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Sample records for a-induced formyl peptide

  1. Role of formyl peptide receptor 2 on the serum amyloid A-induced macrophage foam cell formation.

    PubMed

    Lee, Ha Young; Kim, Sang Doo; Baek, Suk-Hwan; Choi, Joon Hyuk; Bae, Yoe-Sik

    2013-04-05

    Recently we demonstrated that SAA induces macrophage foam cell formation. In this study we show that SAA-induced foam cell formation is inhibited by formyl peptide receptor 2 (FPR2) antagonist WRW(4), as well as by FPR2-targeted siRNA knockdown. SAA-stimulated LOX1 expression was also mediated by FPR2. We also found that SAA-stimulated foam cell formation and LOX1 expression was pertussis toxin-insensitive. In addition, FPR2 is upregulated in peripheral blood mononuclear cells from patients with atherosclerosis. Our findings therefore suggest that SAA stimulates foam cell formation via FPR2 signaling and LOX1 induction, and thus likely contributes to atherogenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Differential activation of polymorphisms of the formyl peptide receptor by formyl peptides

    PubMed Central

    Mills, John S.

    2007-01-01

    We have investigated the role of two polymorphic sites (R190W and N192K) on the binding and activation of the formyl peptide receptor (FPR) by viral and formyl peptides. WEDWVGWI, a peptide with antiviral activity derived from the membrane proximal region of feline immunodeficiency virus, binds with high affinity to FPR. The three tryptophans in the peptide are all essential for FPR binding, just as they were essential for antiviral activity (Giannecchini, S. et al., J. Virol. 77 (2003) 3724). Formyl-NleWEDWVGWI behaved as a weak partial agonist with FPR W190/N192 but a stronger partial agonist with FPR R190/K192 and FPR R190/N192. Formyl-NleNleWEDWVGWI behaved as a full agonist toward all three FPRs but exhibited a much higher EC50 with W190/N192 FPR (300±45 nM) than for R190/K192 FPR (40±3 nM) or R190/N192 (60±8 nM). Formyl-MYKWPWYVWL preferentially activated R190/K192 and R190/N192 FPRs by >5 fold compared to W190/N192 FPR. Formyl-MFEDAVAWF, a peptide derived from a protein in Mycobacterium avium subsp. paratuberculosis and formyl-MFTFEPFPTN, a peptide derived from the N-terminus of chemotaxis inhibitory protein of Staphylococcus aureus with an added N-terminal methionine exhibited the greatest selectivity for R190/K192 and R190/N192 FPRs with ~10 fold lower EC50S than that observed with FPR W190/N192. Thus, individuals with the W190 polymorphism may display a reduced ability to detect certain formyl peptides. PMID:17644322

  3. Formyl peptide receptor chimeras define domains involved in ligand binding.

    PubMed

    Perez, H D; Holmes, R; Vilander, L R; Adams, R R; Manzana, W; Jolley, D; Andrews, W H

    1993-02-05

    We have begun to study the structural requirements for the binding of formyl peptides to their specific receptors. As an initial approach, we constructed C5a-formyl peptide receptor chimeras. Unique (and identical) restriction sites were introduced within the transmembrane domains of these receptors that allowed for the exchange of specific areas. Four types of chimeric receptors were generated. 1) The C5a receptor was progressively substituted by the formyl peptide receptor. 2) The formyl peptide receptor was progressively substituted by the C5a receptor. 3) Specific domains of the C5a receptor were substituted by the corresponding domain of the formyl peptide receptor. 4) Specific domains of the formyl peptide receptor were replaced by the same corresponding domain of the C5a receptor. Wild type and chimeric receptors were transfected into COS 7 cells and their ability to bind formyl peptide determined, taking into account efficiency of transfection and expression of chimeric protein. Based on these results, a ligand binding model is presented in which the second, third, and fourth extracellular (and/or their transmembrane) domains together with the first transmembrane domain form a ligand binding pocket for formyl peptides. It is proposed that the amino-terminal domain plays a role by presumably providing a "lid" to the pocket. The carboxyl-terminal cytoplasmic tail appears to modulate ligand binding by regulating receptor affinity.

  4. Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1.

    PubMed

    Liu, Fu-Chao; Yu, Huang-Ping; Syu, Yu-Ting; Fang, Jia-You; Lin, Chwan-Fwu; Chang, Shih-Hsin; Lee, Yen-Tung; Hwang, Tsong-Long

    2017-07-27

    Formyl peptide receptor 1 (FPR1) mediates bacterial and mitochondrial N-formyl peptides-induced neutrophil activation. Therefore, FPR1 is an important therapeutic target for drugs to treat septic or sterile inflammatory diseases. Honokiol, a major bioactive compound of Magnoliaceae plants, possesses several anti-inflammatory activities. Here, we show that honokiol exhibits an inhibitory effect on FPR1 binding in human neutrophils. Honokiol inhibited superoxide anion generation, reactive oxygen species formation, and elastase release in bacterial or mitochondrial N-formyl peptides (FPR1 agonists)-activated human neutrophils. Adhesion of FPR1-induced human neutrophils to cerebral endothelial cells was also reduced by honokiol. The receptor-binding results revealed that honokiol repressed FPR1-specific ligand N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein binding to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells. However, honokiol did not inhibit FPR2-specific ligand binding to FPR2 in human neutrophils. Furthermore, honokiol inhibited FPR1 agonist-induced calcium mobilization as well as phosphorylation of p38 MAPK, ERK, and JNK in human neutrophils. In conclusion, our data demonstrate that honokiol may have therapeutic potential for treating FPR1-mediated inflammatory diseases.

  5. Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors

    PubMed Central

    Bufe, Bernd; Schumann, Timo; Kappl, Reinhard; Bogeski, Ivan; Kummerow, Carsten; Podgórska, Marta; Smola, Sigrun; Hoth, Markus; Zufall, Frank

    2015-01-01

    Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system. PMID:25605714

  6. Novel role for endogenous mitochondrial formylated peptide-driven formyl peptide receptor 1 signalling in acute respiratory distress syndrome.

    PubMed

    Dorward, David A; Lucas, Christopher D; Doherty, Mary K; Chapman, Gavin B; Scholefield, Emma J; Conway Morris, Andrew; Felton, Jennifer M; Kipari, Tiina; Humphries, Duncan C; Robb, Calum T; Simpson, A John; Whitfield, Phillip D; Haslett, Christopher; Dhaliwal, Kevin; Rossi, Adriano G

    2017-10-01

    Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography-tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1-/- mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  7. Commensal-Epithelial Signaling Mediated via Formyl Peptide Receptors

    PubMed Central

    Wentworth, Christy C.; Jones, Rheinallt M.; Kwon, Young Man; Nusrat, Asma; Neish, Andrew S.

    2010-01-01

    Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteria-mediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IκB or pro-apoptotic phospho-c-Jun NH2-terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-Met-Leu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a Gi-protein inhibitor) abolished commensal-mediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia. PMID:21037077

  8. Commensal-epithelial signaling mediated via formyl peptide receptors.

    PubMed

    Wentworth, Christy C; Jones, Rheinallt M; Kwon, Young Man; Nusrat, Asma; Neish, Andrew S

    2010-12-01

    Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteria-mediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IκB or pro-apoptotic phospho-c-Jun NH(2)-terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-Met-Leu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a G(i)-protein inhibitor) abolished commensal-mediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia.

  9. Formylated MHC Class Ib Binding Peptides Activate Both Human and Mouse Neutrophils Primarily through Formyl Peptide Receptor 1.

    PubMed

    Winther, Malene; Holdfeldt, André; Gabl, Michael; Wang, Ji Ming; Forsman, Huamei; Dahlgren, Claes

    2016-01-01

    Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits.

  10. Formylated MHC Class Ib Binding Peptides Activate Both Human and Mouse Neutrophils Primarily through Formyl Peptide Receptor 1

    PubMed Central

    Winther, Malene; Holdfeldt, André; Gabl, Michael; Wang, Ji Ming; Forsman, Huamei; Dahlgren, Claes

    2016-01-01

    Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits. PMID:27907124

  11. Recruitment of opioid peptide-containing neutrophils is independent of formyl peptide receptors.

    PubMed

    Hackel, D; Stolz, A; Mousa, S A; Brack, A; Rittner, H L

    2011-01-01

    In complete Freund's adjuvants (CFA) inflammation opioid containing neutrophils release opioid peptides upon stimulation and mediate peripheral analgesia. Neutrophil migration is regulated partially by chemokines, but other mediators e.g. formyl peptides could also contribute. In vitro, formyl peptides but not Mycobacterium butyricum (CFA component) induced migration of neutrophils. In contrast, local formyl peptide injection did not induce leukocyte recruitment in vivo due to insufficient up-regulation of adhesion molecule expression. Furthermore, leukocyte recruitment and peripheral opioid-mediated analgesia were unaffected by systemic formyl peptide receptor blockade in CFA inflammation. Thus, while formyl peptides do not regulate migration they directly stimulate opioid peptide release. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Functional characterization of three mouse formyl peptide receptors.

    PubMed

    He, Hui-Qiong; Liao, Dan; Wang, Zhen-Guo; Wang, Zhong-Li; Zhou, Hu-Chen; Wang, Ming-Wei; Ye, Richard D

    2013-02-01

    The evolutionary relationship and functional correlation between human formyl peptide receptors (FPRs) and their mouse counterparts remain incompletely understood. We examined three members of the mouse formyl peptide receptor subfamily (mFprs) and found that they differ in agonist preference and cellular distributions. When stably expressed in transfected rat basophilic leukemia (RBL-2H3) cells, mFpr1 was readily activated by N-formylated peptides derived from Listeria monocytogenes (fMIVTLF), Staphylococcus aureus (fMIFL), and mitochondria (fMMYALF). In contrast, the Escherichia coli-derived fMLF was 1000-fold less potent. The aforementioned peptides were much less efficacious at mFpr2, which responded better to the synthetic hexapeptide WKYMVm, the synthetic agonists Quin-C1 (a substituted quinazolinone), and compound 43 (a nitrosylated pyrazolone derivative). Saturation binding assays showed that mFpr1 and mFpr2 were expressed at similar levels on the cell surface, although their affinity for N-formyl-Met-Leu-Phe-Ile-Ile-Lys-fluorescein isothiocyanate varied by more than 1000-fold [dissociation constant (K(d)) values of 2.8 nM for mFpr1 and 4.8 μM for mFpr2]). Contrary to these receptors, mFpr-rs1 responded poorly to all the previously mentioned peptides that were tested. Fluorescent microscopy revealed an intracellular distribution pattern of mFpr-rs1. On the basis of these results, we conclude that mFpr1 is an ortholog of human FPR1 with certain pharmacologic properties of human FPR2/ALX, whereas mFpr2 has much lower affinity for formyl peptides. The intracellular distribution of mFpr-rs1 suggests an evolutionary correlation with human FPR3.

