosts for binding and activation of dioxygen

    NARCIS (Netherlands)

    Sprakel, Vera Stefanie Irene

    2004-01-01

    Host-guest chemistry involves the binding of a specific substrate in a receptor via molecular recognition based on supramolecular interactions. Metal-containing derivatives of receptors for the selective supramolecular binding of dihydroxybenzene substrates, which receptors model oxygen binding enz

  16. Rapid determination of thyroxine binding proteins of human serum

    Directory of Open Access Journals (Sweden)

    Arima,Terukatsu

    1976-02-01

    Full Text Available A simple method is described for determing thyroxine binding proteins in human serum by electrophoresis at pH 8.6, using cellulose acetate membrane as the supporting medium. The procedure had high reliability in sera of normal subjects, pregnant women and patients with decreased thyroxine binding capacity of thyroxine binding globulin.

  17. Binding of Intrinsic and Extrinsic Features in Working Memory

    Science.gov (United States)

    Ecker, Ullrich K. H.; Maybery, Murray; Zimmer, Hubert D.

    2013-01-01

    There is ongoing debate concerning the mechanisms of feature binding in working memory. In particular, there is controversy regarding the extent to which these binding processes are automatic. The present article demonstrates that binding mechanisms differ depending on whether the to-be-integrated features are perceived as forming a coherent…

  18. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic aci...

  19. Tetrapyrrole binding affinity of the murine and human p22HBP heme-binding proteins.

    Science.gov (United States)

    Micaelo, Nuno M; Macedo, Anjos L; Goodfellow, Brian J; Félix, Vítor

    2010-11-01

    We present the first systematic molecular modeling study of the binding properties of murine (mHBP) and human (hHBP) p22HBP protein (heme-binding protein) with four tetrapyrrole ring systems belonging to the heme biosynthetic pathway: iron protoporphyrin IX (HEMIN), protoporphyrin IX (PPIX), coproporphyrin III (CPIII), coproporphyrin I (CPI). The relative binding affinities predicted by our computational study were found to be similar to those observed experimentally, providing a first rational structural analysis of the molecular recognition mechanism, by p22HBP, toward a number of different tetrapyrrole ligands. To probe the structure of these p22HBP protein complexes, docking, molecular dynamics and MM-PBSA methodologies supported by experimental NMR ring current shift data have been employed. The tetrapyrroles studied were found to bind murine p22HBP with the following binding affinity order: HEMIN> PPIX> CPIII> CPI, which ranged from -22.2 to -6.1 kcal/mol. In general, the protein-tetrapyrrole complexes are stabilized by non-bonded interactions between the tetrapyrrole propionate groups and basic residues of the protein, and by the preferential solvation of the complex compared to the unbound components. PMID:20800521

  20. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  1. 14C-glucose binding assay of the glucose transporter binding sites in muscular cell membrane

    International Nuclear Information System (INIS)

    A method of determining the binding sites of glucose transporter in rat muscular cell membrane was introduced. The crude products of cell membrane form the skeletal muscle of control and insulin treated rats were prepared, and then fractionated in sucrose gradient. Both plasma membrane and microsome membrane were incubated with D-[U-14C] glucose respectively for the measurement of radioactivity and Scatchard plot analysis. It was found that the binding sites of glucose transporter in plasma membrane and intracellular membrane were 5.6 nmol 14C-glucose/mg protein and 8.7 nmol 14C-glucose-mg protein respectively at basic state. Insulin treatment in experimental groups caused approximately 146% increase in plasma membrane fraction and 88% decrease in intracellular membrane fraction. Moreover, the kinetic data of Scatchard plot curve were similar to those of the [3H]-cytochalasin B binding assay. D-[U-14C] glucose binding assay of glucose transporter binding sites in muscular cell membrane is simple, easy and practicable. The D-[U-14C] glucose is commercially available

  2. Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

    OpenAIRE

    Win, Maung Nyan; Klein, Joshua S.; Smolke, Christina D.

    2006-01-01

    RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer se...

  3. PRBP: Prediction of RNA-Binding Proteins Using a Random Forest Algorithm Combined with an RNA-Binding Residue Predictor.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Xiao, Ke; Sun, Xiao

    2015-01-01

    The prediction of RNA-binding proteins is an incredibly challenging problem in computational biology. Although great progress has been made using various machine learning approaches with numerous features, the problem is still far from being solved. In this study, we attempt to predict RNA-binding proteins directly from amino acid sequences. A novel approach, PRBP predicts RNA-binding proteins using the information of predicted RNA-binding residues in conjunction with a random forest based method. For a given protein, we first predict its RNA-binding residues and then judge whether the protein binds RNA or not based on information from that prediction. If the protein cannot be identified by the information associated with its predicted RNA-binding residues, then a novel random forest predictor is used to determine if the query protein is a RNA-binding protein. We incorporated features of evolutionary information combined with physicochemical features (EIPP) and amino acid composition feature to establish the random forest predictor. Feature analysis showed that EIPP contributed the most to the prediction of RNA-binding proteins. The results also showed that the information from the RNA-binding residue prediction improved the overall performance of our RNA-binding protein prediction. It is anticipated that the PRBP method will become a useful tool for identifying RNA-binding proteins. A PRBP Web server implementation is freely available at http://www.cbi.seu.edu.cn/PRBP/.

  4. Zooming into the binding groove of HLA molecules : which positions and which substitutions change peptide binding most?

    NARCIS (Netherlands)

    van Deutekom, Hanneke W M; Kesmir, C.

    2015-01-01

    Human leukocyte antigen (HLA) genes are the most polymorphic genes in the human genome. Almost all polymorphic residues are located in the peptide-binding groove, resulting in different peptide-binding preferences. Whether a single amino acid change can alter the peptide-binding repertoire of an HLA

  5. Dual chain synthetic heparin-binding growth factor analogs

    Science.gov (United States)

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua

    2009-10-06

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  6. Dual chain synthetic heparin-binding growth factor analogs

    Energy Technology Data Exchange (ETDEWEB)

    Zamora, Paul O. (Gaithersburg, MD); Pena, Louis A. (Poquott, NY); Lin, Xinhua (Plainview, NY)

    2012-04-24

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  7. Binding of collagen to Staphylococcus aureus Cowan 1.

    OpenAIRE

    Speziale, P; Raucci, G; Visai, L.; Switalski, L M; Timpl, R; Höök, M

    1986-01-01

    Collagen binds to a receptor protein present on the surfaces of Staphylococcus aureus cells. Binding of 125I-labeled type II collagen to its bacterial receptor is reversible, and Scatchard plot analysis indicates the presence of one class of receptor that occurs on an average of 3 X 10(4) copies per cell and binds type II collagen with a Kd of 10(-7) M. Studies on the specificity of collagen cell binding indicate that the receptor does not recognize noncollagenous proteins but binds all of th...

  8. Dengue virus binding and replication by platelets.

    Science.gov (United States)

    Simon, Ayo Y; Sutherland, Michael R; Pryzdial, Edward L G

    2015-07-16

    Dengue virus (DENV) infection causes ∼200 million cases of severe flulike illness annually, escalating to life-threatening hemorrhagic fever or shock syndrome in ∼500,000. Although thrombocytopenia is typical of both mild and severe diseases, the mechanism triggering platelet reduction is incompletely understood. As a probable initiating event, direct purified DENV-platelet binding was followed in the current study by quantitative reverse transcription-polymerase chain reaction and confirmed antigenically. Approximately 800 viruses specifically bound per platelet at 37°C. Fewer sites were observed at 25°C, the blood bank storage temperature (∼350 sites), or 4°C, known to attenuate virus cell entry (∼200 sites). Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and heparan sulfate proteoglycan were implicated as coreceptors because only the combination of anti-DC-SIGN and low-molecular-weight heparin prevented binding. Interestingly, at 37°C and 25°C, platelets replicated the positive sense single-stranded RNA genome of DENV by up to ∼4-fold over 7 days. Further time course experiments demonstrated production of viral NS1 protein, which is known to be highly antigenic in patient serum. The infectivity of DENV intrinsically decayed in vitro, which was moderated by platelet-mediated generation of viable progeny. This was shown using a transcription inhibitor and confirmed by freeze-denatured platelets being incapable of replicating the DENV genome. For the first time, these data demonstrate that platelets directly bind DENV saturably and produce infectious virus. Thus, expression of antigen encoded by DENV is a novel consideration in the pathogen-induced thrombocytopenia mechanism. These results furthermore draw attention to the possibility that platelets may produce permissive RNA viruses in addition to DENV.

  9. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  10. Metal ion binding to iron oxides

    Science.gov (United States)

    Ponthieu, M.; Juillot, F.; Hiemstra, T.; van Riemsdijk, W. H.; Benedetti, M. F.

    2006-06-01

    The biogeochemistry of trace elements (TE) is largely dependent upon their interaction with heterogeneous ligands including metal oxides and hydrous oxides of iron. The modeling of TE interactions with iron oxides has been pursued using a variety of chemical models. The objective of this work is to show that it is possible to model the adsorption of protons and TE on a crystallized oxide (i.e., goethite) and on an amorphous oxide (HFO) in an identical way. Here, we use the CD-MUSIC approach in combination with valuable and reliable surface spectroscopy information about the nature of surface complexes of the TE. The other objective of this work is to obtain generic parameters to describe the binding of the following elements (Cd, Co, Cu, Ni, Pb, and Zn) onto both iron oxides for the CD-MUSIC approach. The results show that a consistent description of proton and metal ion binding is possible for goethite and HFO with the same set of model parameters. In general a good prediction of almost all the collected experimental data sets corresponding to metal ion binding to HFO is obtained. Moreover, dominant surface species are in agreement with the recently published surface complexes derived from X-ray absorption spectroscopy (XAS) data. Until more detailed information on the structure of the two iron oxides is available, the present option seems a reasonable approximation and can be used to describe complex geochemical systems. To improve our understanding and modeling of multi-component systems we need more data obtained at much lower metal ion to iron oxide ratios in order to be able to account eventually for sites that are not always characterized in spectroscopic studies.

  11. Liver fatty acid-binding protein binds monoacylglycerol in vitro and in mouse liver cytosol.

    Science.gov (United States)

    Lagakos, William S; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E; Storch, Judith

    2013-07-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  12. Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol*

    Science.gov (United States)

    Lagakos, William S.; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E.; Storch, Judith

    2013-01-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  13. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  14. Thyroxine binding to serum thyronine-binding globulin in thyroidectomized adult and normal neonatal rats

    International Nuclear Information System (INIS)

    The amount of tracer [125I]T4 bound to serum thyronine-binding globulin (TBG) was measured by polyacrylamide gel electrophoresis in adult thyroidectomized (TX) rats and normal 1-day to 4-week-old rat puts. Thyroidectomy was associated with the appearance of significant amounts of [125I]T4 binding to serum TBG in lean rats, but not in obese Zucker rats. Treatment of the TX rats in vivo with replacement doses of T4 prevented this increase in TBG binding, but enrichment of serum from TX rats with T4 did not. Significant amounts of tracer [125I]T4 binding to TBG was present in serum from 1- to 3-week-old normal rat pups, but not in 1-day- or 4-week-old pups. There were significantly higher levels of TBG binding of [125I]T4 in serum from 2-week-old rat pups raised in litters of 16 pups compared to those raised in litters of 4 pups. All manipulations that result in the appearance of TBG in rat serum also result in either weight loss or a slowing in the rate of growth, suggesting that the appearance of TBG in rat serum has a nutritional component. This possibility is further supported by the observations that increases in TBG binding of [125I]T4 are not found in obese Zucker rats fed a low protein-high carbohydrate diet for 14 days or fasted for 7 days, or after thyroidectomy, perhaps owing to the large stores of fuel in the obese rat

  15. Oligomerization of Mannan-binding Lectin Dictates Binding Properties and Complement Activation.

    Science.gov (United States)

    Kjaer, T R; Jensen, L; Hansen, A; Dani, R; Jensenius, J C; Dobó, J; Gál, P; Thiel, S

    2016-07-01

    The complement system is a part of the innate immune system and is involved in recognition and clearance of pathogens and altered-self structures. The lectin pathway of the complement system is initiated when soluble pattern recognition molecules (PRMs) with collagen-like regions bind to foreign or altered self-surfaces. Associated with the collagen-like stems of these PRMs are three mannan-binding lectin (MBL)-associated serine proteases (MASPs) and two MBL-associated proteins (MAps). The most studied of the PRMs, MBL, is present in serum mainly as trimeric and tetrameric oligomers of the structural subunit. We hypothesized that oligomerization of MBL may influence both the potential to bind to micro organisms and the interaction with the MASPs and MAps, thus influencing the ability to initiate complement activation. When testing binding at 37 °C, we found higher binding of tetrameric MBL to Staphylococcus aureus (S. aureus) than trimeric and dimeric MBL. In serum, we found that tetrameric MBL was the main oligomeric form present in complexes with the MASPs and MAp44. Such preference was confirmed using purified forms of recombinant MBL (rMBL) oligomers, where tetrameric rMBL interacted stronger with all of the MASPs and MAp44, compared to trimeric MBL. As a direct consequence of the weaker interaction with the MASPs, we found that trimeric rMBL was inferior to tetrameric rMBL in activating the complement system. Our data suggest that the oligomeric state of MBL is crucial both for the binding properties and the effector function of MBL.

  16. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  17. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  18. Systematic Calculations of Total Atomic Binding Energies

    International Nuclear Information System (INIS)

    We have calculated total atomic binding energies of 3- to 91-electron ions of all atoms with Z=3 to 118, in the Dirac-Fock model, for applications to atomic mass determination from highly-charged ions. In this process we have determined the ground-state configuration of many ions for which it was not known. We also provide total electronic correlation including Breit correlation for iso-electronic series of beryllium, neon, magnesium and argon, using the multiconfiguration Dirac-Fock approach.

  19. Binding of Actinomyces naeslundii to glycosphingolipids.

    OpenAIRE

    Brennan, M J; Joralmon, R A; Cisar, J O; Sandberg, A L

    1987-01-01

    The type 2 fimbrial lectin of Actinomyces naeslundii WVU45 mediated the binding of this bacterium to glycosphingolipids chromatographed on thin-layer silica gel plates. Radioiodinated bacteria attached to GM1, GD1b, and globoside. After chromatograms were treated with sialidase, the bacteria also bound to GD1a and GT1b. The actinomyces lectin apparently recognized the Gal beta 3GalNAc termini of these gangliosides and the GalNAc beta 3Gal terminus of globoside, suggesting that glycolipids con...

  20. Where metal ions bind in proteins.

    OpenAIRE

    Yamashita, M M; Wesson, L.; Eisenman, G.; Eisenberg, D.

    1990-01-01

    The environments of metal ions (Li+, Na+, K+, Ag+, Cs+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+) in proteins and other metal-host molecules have been examined. Regardless of the metal and its precise pattern of ligation to the protein, there is a common qualitative feature to the binding site: the metal is ligated by a shell of hydrophilic atomic groups (containing oxygen, nitrogen, or sulfur atoms) and this hydrophilic shell is embedded within a larger shell of hydrophobic atomic groups (containing car...

  1. DNS and BIND on IPv6

    CERN Document Server

    Liu, Cricket

    2011-01-01

    If you're preparing to roll out IPv6 on your network, this concise book provides the essentials you need to support this protocol with DNS. You'll learn how DNS was extended to accommodate IPv6 addresses, and how you can configure a BIND name server to run on the network. This book also features methods for troubleshooting problems with IPv6 forward- and reverse-mapping, and techniques for helping islands of IPv6 clients communicate with IPv4 resources. Topics include: DNS and IPv6-Learn the structure and representation of IPv6 addresses, and the syntaxes of AAAA and PTR records in the ip6.a

  2. Binding of Glutamate to the Umami Receptor

    OpenAIRE

    Lopez Cacales, J.; Oliviera Costa, S.; de Groot, B.; Walters, D

    2010-01-01

    Abstract The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. It detects glutamate in humans, and is a more general amino acid detector in other species. We have constructed homology models of the ligand binding domains of the human umami receptor (based on crystallographic structures of the metabotropic glutamate receptor of the central nervous system). We have carried out molecular dynamics simulations of the ligand bi...

  3. Binding characterization, synthesis and biological evaluation of RXRα antagonists targeting the coactivator binding site.

    Science.gov (United States)

    Xu, Dingyu; Guo, Shangjie; Chen, Ziwen; Bao, Yuzhou; Huang, Fengyu; Xu, Dan; Zhang, Xindao; Zeng, Zhiping; Zhou, Hu; Zhang, Xiaokun; Su, Ying

    2016-08-15

    Previously we identified the first retinoid X receptor-alpha (RXRα) modulators that regulate the RXRα biological function via binding to the coregulator-binding site. Here we report the characterization of the interactions between the hit molecule and RXRα through computational modeling, mutagenesis, SAR and biological evaluation. In addition, we reported studies of additional new compounds and identified a molecule that mediated the NF-κB pathway by inhibiting the TNFα-induced IκBα degradation and p65 nuclear translocation. PMID:27450787

  4. Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Lindblom, Karin;

    2009-01-01

    The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but...... biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins)....

  5. A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

    Directory of Open Access Journals (Sweden)

    Brian Krumm

    Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

  6. Characterization of the DNA binding properties of polyomavirus capsid protein

    Science.gov (United States)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  7. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  8. Methyl-CpG binding proteins in the nervous system

    Institute of Scientific and Technical Information of China (English)

    Guoping FAN; Leah HUTNICK

    2005-01-01

    Classical methyl-CpG binding proteins contain the conserved DNA binding motif methyl-cytosine binding domain (MBD), which preferentially binds to methylated CpG dinucleotides. These proteins serve as transcriptional repressors,mediating gene silencing via DNA cytosine methylation. Mutations in methyl-CpG binding protein 2 (MeCP2) have been linked to the human mental retardation disorder Rett syndrome, suggesting an important role for methyl-CpG binding proteins in brain development and function. This mini-review summarizes the recent advances in studying the diverse functions of MeCP2 as a prototype for other methyl-CpG binding proteins in the development and function of the vertebrate nervous system.

  9. Autoradiographic localization of endothelin-1 binding sites in porcine skin

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Y.D.; Springall, D.R.; Wharton, J.; Polak, J.M. (Royal Postgraduate Medical School, London (England))

    1991-01-01

    Autoradiographic techniques and {sup 125}I-labeled endothelin-1 were used to study the distribution of endothelin-1 binding sites in porcine skin. Specific endothelin-1 binding sites were localized to blood vessels (capillaries, deep cutaneous vascular plexus, arteries, and arterioles), the deep dermal and connective tissue sheath of hair follicles, sebaceous and sweat glands, and arrector pili muscle. Specific binding was inhibited by endothelin-2 and endothelin-3 as well as endothelin-1. Non-specific binding was found in the epidermis and the medulla of hair follicles. No binding was found in connective tissue or fat. These vascular binding sites may represent endothelin receptors, in keeping with the known cutaneous vasoconstrictor actions of the peptide. If all binding sites are receptors, the results suggest that endothelin could also regulate the function of sweat glands and may have trophic effects in the skin.

  10. Cooperative binding modes of Cu(II) in prion protein

    Science.gov (United States)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  11. Binding of Diphtheria Toxin to Phospholipids in Liposomes

    Science.gov (United States)

    Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

    1980-04-01

    Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

  12. Binding of anandamide to bovine serum albumin

    DEFF Research Database (Denmark)

    Bojesen, I.N.; Hansen, Harald S.

    2003-01-01

    with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0°C, 10°C, 23°C, and 37°C. The equilibrium dissociation constant (K ) increases with temperature from 6.87 ± 0.53 nM at 0°C to 54.92 ± 1.91 nM at 37°C. Regression analyses of the data suggest......The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because...... a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium...

  13. Reflection-Based Python-C++ Bindings

    International Nuclear Information System (INIS)

    Python is a flexible, powerful, high-level language with excellent interactive and introspective capabilities and a very clean syntax. As such, it can be a very effective tool for driving physics analysis. Python is designed to be extensible in low-level C-like languages, and its use as a scientific steering language has become quite widespread. To this end, existing and custom-written C or C++ libraries are bound to the Python environment as so-called