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Control Peptide (control + peptide)
Selected AbstractsIn vivo inhibition of antiphospholipid antibody-induced pathogenicity utilizing the antigenic target peptide domain I of ,2 -glycoprotein I: proof of conceptJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 5 2009Y. IOANNOU Summary.,Objectives:,In the antiphospholipid syndrome (APS), the immunodominant epitope for the majority of circulating pathogenic antiphospholipid antibodies (aPLs) is the N-terminal domain I (DI) of ,2 -glycoprotein I. We have previously shown that recombinant DI inhibits the binding of aPLs in fluid phase to immobilized native antigen, and that this inhibition is greater with the DI(D8S/D9G) mutant and absent with the DI(R39S) mutant. Hence, we hypothesized that DI and DI(D8S/D9G) would inhibit aPL-induced pathogenicity in vivo. Methods:,C57BL/6 mice (n = 5, each group) were injected with purified IgG derived from APS patients (IgG-APS, 500 ,g) or IgG from normal healthy serum (IgG-NHS) and either recombinant DI, DI(R39S), DI(D8S/D9G), or an irrelevant control peptide (at 10,40 ,g). Outcome variables measured were femoral vein thrombus dynamics in treated and control groups following standardized vessel injury, expression of vascular cell adhesion molecule-1 (VCAM-1) on the aortic endothelial surface, and tissue factor (TF) activity in murine macrophages. Results:,IgG-APS significantly increased thrombus size as compared with IgG-NHS. The IgG-APS thrombus enhancement effect was abolished in mice pretreated with recombinant DI (P , 0.0001) and DI(D8S/D9G) (P , 0.0001), but not in those treated with DI(R39S) or control peptide. This inhibitory effect by DI was dose-dependent, and at lower doses DI(D8S/D9G) was a more potent inhibitor of thrombosis than wild-type DI (P , 0.01). DI also inhibited IgG-APS induction of VCAM-1 on the aortic endothelial surface and TF production by murine macrophages. Conclusion:,Our findings in this proof-of-concept study support the development of recombinant DI or the novel variant DI(D8S/D9G) as a potential future therapeutic agent for APS. [source] Protease-activated receptor-4 (PAR4): a role as inhibitor of visceral pain and hypersensitivityNEUROGASTROENTEROLOGY & MOTILITY, Issue 11 2009C. Augé Abstract, Protease-activated receptor-4 (PAR4) belongs to the family of receptors activated by the proteolytic cleavage of their extracellular N-terminal domain and the subsequent binding of the newly released N-terminus. While largely expressed in the colon, the role of PAR4 in gut functions has not been defined. We have investigated the effects of PAR4 agonist on colonic sensations and sensory neuron signalling, and its role in visceral pain. We observed that a single administration of the PAR4 agonist peptide (AYPGKF-NH2), but not the control peptide (YAPGKF-NH2) into the colon lumen of mice significantly reduced the visceromotor response to colorectal distension at different pressures of distension. Further, intracolonic administration of the PAR4 agonist, but not the control peptide, was able to significantly inhibit PAR2 agonist- and transcient receptor potential vanilloid-4 (TRPV4) agonist-induced allodynia and hyperalgesia in response to colorectal distension. Protease-activated receptor-4 was detected in sensory neurons projecting from the colon, and isolated from the dorsal root ganglia, where it co-expressed with PAR2 and TRPV4. In total sensory neurons, PAR4 agonist exposure inhibited free intracellular calcium mobilization induced by the pro-nociceptive agonists of PAR2 and TRPV4. Finally, PAR4 -deficient mice experienced increased pain behaviour in response to intracolonic administration of mustard oil, compared with wild-type littermates. These results show that PAR4 agonists modulate colonic nociceptive response, inhibit colonic hypersensitivity and primary afferent responses to pro-nociceptive mediators. Endogenous activation of PAR4 also plays a major role in controlling visceral pain. These results identify PAR4 as a previously unknown modulator of visceral nociception. [source] Analysis of the factors that stabilize a designed two-stranded antiparallel ,-sheetPROTEIN SCIENCE, Issue 6 2002Juan F. Espinosa Abstract Autonomously folding ,-hairpins (two-strand antiparallel ,-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a ,-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in ,-hairpin stability by comparing 1 with DP, N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the DPG segment decreases the entropic cost of ,-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of DPG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed ,-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins. [source] Triggering of proteinase-activated receptor 4 leads to joint pain and inflammation in miceARTHRITIS & RHEUMATISM, Issue 3 2009Jason J. McDougall Objective To investigate the role of proteinase-activated receptor 4 (PAR-4) in mediating joint inflammation and pain in mice. Methods Knee joint blood flow, edema, and pain sensitivity (as induced by thermal and mechanical stimuli) were assessed in C57BL/6 mice following intraarticular injection of either the selective PAR-4 agonist AYPGKF-NH2 or the inactive control peptide YAPGKF-NH2. The mechanism of action of AYPGKF-NH2 was examined by pretreatment of each mouse with either the PAR-4 antagonist pepducin P4pal-10 or the bradykinin antagonist HOE 140. Finally, the role of PAR-4 in mediating joint inflammation was tested by pretreating mice with acutely inflamed knees with pepducin P4pal-10. Results PAR-4 activation caused a long-lasting increase in joint blood flow and edema formation, which was not seen following injection of the control peptide. The PAR-4,activating peptide was also found to be pronociceptive in the joint, where it enhanced sensitivity to a noxious thermal stimulus and caused mechanical allodynia and hyperalgesia. The proinflammatory and pronociceptive effects of AYPGKF-NH2 could be inhibited by pepducin P4pal-10 and HOE 140. Finally, pepducin P4pal-10 ameliorated the clinical and physiologic signs of acute joint inflammation. Conclusion This study demonstrates that local activation of PAR-4 leads to proinflammatory changes in the knee joint that are dependent on the kallikrein,kinin system. We also show for the first time that PARs are involved in the modulation of joint pain, with PAR-4 being pronociceptive in this tissue. Thus, blockade of articular PAR-4 may be a useful means of controlling joint inflammation and pain. [source] CXCR4 chemokine receptors (CD184) and ,4,1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis)BRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2003Jan A. Burger Summary. Marrow stromal cells play an important role in regulating the development and proliferation of haematopoietic stem cells (HSC) within the marrow microenvironment. However, the molecular mechanisms of stem cell,stromal cell interactions are not fully understood. We observed that mobilized peripheral blood and cord-blood-derived CD34+ progenitor cells, or CD34+ acute myeloid leukaemia (AML) cells spontaneously migrated beneath marrow stromal cells, an in vitro migration phenomenon termed pseudoemperipolesis. In contrast, the CD34+ myeloid leukaemia cell line, Kasumi-1, did not display pseudoemperipolesis. Cord blood CD34+ cells had a higher capacity than granulocyte-colony-stimulating-factor-mobilized CD34+ cells for pseudoemperipolesis (28·7 ± 12%vs 18·1 ± 6·1% of input cells within 24 h, mean ± SD, n = 8), whereas 9·4 ± 12·6% (mean ± SD, n = 10) of input AML cells displayed this phenomenon. Pseudoemperipolesis of CD34+ progenitor and AML cells was significantly inhibited by pertussis toxin and antibodies to the CXCR4 chemokine receptor (CXCR4, CD184), but not control antibodies. Moreover, CD34+ and AML cell migration was significantly inhibited by a CS1 peptide that blocks ,4,1 integrin binding, but not by a control peptide, in which the fibronectin binding motif was scrambled. Pseudoemperipolesis was associated with an increased proliferation of migrated CD34+ progenitor cells but not AML cells within the stromal layer, demonstrated by cell cycle analysis and cell division tracking. We conclude that ,4,1 integrin binding and CXCR4 chemokine receptor activation are prerequisites for the migration of CD34+ haematopoietic progenitors and AML cells beneath marrow stromal cells. These observations suggest a central role of marrow stromal cells for HSC trafficking and homing within the marrow microenvironment. [source] | ||||||||||