  13. Functional Characterization of Three Mouse Formyl Peptide Receptors

    PubMed Central

    He, Hui-Qiong; Liao, Dan; Wang, Zhen-Guo; Wang, Zhong-Li; Zhou, Hu-Chen; Wang, Ming-Wei

    2013-01-01

    The evolutionary relationship and functional correlation between human formyl peptide receptors (FPRs) and their mouse counterparts remain incompletely understood. We examined three members of the mouse formyl peptide receptor subfamily (mFprs) and found that they differ in agonist preference and cellular distributions. When stably expressed in transfected rat basophilic leukemia (RBL-2H3) cells, mFpr1 was readily activated by N-formylated peptides derived from Listeria monocytogenes (fMIVTLF), Staphylococcus aureus (fMIFL), and mitochondria (fMMYALF). In contrast, the Escherichia coli–derived fMLF was 1000-fold less potent. The aforementioned peptides were much less efficacious at mFpr2, which responded better to the synthetic hexapeptide WKYMVm, the synthetic agonists Quin-C1 (a substituted quinazolinone), and compound 43 (a nitrosylated pyrazolone derivative). Saturation binding assays showed that mFpr1 and mFpr2 were expressed at similar levels on the cell surface, although their affinity for N-formyl-Met-Leu-Phe-Ile-Ile-Lys-fluorescein isothiocyanate varied by more than 1000-fold [dissociation constant (Kd) values of 2.8 nM for mFpr1 and 4.8 μM for mFpr2]). Contrary to these receptors, mFpr-rs1 responded poorly to all the previously mentioned peptides that were tested. Fluorescent microscopy revealed an intracellular distribution pattern of mFpr-rs1. On the basis of these results, we conclude that mFpr1 is an ortholog of human FPR1 with certain pharmacologic properties of human FPR2/ALX, whereas mFpr2 has much lower affinity for formyl peptides. The intracellular distribution of mFpr-rs1 suggests an evolutionary correlation with human FPR3. PMID:23160941

  14. The potential impacts of formyl peptide receptor 1 in inflammatory diseases.

    PubMed

    Yang, Shun-Chin; Hwang, Tsong-Long

    2016-06-01

    Neutrophils play a critical role in acute and chronic inflammatory diseases. N-formyl peptides, which originate from bacterial peptides or mitochondrial proteins bind with a high binding affinity to formyl peptide receptor 1 (FPR1). N-formyl peptide-FPR1 is involved in the pathogenesis of sterile and infectious inflammatory processes and causes phagocytosis of pathogens or injured cells by neutrophils. Excessive activation of neutrophils by binding of N-formyl peptides is associated with tissue injury requiring drugs that block FPR1-dependent signaling. Here, we review the roles of FPR1 as a critical regulator of inflammatory processes and its involvement in pathological conditions.

  15. Mitochondrial N-formyl peptides cause airway contraction and lung neutrophil infiltration via formyl peptide receptor activation.

    PubMed

    Wenceslau, Camilla Ferreira; Szasz, Theodora; McCarthy, Cameron G; Baban, Babak; NeSmith, Elizabeth; Webb, R Clinton

    2016-04-01

    Respiratory failure is a common characteristic of systemic inflammatory response syndrome (SIRS) and sepsis. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns (DAMPs). Mitochondrial N-formyl peptides (F-MITs) are DAMPs that share similarities with bacterial N-formylated peptides, and are potent immune system activators. Recently, we observed that hemorrhagic shock-induced increases in plasma levels of F-MITs associated with lung damage, and that antagonism of formyl peptide receptors (FPR) ameliorated hemorrhagic shock-induced lung injury in rats. Corroborating these data, in the present study, it was observed that F-MITs expression is higher in plasma samples from trauma patients with SIRS or sepsis when compared to control trauma group. Therefore, to better understand the role of F-MITs in the regulation of lung and airway function, we studied the hypothesis that F-MITs lead to airway contraction and lung inflammation. We observed that F-MITs induced concentration-dependent contraction in trachea, bronchi and bronchioles. However, pre-treatment with mast cells degranulator or FPR antagonist decreased this response. Finally, intratracheal challenge with F-MITs increased neutrophil elastase expression in lung and inducible nitric oxide synthase and cell division control protein 42 expression in all airway segments. These data suggest that F-MITs could be a putative target to treat respiratory failure in trauma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. New development in studies of formyl-peptide receptors: critical roles in host defense

    PubMed Central

    Li, Liangzhu; Chen, Keqiang; Xiang, Yi; Yoshimura, Teizo; Su, Shaobo; Zhu, Jianwei; Bian, Xiu-wu; Wang, Ji Ming

    2016-01-01

    Formyl-peptide receptors are a family of 7 transmembrane domain, Gi-protein-coupled receptors that possess multiple functions in many pathophysiologic processes because of their expression in a variety of cell types and their capacity to interact with a variety of structurally diverse, chemotactic ligands. Accumulating evidence demonstrates that formyl-peptide receptors are critical mediators of myeloid cell trafficking in the sequential chemotaxis signal relays in microbial infection, inflammation, and immune responses. Formyl-peptide receptors are also involved in the development and progression of cancer. In addition, one of the formyl-peptide receptor family members, Fpr2, is expressed by normal mouse-colon epithelial cells, mediates cell responses to microbial chemotactic agonists, participates in mucosal development and repair, and protects against inflammation-associated tumorigenesis. These novel discoveries greatly expanded the current understanding of the role of formyl-peptide receptors in host defense and as potential molecular targets for the development of therapeutics. PMID:26701131

  17. Formyl Peptide Receptors in Cellular Differentiation and Inflammatory Diseases.

    PubMed

    Lee, Ha Young; Lee, Mingyu; Bae, Yoe-Sik

    2017-06-01

    Formyl peptide receptors (FPRs) are a family of classical chemoattractant receptors. Although FPRs are mainly expressed in phagocytic innate immune cells including monocytes/macrophages and neutrophils, recent reports demonstrated that additional different cell types such as T-lymphocytes and several non-immune cells also express functional FPRs. FPRs were first reported as a specific receptor to detect bacteria-derived N-formyl peptides. However, accumulating evidence has shown that FPRs can recognize various ligands derived from pathogens, mitochondria, and host. This review summarizes studies on some interesting endogenous agonists for FPRs. Here, we discuss functional roles of FPRs and their ligands concerning the regulation of cellular differentiation focusing on myeloid lineage cells. Accumulating evidence also suggests that FPRs may contribute to the control of inflammatory diseases. Here, we briefly review the current understanding of the functional role of FPRs and their ligands in inflammatory disorders in some animal disease models. J. Cell. Biochem. 118: 1300-1307, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  18. Structural determinants for the interaction of formyl peptide receptor 2 with peptide ligands.

    PubMed

    He, Hui-Qiong; Troksa, Erica L; Caltabiano, Gianluigi; Pardo, Leonardo; Ye, Richard D

    2014-01-24

    Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-281(7.32) is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-281(7.32) might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D281(7.32)G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides.

  19. Structural Determinants for the Interaction of Formyl Peptide Receptor 2 with Peptide Ligands*

    PubMed Central

    He, Hui-Qiong; Troksa, Erica L.; Caltabiano, Gianluigi; Pardo, Leonardo; Ye, Richard D.

    2014-01-01

    Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-2817.32 is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-2817.32 might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D2817.32G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides. PMID:24285541

  20. Monocyte Activation by Necrotic Cells Is Promoted by Mitochondrial Proteins and Formyl Peptide Receptors

    PubMed Central

    Crouser, Elliott D.; Shao, Guohong; Julian, Mark W.; Macre, Jennifer E.; Shadel, Gerald S.; Tridandapani, Susheela; Huang, Qin; Wewers, Mark D.

    2009-01-01

    Objective Necrotic cells evoke potent innate immune responses through unclear mechanisms. The mitochondrial fraction of the cell retains constituents of its bacterial ancestors, including N-formyl peptides, which are potentially immunogenic. Thus, we hypothesized that the mitochondrial fraction of the cell, particularly N-formyl peptides, contributes significantly to the activation of monocytes by necrotic cells. Design Human peripheral blood monocytes were incubated with necrotic cell fractions and mitochondrial proteins in order to investigate their potential for immune cell activation. Setting University medical center research laboratory. Subjects Healthy human adults served as blood donors. Measurements and Main Results Human blood monocyte activation was measured after treatment with cytosolic, nuclear and mitochondrial fractions of necrotic HepG2 cells or necrotic HepG2 cells depleted of N-formyl peptides [Rho(0) cells]. The specific role of the high affinity formyl peptide receptor (FPR) was then tested using specific pharmacological inhibitors and RNA-silencing. The capacity of mitochondrial N-formyl peptides to activate monocytes was confirmed using a synthetic peptide conforming to the N-terminus of mitochondrial NADH subunit 6. The results demonstrated that mitochondrial cell fractions most potently activated monocytes, and IL-8 was selectively released at low protein concentrations. Mitochondria from Rho(0) cells induced minimal monocyte IL-8 release, and specific pharmacological inhibitors and RNA-silencing confirmed that FPR contributes significantly to monocyte IL-8 responses to both necrotic cells and mitochondrial proteins. N-formyl peptides alone did not induce monocyte IL-8 release; whereas, the combination of mitochondrial N-formyl peptides and mitochondrial transcription factor A (TFAM) dramatically increased IL-8 release from monocytes. Likewise, HMGB1, the nuclear homologue of TFAM, did not induce monocyte IL-8 release unless combined with

  1. Chemotherapy-induced antitumor immunity requires formyl peptide receptor 1.

    PubMed

    Vacchelli, Erika; Ma, Yuting; Baracco, Elisa E; Sistigu, Antonella; Enot, David P; Pietrocola, Federico; Yang, Heng; Adjemian, Sandy; Chaba, Kariman; Semeraro, Michaela; Signore, Michele; De Ninno, Adele; Lucarini, Valeria; Peschiaroli, Francesca; Businaro, Luca; Gerardino, Annamaria; Manic, Gwenola; Ulas, Thomas; Günther, Patrick; Schultze, Joachim L; Kepp, Oliver; Stoll, Gautier; Lefebvre, Céline; Mulot, Claire; Castoldi, Francesca; Rusakiewicz, Sylvie; Ladoire, Sylvain; Apetoh, Lionel; Bravo-San Pedro, José Manuel; Lucattelli, Monica; Delarasse, Cécile; Boige, Valérie; Ducreux, Michel; Delaloge, Suzette; Borg, Christophe; André, Fabrice; Schiavoni, Giovanna; Vitale, Ilio; Laurent-Puig, Pierre; Mattei, Fabrizio; Zitvogel, Laurence; Kroemer, Guido

    2015-11-20

    Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer. We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1(-/-) mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity. Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes. Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses.

  2. Epigenetic regulation of the formyl peptide receptor 2 gene.

    PubMed

    Simiele, Felice; Recchiuti, Antonio; Patruno, Sara; Plebani, Roberto; Pierdomenico, Anna Maria; Codagnone, Marilina; Romano, Mario

    2016-10-01

    Lipoxin (LX) A4, a main stop signal of inflammation, exerts potent bioactions by activating a specific G protein-coupled receptor, termed formyl peptide receptor 2 and recently renamed ALX/FPR2. Knowledge of the regulatory mechanisms that drive ALX/FPR2 gene expression is key for the development of innovative anti-inflammatory pharmacology. Here, we examined chromatin patterns of the ALX/FPR2 gene. We report that in MDA-MB231 breast cancer cells, the ALX/FPR2 gene undergoes epigenetic silencing characterized by low acetylation at lysine 27 and trimethylation at lysine 4, associated with high methylation at lysine 27 of histone 3. This pattern, which is consistent with transcriptionally inaccessible chromatin leading to low ALX/FPR2 mRNA and protein expression, is reversed in polymorphonuclear leukocytes that express high ALX/FPR2 levels. Activation of p300 histone acetyltransferase and inhibition of DNA methyltransferase restored chromatin accessibility and significantly increased ALX/FPR2 mRNA transcription and protein levels in MDA-MB231 cells, as well as in pulmonary artery endothelial cells. In both cells types, changes in the histone acetylation/methylation status enhanced ALX/FPR2 signaling in response to LXA4. Collectively, these results uncover unappreciated epigenetic regulation of ALX/FPR2 expression that can be exploited for innovative approaches to inflammatory disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Expression of Formyl-peptide Receptors in Human Lung Carcinoma.

    PubMed

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; Lucariello, Angela; De Luca, Antonio; De Rosa, Nicolina; Mazzarella, Gennaro; Bianco, Andrea; Ammendola, Rosario

    2015-05-01

    Formyl-peptide receptors (FPRs) are expressed in several tissues and cell types. The identification of markers involved in cell growth may further allow for molecular profiling of lung cancer. We investigated the possible role of FPRs as molecular markers in several types of lung carcinomas which is the main cause of cancer death worldwide. Tumor tissue samples were collected from six patients affected by lung cancer. Biopsies were analyzed for expression of FPR isoforms both in tumoral and peritumoral tissue by real-time polymerase chain reaction (PCR), western blot and immunofluorescence. Real-time PCR, western blot and immunofluorescence analyses showed that FPR expression is lower in types of human lung cancer tissues when compared to the surrounding peritumoral tissues. The study of the mechanistic basis for the control of FPR expression in normal peritumoral versus tumoral tissues could provide the basis for new diagnostic and therapeutic interventions. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  4. Mycobacteria Attenuate Nociceptive Responses by Formyl Peptide Receptor Triggered Opioid Peptide Release from Neutrophils

    PubMed Central

    Voigt, Philipp; Mousa, Shaaban; Stolz, Andrea; Labuz, Dominika; Schäfer, Michael; Schaefer, Michael; Stein, Christoph; Brack, Alexander

    2009-01-01

    In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, β-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR

  5. The Role of Formylated Peptides and Formyl Peptide Receptor 1 in Governing Neutrophil Function during Acute Inflammation

    PubMed Central

    Dorward, David A.; Lucas, Christopher D.; Chapman, Gavin B.; Haslett, Christopher; Dhaliwal, Kevin; Rossi, Adriano G.

    2015-01-01

    Neutrophil migration to sites of inflammation and the subsequent execution of multiple functions are designed to contain and kill invading pathogens. These highly regulated and orchestrated processes are controlled by interactions between numerous receptors and their cognate ligands. Unraveling and identifying those that are central to inflammatory processes may represent novel therapeutic targets for the treatment of neutrophil-dominant inflammatory disorders in which dysregulated neutrophil recruitment, function, and elimination serve to potentiate rather than resolve an initial inflammatory insult. The first G protein–coupled receptor to be described on human neutrophils, formyl peptide receptor 1 (FPR1), is one such receptor that plays a significant role in the execution of these functions through multiple intracellular signaling pathways. Recent work has highlighted important observations with regard to both receptor function and the importance and functional relevance of FPR1 in the pathogenesis of a range of both sterile and infective inflammatory conditions. In this review, we explore the multiple components of neutrophil migration and function in both health and disease, with a focus on the role of FPR1 in these processes. The current understanding of FPR1 structure, function, and signaling is examined, alongside discussion of the potential importance of FPR1 in inflammatory diseases suggesting that FPR1 is a key regulator of the inflammatory environment. PMID:25791526

  6. New development in studies of formyl-peptide receptors: critical roles in host defense.

    PubMed

    Li, Liangzhu; Chen, Keqiang; Xiang, Yi; Yoshimura, Teizo; Su, Shaobo; Zhu, Jianwei; Bian, Xiu-wu; Wang, Ji Ming

    2016-03-01

    Formyl-peptide receptors are a family of 7 transmembrane domain, Gi-protein-coupled receptors that possess multiple functions in many pathophysiologic processes because of their expression in a variety of cell types and their capacity to interact with a variety of structurally diverse, chemotactic ligands. Accumulating evidence demonstrates that formyl-peptide receptors are critical mediators of myeloid cell trafficking in the sequential chemotaxis signal relays in microbial infection, inflammation, and immune responses. Formyl-peptide receptors are also involved in the development and progression of cancer. In addition, one of the formyl-peptide receptor family members, Fpr2, is expressed by normal mouse-colon epithelial cells, mediates cell responses to microbial chemotactic agonists, participates in mucosal development and repair, and protects against inflammation-associated tumorigenesis. These novel discoveries greatly expanded the current understanding of the role of formyl-peptide receptors in host defense and as potential molecular targets for the development of therapeutics. © Society for Leukocyte Biology.

  7. The Formyl Peptide Receptors: Diversity of Ligands and Mechanism for Recognition.

    PubMed

    He, Hui-Qiong; Ye, Richard D

    2017-03-13

    The formyl peptide receptors (FPRs) are G protein-coupled receptors that transduce chemotactic signals in phagocytes and mediate host-defense as well as inflammatory responses including cell adhesion, directed migration, granule release and superoxide production. In recent years, the cellular distribution and biological functions of FPRs have expanded to include additional roles in homeostasis of organ functions and modulation of inflammation. In a prototype, FPRs recognize peptides containing N-formylated methionine such as those produced in bacteria and mitochondria, thereby serving as pattern recognition receptors. The repertoire of FPR ligands, however, has expanded rapidly to include not only N-formyl peptides from microbes but also non-formyl peptides of microbial and host origins, synthetic small molecules and an eicosanoid. How these chemically diverse ligands are recognized by the three human FPRs (FPR1, FPR2 and FPR3) and their murine equivalents is largely unclear. In the absence of crystal structures for the FPRs, site-directed mutagenesis, computer-aided ligand docking and structural simulation have led to the identification of amino acids within FPR1 and FPR2 that interact with several formyl peptides. This review article summarizes the progress made in the understanding of FPR ligand diversity as well as ligand recognition mechanisms used by these receptors.

  8. Adaptive evolution of formyl peptide receptors in mammals.

    PubMed

    Muto, Yoshinori; Guindon, Stéphane; Umemura, Toshiaki; Kőhidai, László; Ueda, Hiroshi

    2015-02-01

    The formyl peptide receptors (FPRs) are a family of chemoattractant receptors with important roles in host defense and the regulation of inflammatory reactions. In humans, three FPR paralogs have been identified (FPR1, FPR2, and FPR3) and may have functionally diversified by gene duplication and adaptive evolution. However, the evolutionary mechanisms operating in the diversification of FPR family genes and the changes in selection pressures have not been characterized to date. Here, we have made a comprehensive evolutionary analysis of FPR genes from mammalian species. Phylogenetic analysis showed that an early duplication was responsible for FPR1 and FPR2/FPR3 splitting, and FPR3 originated from the latest duplication event near the origin of primates. Codon-based tests of positive selection reveal interesting patterns in FPR1 and FPR2 versus FPR3, with the first two genes showing clear evidence of positive selection at some sites while the majority of them evolve under strong negative selection. In contrast, our results suggest that the selective pressure may be relaxed in the FPR3 lineage. Of the six amino acid sites inferred to evolve under positive selection in FPR1 and FPR2, four sites were located in extracellular loops of the protein. The electrostatic potential of the extracellular surface of FPR might be affected more frequently with amino acid substitutions in positively selected sites. Thus, positive selection of FPRs among mammals may reflect a link between changes in the sequence and surface structure of the proteins and is likely to be important in the host's defense against invading pathogens.

  9. FITC Conjugation Markedly Enhances Hepatic Clearance of N-Formyl Peptides.

    PubMed

    Øie, Cristina Ionica; Snapkov, Igor; Elvevold, Kjetil; Sveinbjørnsson, Baldur; Smedsrød, Bård

    2016-01-01

    In both septic and aseptic inflammation, N-formyl peptides may enter the circulation and induce a systemic inflammatory response syndrome similar to that observed during septic shock. The inflammatory response is brought about by the binding of N-formyl peptide to formyl peptide receptors (FPRs), specific signaling receptors expressed on myeloid as well as non-myeloid cells involved in the inflammatory process. N-formyl peptides conjugated with fluorochromes, such as fluorescein isothiocyanate (FITC) are increasingly experimentally used to identify tissues involved in inflammation. Hypothesizing that the process of FITC-conjugation may transfer formyl peptide to a ligand that is efficiently cleared from the circulation by the natural powerful hepatic scavenging regime we studied the biodistribution of intravenously administered FITC-fNLPNTL (Fluorescein-isothiocyanate- N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys) in mice. Our findings can be summarized as follows: i) In contrast to unconjugated fNLPNTL, FITC-fNLPNTL was rapidly taken up in the liver; ii) Mouse and human liver sinusoidal endothelial cells (LSECs) and hepatocytes express formyl peptide receptor 1 (FRP1) on both mRNA (PCR) and protein (Western blot) levels; iii) Immunohistochemistry showed that mouse and human liver sections expressed FRP1 in LSECs and hepatocytes; and iv) Uptake of FITC-fNLPNTL could be largely blocked in mouse and human hepatocytes by surplus-unconjugated fNLPNTL, thereby suggesting that the hepatocytes in both species recognized FITC-fNLPNTL and fNLPNTL as indistinguishable ligands. This was in contrast to the mouse and human LSECs, in which the uptake of FITC-fNLPNTL was mediated by both FRP1 and a scavenger receptor, specifically expressed on LSECs. Based on these results we conclude that a significant proportion of FITC-fNLPNTL is taken up in LSECs via a scavenger receptor naturally expressed in these cells. This calls for great caution when using FITC-fNLPNTL and other chromogen

  10. FITC Conjugation Markedly Enhances Hepatic Clearance of N-Formyl Peptides

    PubMed Central

    Elvevold, Kjetil; Sveinbjørnsson, Baldur; Smedsrød, Bård

    2016-01-01

    In both septic and aseptic inflammation, N-formyl peptides may enter the circulation and induce a systemic inflammatory response syndrome similar to that observed during septic shock. The inflammatory response is brought about by the binding of N-formyl peptide to formyl peptide receptors (FPRs), specific signaling receptors expressed on myeloid as well as non-myeloid cells involved in the inflammatory process. N-formyl peptides conjugated with fluorochromes, such as fluorescein isothiocyanate (FITC) are increasingly experimentally used to identify tissues involved in inflammation. Hypothesizing that the process of FITC-conjugation may transfer formyl peptide to a ligand that is efficiently cleared from the circulation by the natural powerful hepatic scavenging regime we studied the biodistribution of intravenously administered FITC-fNLPNTL (Fluorescein-isothiocyanate- N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys) in mice. Our findings can be summarized as follows: i) In contrast to unconjugated fNLPNTL, FITC-fNLPNTL was rapidly taken up in the liver; ii) Mouse and human liver sinusoidal endothelial cells (LSECs) and hepatocytes express formyl peptide receptor 1 (FRP1) on both mRNA (PCR) and protein (Western blot) levels; iii) Immunohistochemistry showed that mouse and human liver sections expressed FRP1 in LSECs and hepatocytes; and iv) Uptake of FITC-fNLPNTL could be largely blocked in mouse and human hepatocytes by surplus-unconjugated fNLPNTL, thereby suggesting that the hepatocytes in both species recognized FITC-fNLPNTL and fNLPNTL as indistinguishable ligands. This was in contrast to the mouse and human LSECs, in which the uptake of FITC-fNLPNTL was mediated by both FRP1 and a scavenger receptor, specifically expressed on LSECs. Based on these results we conclude that a significant proportion of FITC-fNLPNTL is taken up in LSECs via a scavenger receptor naturally expressed in these cells. This calls for great caution when using FITC-fNLPNTL and other chromogen

  11. Immunotherapeutic potential of N-formylated peptides of ESAT-6 and glutamine synthetase in experimental tuberculosis.

    PubMed

    Mir, Shabir Ahmad; Sharma, Sadhna

    2014-02-01

    Recent understanding of the pathogenesis of tuberculosis allows the possible application of immunotherapy for the treatment of tuberculosis. Therapies that would upregulate the host anti mycobacterial innate and/or adaptive immune response have been supposed to be useful in the treatment of tuberculosis. Since N-formyl peptides are products of bacterial metabolism, and their binding to a specific phagocyte receptor (FPR) induces chemotaxis and activation of phagocytes that are critical effectors in our innate immune system, it is reasonable to assume that the interaction between these two counterparts (i.e. formylated peptides and FPR) is also important in host defence against M. tuberculosis. In the present study the direct immunotherapeutic potential of N-formylated peptides of two non-classically secreted proteins (early secreted antigenic target-6 and glutamine synthetase) of M. tuberculosis H37Rv was evaluated. Treatment of M. tuberculosis H37Rv infected mice with N-formylated peptides of early secreted antigenic target-6 (ESAT-6) and glutamine synthetase (GS) markedly reduced the bacilli load in their lungs (p < 0.001) and spleen (p < 0.01) as compared to the untreated mice. In addition, the histopathological changes were observed to be in correlation with the CFU data with minor areas of consolidation in the lung sections of N-formylated peptide treated infected mice as compared to those of the untreated mice. Further, these N-formylated peptides were able to confer an additional therapeutic effect when given in combination with the anti tuberculosis drugs and hence can be used as an adjunct to the conventional chemotherapy against tuberculosis.

  12. Mitochondrial-derived N-formyl peptides: novel links between trauma, vascular collapse and sepsis.

    PubMed

    Wenceslau, C F; McCarthy, C G; Goulopoulou, S; Szasz, T; NeSmith, E G; Webb, R C

    2013-10-01

    Sepsis is a major cause of mortality and morbidity in trauma patients despite aggressive treatment. Traumatic injury may trigger infective or non-infective systemic inflammatory response syndrome (SIRS) and sepsis. Sepsis and SIRS are accompanied by an inability to regulate the inflammatory response but the cause of this perturbation is still unknown. The major pathophysiological characteristic of sepsis is the vascular collapse (i.e., loss of control of vascular tone); however, at the cellular level the final mediator of extreme vasodilatation has yet to be identified. After trauma, cellular injury releases endogenous damage-associated molecular patterns (DAMPs) that activate the innate immune system. Mitochondrial DAMPs express at least two molecular signatures, N-formyl peptides and mitochondrial DNA that act on formyl peptide receptors (FPRs) and Toll-like receptor 9, respectively. N-Formyl peptides are potent immunocyte activators and, once released in the circulation, they induce modulation of vascular tone by cellular mechanisms that are not completely understood. We have observed that N-formyl peptides from bacterial (FMLP) and mitochondrial (FMIT) sources induce FPR-mediated vasodilatation in resistance arteries. Accordingly, we propose that tissue and cellular trauma induces the release of N-formyl peptides from mitochondria triggering inflammation and vascular collapse via activation of FPR and contributing to the development of sepsis. The proposed hypothesis provides clinically significant information linking trauma, mitochondrial N-formyl peptides and inflammation to vascular collapse and sepsis. If our hypothesis is true, it may lead to new strategies in the management of sepsis that can help clinicians effectively manage non-infectious and infectious inflammatory responses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Propofol inhibits superoxide production, elastase release, and chemotaxis in formyl peptide-activated human neutrophils by blocking formyl peptide receptor 1.

    PubMed

    Yang, Shun-Chin; Chung, Pei-Jen; Ho, Chiu-Ming; Kuo, Chan-Yen; Hung, Min-Fa; Huang, Yin-Ting; Chang, Wen-Yi; Chang, Ya-Wen; Chan, Kwok-Hon; Hwang, Tsong-Long

    2013-06-15

    Neutrophils play a critical role in acute and chronic inflammatory processes, including myocardial ischemia/reperfusion injury, sepsis, and adult respiratory distress syndrome. Binding of formyl peptide receptor 1 (FPR1) by N-formyl peptides can activate neutrophils and may represent a new therapeutic target in either sterile or septic inflammation. Propofol, a widely used i.v. anesthetic, has been shown to modulate immunoinflammatory responses. However, the mechanism of propofol remains to be established. In this study, we showed that propofol significantly reduced superoxide generation, elastase release, and chemotaxis in human neutrophils activated by fMLF. Propofol did not alter superoxide generation or elastase release in a cell-free system. Neither inhibitors of γ-aminobutyric acid receptors nor an inhibitor of protein kinase A reversed the inhibitory effects of propofol. In addition, propofol showed less inhibitory effects in non-FPR1-induced cell responses. The signaling pathways downstream from FPR1, involving calcium, AKT, and ERK1/2, were also competitively inhibited by propofol. These results show that propofol selectively and competitively inhibits the FPR1-induced human neutrophil activation. Consistent with the hypothesis, propofol inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analog of fMLF, to FPR1 in human neutrophils, differentiated THP-1 cells, and FPR1-transfected human embryonic kidney-293 cells. To our knowledge, our results identify, for the first time, a novel anti-inflammatory mechanism of propofol by competitively blocking FPR1 in human neutrophils. Considering the importance of N-formyl peptides in inflammatory processes, our data indicate that propofol may have therapeutic potential to attenuate neutrophil-mediated inflammatory diseases by blocking FPR1.

  14. Identification of formyl peptides from Listeria monocytogenes and Staphylococcus aureus as potent chemoattractants for mouse neutrophils 1

    PubMed Central

    Southgate, Erica L.; He, Rong L.; Gao, Ji-Liang; Murphy, Philip M.; Nanamori, Masakatsu; Ye, Richard D.

    2009-01-01

    The prototypic formyl peptide N-formyl-Met-Leu-Phe (fMLF) is a major chemoattractant found in Escherichia coli culture supernatants and a potent agonist at human formyl peptide receptor (FPR) 1. Consistent with this, fMLF induces bactericidal functions in human neutrophils at nanomolar concentrations. However, it is a much less potent agonist for mouse FPR (mFPR) 1 and mouse neutrophils, requiring micromolar concentrations for cell activation. To determine whether other bacteria produce more potent agonists for mFPR1, we examined formyl peptides from Listeria monocytogenes and Staphylococcus aureus for their abilities to activate mouse neutrophils. A pentapeptide (N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL)) from L. monocytogenes and a tetrapeptide (N-formyl-Met-Ile-Phe-Leu (fMIFL)) from S. aureus were found to induce mouse neutrophil chemotaxis at 1-10 nM and superoxide production at 10-100 nM, similar to the potency of fMLF on human neutrophils. Using transfected cell lines expressing mFPR1 and mFPR2, which are major forms of FPRs in mouse neutrophils, we found that mFPR1 is responsible for the high potency of fMIVIL and fMIFL. In comparison, activation of mFPR2 requires micromolar concentrations of the two peptides. Genetic deletion of mfpr1 resulted in abrogation of neutrophil superoxide production and degranulation in response to fMIVIL and fMIFL, further demonstrating that mFPR1 is the primary receptor for detection of these formyl peptides. In conclusion, the formyl peptides from L. monocytogenes and S. aureus are 100-fold more potent than fMLF in activating mouse neutrophils. The ability of mFPR1 to detect bacterially derived formyl peptides indicates that this important host defense mechanism is conserved in mice. PMID:18606697

  15. Non-peptide ligand binding to the formyl peptide receptor FPR2--A comparison to peptide ligand binding modes.

    PubMed

    Stepniewski, Tomasz M; Filipek, Slawomir

    2015-07-15

    Ligands of the FPR2 receptor initiate many signaling pathways including activation of phospholipase C, protein kinase C, the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/protein kinase B pathway. The possible actions include also calcium flux, superoxide generation, as well as migration and proliferation of monocytes. FPR2 activation may induce a pro- and anti-inflammatory effect depending on the ligand type. It is also found that this receptor is involved in tumor growth. Most of currently known FPR2 ligands are agonists since they were designed based on N-formyl peptides, which are natural agonists of formyl receptors. Since the non-peptide drugs are indispensable for effective treatment strategies, we performed a docking study of such ligands employing a generated dual template homology model of the FPR2 receptor. The study revealed different binding modes of particular classes of these drugs. Based on the obtained docking poses we proposed a detailed location of three hydrophobic pockets in orthosteric binding site of FPR2. Our model emphasizes the importance of aromatic stacking, especially with regard to residues His102(3.29) and Phe257(6.51), for binding of FPR2 ligands. We also identified other residues important for non-peptide ligand binding in the binding site of FPR2. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Peptides derived from HIV-1, HIV-2, Ebola virus, SARS coronavirus and coronavirus 229E exhibit high affinity binding to the formyl peptide receptor

    PubMed Central

    Mills, John S.

    2007-01-01

    Peptides derived from the membrane proximal region of fusion proteins of human immunodeficiency viruses 1 and 2, Coronavirus 229 E, severe acute respiratory syndrome coronavirus and Ebola virus were all potent antagonists of the formyl peptide receptor expressed in Chinese hamster ovary cells. Binding of viral peptides was affected by the naturally occurring polymorphisms at residues 190 and 192, which are located at second extracellular loop-transmembrane helix 5 interface. Substitution of R190 with W190 enhanced the affinity for a severe acute respiratory syndrome coronavirus peptide 6 fold but reduced the affinity for N-formyl-Nle–Leu-Phe by 2.5 fold. A 12 mer peptide derived from coronavirus 229E (ETYIKPWWVWL) was the most potent antagonist of the formyl peptide receptor W190 with a Ki of 230 nM. Fluorescently labeled ETYIKPWWVWL was effectively internalized by all three variants with EC50 of ~25 nM. An HKU-1 coronavirus peptide, MYVKWPWYVWL, was a potent antagonist but N-formyl-MYVKWPWYVWL was a potent agonist. ETYIKPWWVWL did not stimulate GTPγS binding but inhibited the stimulation by formyl-NleLeuPhe. It also blocked β arrestin translocation and receptor downregulation induced by formyl-Nle–Leu–Phe. This indicates that formyl peptide receptor may be important in viral infections and that variations in its sequence among individuals may affect their likelihood of viral and bacterial infections. PMID:16842982

  17. Mitochondrial N-formyl peptides induce cardiovascular collapse and sepsis-like syndrome

    PubMed Central

    McCarthy, Cameron G.; Szasz, Theodora; Goulopoulou, Styliani; Webb, R. Clinton

    2015-01-01

    Fifty percent of trauma patients who present sepsis-like syndrome do not have bacterial infections. This condition is known as systemic inflammatory response syndrome (SIRS). A unifying factor of SIRS and sepsis is cardiovascular collapse. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns. Mitochondrial N-formyl peptides (F-MIT) are damage-associated molecular patterns that share similarities with bacterial N-formylated peptides and are potent immune system activators. The goal of this study was to investigate whether F-MIT trigger SIRS, including hypotension and vascular collapse via formyl peptide receptor (FPR) activation. We evaluated cardiovascular parameters in Wistar rats treated with FPR or histamine receptor antagonists and inhibitors of the nitric oxide pathway before and after F-MIT infusion. F-MIT, but not nonformylated peptides or mitochondrial DNA, induced severe hypotension via FPR activation and nitric oxide and histamine release. Moreover, F-MIT infusion induced hyperthermia, blood clotting, and increased vascular permeability. To evaluate the role of leukocytes in F-MIT-induced hypotension, neutrophil, basophil, or mast cells were depleted. Depletion of basophils, but not neutrophils or mast cells, abolished F-MIT-induced hypotension. Rats that underwent hemorrhagic shock increased plasma levels of mitochondrial formylated proteins associated with lung damage and antagonism of FPR ameliorated hemorrhagic shock-induced lung injury. Finally, F-MIT induced vasodilatation in isolated resistance arteries via FPR activation; however, F-MIT impaired endothelium-dependent relaxation in the presence of blood. These data suggest that F-MIT may be the link among trauma, SIRS, and cardiovascular collapse. PMID:25637548

  18. Mitochondrial N-formyl peptides induce cardiovascular collapse and sepsis-like syndrome.

    PubMed

    Wenceslau, