ABSTRACT
Forkhead transcription factors (TFs) often dimerize outside their extensive family, whereas bHLH transcription factors typically dimerize with E12/E47. Based on structural similarities, we predicted that a member of the former, Forkhead Box P1 (FOXP1), might heterodimerize with a member of the latter, MYOD1 (MyoD). Data shown here support this hypothesis and further demonstrate the specificity of this forkhead/myogenic interaction among other myogenic regulatory factors. We found that FOXP1-MyoD heterodimerization compromises the ability of MyoD to bind to E-boxes and to transactivate E box- containing promoters. We observed that FOXP1 is required for the full ability of MyoD to convert fibroblasts into myotubules. We provide a model in which FOXP1 displaces ID and E12/E47 to repress MyoD during the proliferative phase of myoblast differentiation. These data identify FOXP1 as a hitherto unsuspected transcriptional repressor of MyoD. We suggest that isolation of paired E-box and forkhead sites within 1 turn helical spacings provides potential for cooperative interactions among heretofore distinct classes of transcription factors.
ABSTRACT
rhPTH1-34 is clinically used for osteoporosis treatment. However, this peptide drug has no oral bioavailability because of proteolysis and low membrane permeability in gastrointestinal gut. This study explored the possibility of absorption enhancement for rhPTH1-34 through the oral delivery of the microemulsion. The microemulsion (85:15, oil/water) consisting of Labrasol, Crodamol GTCC, Solutol HS 15, d-α-tocopheryl acetate (6:2:1:1, w/w) and saline water was developed and characterized, including particle size, morphology, drug loading efficiency and permeability, stability and pharmacokinetics. The microemulsion showed high drug loading efficiency (83%) and permeability, and significantly higher resistance to proteolysis in vitro study. The relative oral bioavailability was 5.4% and 12.0% when delivered to gastric and ileum. Besides, osteoporosis rats were induced and treated with oral rhPTH1-34 microemulsion (0.05 mg/kg), injection (0.01 mg/kg) and vehicle, respectively, for 8 weeks. The proximal tibia bone mineral content and density in oral rats (0.188 ± 0.008 g, 0.283 ± 0.014 g/cm(2)) was significantly increased compared to the control rats (0.169 ± 0.006 g, 0.266 ± 0.011 g/cm(2)), reaching to the sham rats. And the proximal tibia microstructure of oral rats was improved greatly, approaching sham level too. These findings revealed that oral microemulsion may represent an effective oral delivery system for rhPTH1-34.
Subject(s)
Bone Density Conservation Agents/administration & dosage , Drug Carriers , Teriparatide/administration & dosage , Administration, Oral , Animals , Biological Availability , Bone Density/drug effects , Bone Density Conservation Agents/chemistry , Caco-2 Cells , Chemistry, Pharmaceutical , Disease Models, Animal , Drug Compounding , Drug Stability , Emulsions , Female , Gastric Mucosa/metabolism , Humans , Ileum/metabolism , Intestinal Absorption , Male , Nanotechnology , Osteoporosis/drug therapy , Osteoporosis/pathology , Ovariectomy , Particle Size , Peptide Hydrolases/metabolism , Permeability , Rats , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosage , Solubility , Technology, Pharmaceutical/methods , Teriparatide/chemistry , Tibia/drug effects , Tibia/pathologyABSTRACT
Auxin is a pivotal plant hormone that controls many aspects of plant growth and development. Perceived by a small family of F-box proteins including transport inhibitor response 1 (TIR1), auxin regulates gene expression by promoting SCF ubiquitin-ligase-catalysed degradation of the Aux/IAA transcription repressors, but how the TIR1 F-box protein senses and becomes activated by auxin remains unclear. Here we present the crystal structures of the Arabidopsis TIR1-ASK1 complex, free and in complexes with three different auxin compounds and an Aux/IAA substrate peptide. These structures show that the leucine-rich repeat domain of TIR1 contains an unexpected inositol hexakisphosphate co-factor and recognizes auxin and the Aux/IAA polypeptide substrate through a single surface pocket. Anchored to the base of the TIR1 pocket, auxin binds to a partially promiscuous site, which can also accommodate various auxin analogues. Docked on top of auxin, the Aux/IAA substrate peptide occupies the rest of the TIR1 pocket and completely encloses the hormone-binding site. By filling in a hydrophobic cavity at the protein interface, auxin enhances the TIR1-substrate interactions by acting as a 'molecular glue'. Our results establish the first structural model of a plant hormone receptor.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , F-Box Proteins/chemistry , F-Box Proteins/metabolism , Indoleacetic Acids/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Arabidopsis/enzymology , Arabidopsis/metabolism , Binding Sites , Crystallography, X-Ray , Indoleacetic Acids/chemistry , Models, Molecular , Phytic Acid/metabolism , Plant Growth Regulators/chemistry , Plant Growth Regulators/metabolism , Protein Structure, Tertiary , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Cryptosporidiosis, a protozoan disease, is caused by Cryptosporidium parvum in animals and humans. To study the humoral and cellular immune responses induced by DNA vaccine expressing the sporozoite surface protein, CP15/60, of Cryptosporidium parvum, the recombinant plasmid containing the CP15/60 gene was injected into tibialis a interior muscle of BALB/c mice. The mice were subsequently given booster doses twice at 3-week intervals. The humoral and cellular immune responses were detected at different times after immunization. The mice were then challenged by inoculation of 1 x 10(6) oocysts of C. parvum. The experimental results have shown that the recombinant plasmid can induce corresponding specific immune responses and thus protect the mice from challenge of the oocysts, suggesting that the recombinant plasmid could be a potential candidate of DNA vaccine.
