Nociceptive sensitization by the secretory protein Bv8.
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1 The small protein Bv8, isolated from amphibian skin, belongs to a novel family of secretory proteins (Bv8-Prokineticin family, SWISS-PROT: Q9PW66) whose orthologues have been conserved throughout evolution, from invertebrates to humans. 2 When injected intravenously or subcutaneously (from 0.06 to 500 pmol kg(-1)) or intrathecally (from 6 fmol to 250 pmol) in rats, Bv8 produced an intense systemic nociceptive sensitization to mechanical and thermal stimuli applied to the tail and paws. 3 Topically delivered into one rat paw, 50 fmol of Bv8 decreased by 50% the nociceptive threshold to pressure in the injected paw without affecting the threshold in the contralateral paw. 4 The two G-protein coupled prokineticin receptors, PK-R1 and PK-R2, were expressed in rat dorsal root ganglia (DRG) and in dorsal quadrants of spinal cord (DSC) and bound Bv8 and the mammalian orthologue, EG-VEGF, with high affinity. In DSC, PK-R1 was more abundant than PK-R2, whereas both receptors were equally expressed in DRG. IC(50) of Bv8 and EG-VEGF to inhibit [(125)I]-Bv8 binding to rat DRG and DSC were 4.1+/-0.4 nM Bv8 and 76.4+/-7.6 nM EG-VEGF, in DRG; 7.3+/-0.9 nM Bv8 and 330+/-41 nM EG-VEGF, in DSC. 5 In the small diameter neurons (<30 microm) of rat DRG cultures, Bv8 concentrations, ranging from 0.2 to 10 nM, raised [Ca(2+)](i) in a dose-dependent manner. 6 These data suggest that Bv8, through binding to PK receptors of DSC and primary sensitive neurons, results in intense sensitization of peripheral nociceptors to thermal and mechanical stimuli. (+info)
Identification of a novel putative signaling center, the tertiary enamel knot in the postnatal mouse molar tooth.
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The final shape of the molar tooth crown is thought to be regulated by the transient epithelial signaling centers in the cusp tips, the secondary enamel knots (SEKs), which are believed to disappear after initiation of the cusp growth. We investigated the developmental fate of the signaling center using the recently characterized Slit1 enamel knot marker as a lineage tracer during morphogenesis of the first molar and crown calcification in the mouse. In situ hybridization analysis showed that after Fgf4 downregulation in the SEK, Slit1 expression persisted in the deep compartment of the knot. After the histological disappearance of the SEK, Slit1 expression was evident in a novel epithelial cell cluster, which we call the tertiary enamel knot (TEK) next to the enamel-free area (EFA)-epithelium at the cusp tips. In embryonic tooth, Slit1 was also observed in the stratum intermedium (SI) and stellate reticulum cells between the parallel SEKs correlating to the area where the inner enamel epithelium cells do not proliferate. After birth, the expression of Slit1 persisted in the SI cells of the transverse connecting lophs of the parallel cusps above the EFA-cells. These results demonstrate the presence of a novel putative signaling center, the TEK, in the calcifying tooth. Moreover, our results suggest that Slit1 signaling may be involved in the regulation of molar tooth shape by regulating epithelial cell proliferation and formation of EFA of the crown. (+info)
Mechanism of corticotropin-releasing factor type I receptor regulation by nonpeptide antagonists.
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Mechanisms of nonpeptide ligand action at family B G protein-coupled receptors are largely unexplored. Here, we evaluated corticotropin-releasing factor 1 (CRF(1)) receptor regulation by nonpeptide antagonists. The antagonist mechanism was investigated at the G protein-coupled (RG) and uncoupled (R) states of the receptor in membranes from Ltk(-) cells expressing the cloned human CRF(1) receptor. R was detected with the antagonist (125)I-astressin with 30 microM guanosine 5'-O-(3-thiotriphosphate present, and RG detected using (125)I-sauvagine. At the R state, nonpeptide antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 only partially inhibited (125)I-astressin binding (22-32% maximal inhibition). NBI 35965 accelerated (125)I-astressin dissociation and only partially increased the IC(50) value of unlabeled sauvagine, CRF, and urocortin for displacing (125)I-astressin binding (by 4.0-7.1-fold). Reciprocal effects at the R state were demonstrated using [(3)H]NBI 35965: agonist peptides only partially inhibited binding (by 13-40%) and accelerated [(3)H]NBI 35965 dissociation. These data are quantitatively consistent with nonpeptide antagonist and peptide ligand binding spatially distinct sites, with mutual, weak negative cooperativity (allosteric inhibition) between their binding. At the RG state the compounds near fully inhibited (125)I-sauvagine binding at low radioligand concentrations (79-94 pM). NBI 35965 did not completely inhibit (125)I-sauvagine binding at high radioligand concentrations (82 +/- 1%, 1.3-2.1 nM) and slowed dissociation of (125)I-sauvagine and (125)I-CRF. The antagonist effect at RG is consistent with either strong allosteric inhibition or competitive inhibition at one of the peptide agonist binding sites. These findings demonstrate a novel effect of R-G interaction on the inhibitory activity of nonpeptide antagonists: Although the compounds are weak inhibitors of peptide binding to the R state, they strongly inhibit peptide agonist binding to RG. Strong inhibition at RG explains the antagonist properties of the compounds. (+info)
A soluble form of the first extracellular domain of mouse type 2beta corticotropin-releasing factor receptor reveals differential ligand specificity.
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The heptahelical receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2, display different specificities for CRF family ligands: CRF and urocortin I bind to CRFR1 with high affinity, whereas urocortin II and III bind to this receptor with very low affinities. In contrast, all the urocortins bind with high affinities, and CRF binds with lower affinity to CRFR2. The first extracellular domain (ECD1) of CRFR1 is important for ligand recognition. Here, we characterize a bacterially expressed soluble protein, ECD1-CRFR2beta, corresponding to the ECD1 of mouse CRFR2beta. The K(i) values for binding to ECD1-CRFR2beta are: astressin = 10.7 (5.4-21.1) nm, urocortin I = 6.4 (4.7-8.7) nm, urocortin II = 6.9 (5.8-8.3) nm, CRF = 97 (22-430) nm, urocortin III = sauvagine >200 nm. These affinities are similar to those for binding to a chimeric receptor in which the ECD1 of CRFR2beta replaces the ECD of the type 1B activin receptor (ALK4). The ECD1-CRFR2beta possesses a disulfide arrangement identical to that of the ECD1 of CRFR1, namely Cys(45)-Cys(70), Cys(60)-Cys(103), and Cys(84)-Cys(118). As determined by circular dichroism, ECD1-CRFR2beta undergoes conformational changes upon binding astressin. These data reinforce the importance of the ECD1 of CRF receptors for ligand recognition and raise the interesting possibility that different ligands having similar affinity for the full-length receptor may, nevertheless, have different affinities for microdomains of the receptor. (+info)
nNOS inhibition, antimicrobial and anticancer activity of the amphibian skin peptide, citropin 1.1 and synthetic modifications. The solution structure of a modified citropin 1.1.
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A large number of bioactive peptides have been isolated from amphibian skin secretions. These peptides have a variety of actions including antibiotic and anticancer activities and the inhibition of neuronal nitric oxide synthase. We have investigated the structure-activity relationship of citropin 1.1, a broad-spectrum antibiotic and anticancer agent that also causes inhibition of neuronal nitric oxide synthase, by making a number of synthetically modified analogues. Citropin 1.1 has been shown previously to form an amphipathic alpha-helix in aqueous trifluoroethanol. The results of the structure-activity studies indicate the terminal residues are important for bacterial activity and increasing the overall positive charge, while maintaining an amphipathic distribution of residues, increases activity against Gram-negative organisms. Anticancer activity generally mirrors antibiotic activity suggesting a common mechanism of action. The N-terminal residues are important for inhibition of neuronal nitric oxide synthase, as is an overall positive charge greater than three. The structure of one of the more active synthetic modifications (A4K14-citropin 1.1) was determined in aqueous trifluoroethanol, showing that this peptide also forms an amphipathic alpha-helix. (+info)
Receptor occupancy of nonpeptide corticotropin-releasing factor 1 antagonist DMP696: correlation with drug exposure and anxiolytic efficacy.
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4-(1,3-Dimethoxyprop-2-ylamine)-2,7-dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5- a]-1,3,5-triazine (DMP696) is a highly selective and potent, nonpeptide corticotropin-releasing factor 1 (CRF(1)) antagonist. In this study, we measured in vivo CRF(1) receptor occupancy of DMP696 by using ex vivo ligand binding and quantitative autoradiography and explored the relationship of receptor occupancy with plasma and brain exposure and behavioral efficacy. In vitro affinity (IC(50)) of DMP696 to brain CRF(1) receptors measured using the brain section binding autoradiography in this study is similar to that assessed using homogenized cell membrane assays previously. The ex vivo binding assay was validated by demonstrating that potential underestimation of receptor occupancy with this procedure could be minimized by identifying an appropriate in vitro incubation time (40 min) based upon the dissociation kinetics of DMP696. Orally administrated DMP696 dose dependently occupied CRF(1) receptors in the brain, with ~60% occupancy at 3 mg/kg. In the defensive withdrawal test of anxiety, this dose of DMP696 produced approximately 50% reduction in the exit latency. The time course of plasma and brain drug levels paralleled that of receptor occupancy, with peak exposure at 90 min after dosing. The plasma-free concentration of DMP696 corresponding to 50% CRF(1) receptor occupancy (in vivo IC(50), 1.22 nM) was similar to the in vitro IC(50) (~1.0 nM). Brain concentrations of DMP696 were over 150-fold higher than the plasma-free levels. In conclusion, doses of DMP696 occupying over 50% brain CRF(1) receptors are consistent with doses producing anxiolytic efficacy in the defense withdrawal test of anxiety, and the IC(50) value estimated in vivo based on plasma-free drug concentrations is consistent with the in vitro IC(50) value. (+info)
Antimicrobial peptides from hylid and ranin frogs originated from a 150-million-year-old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain.
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The dermal glands of frogs produce antimicrobial peptides that protect the skin against noxious microorganisms and assist in wound repair. The sequences of these peptides are very dissimilar, both within and between species, so that the 5000 living anuran frogs may produce approximately 100 000 different antimicrobial peptides. The antimicrobial peptides of South American hylid frogs are derived from precursors, the preprodermaseptins, whose signal peptides and intervening sequences are remarkably conserved, but their C-terminal domains are markedly diverse, resulting in mature peptides with different lengths, sequences and antimicrobial spectra. We have used the extreme conservation in the preproregion of preprodermaseptin transcripts to identify new members of this family in Australian and South American hylids. All these peptides are cationic, amphipathic and alpha-helical. They killed a broad spectrum of microorganisms and acted in synergy. 42 preprodermaseptin gene sequences from 10 species of hylid and ranin frogs were analyzed in the context of their phylogeny and biogeography and of geophysical models for the fragmentation of Gondwana to examine the strategy that these frogs have evolved to generate an enormous array of peptide antibiotics. The hyperdivergence of modern antimicrobial peptides and the number of peptides per species result from repeated duplications of a approximately 150-million-year-old ancestral gene and accelerated mutations of the mature peptide domain, probably involving a mutagenic, error-prone, DNA polymerase similar to Escherichia coli Pol V. The presence of antimicrobial peptides with such different structures and spectra of action represents the successful evolution of multidrug defense by providing frogs with maximum protection against infectious microbes and minimizing the chance of microorganisms developing resistance to individual peptides. The hypermutation of the antimicrobial domain by a targeted mutagenic polymerase that can generate many sequence changes in a few steps may have a selective survival value when frogs colonizing a new ecological niche encounter different microbial predators. (+info)
Conformation, orientation, and adsorption kinetics of dermaseptin B2 onto synthetic supports at aqueous/solid interface.
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The antimicrobial activity of cationic amphipathic peptides is due mainly to the adsorption of peptides onto target membranes, which can be modulated by such physicochemical parameters as charge and hydrophobicity. We investigated the structure of dermaseptin B2 (Drs B2) at the aqueous/synthetic solid support interface and its adsorption kinetics using attenuated total reflection Fourier transform infrared spectroscopy and surface plasmon resonance. We determined the conformation and affinity of Drs B2 adsorbed onto negatively charged (silica or dextran) and hydrophobic supports. Synthetic supports of differing hydrophobicity were obtained by modifying silica or gold with omega-functionalized alkylsilanes (bromo, vinyl, phenyl, methyl) or alkylthiols. The peptide molecules adsorbed onto negatively charged supports mostly had a beta-type conformation. In contrast, a monolayer of Drs B2, mainly in the alpha-helical conformation, was adsorbed irreversibly onto the hydrophobic synthetic supports. The conformational changes during formation of the adsorbed monolayer were monitored by two-dimensional Fourier transform infrared spectroscopy correlation; they showed the influence of peptide-peptide interactions on alpha-helix folding on the most hydrophobic support. The orientation of the alpha-helical Drs B2 with respect to the hydrophobic support was determined by polarized attenuated total reflection; it was around 15 +/- 5 degrees. This orientation was confirmed and illustrated by a molecular dynamics study. These combined data demonstrate that specific chemical environments influence the structure of Drs B2, which could explain the many functions of antimicrobial peptides. (+info)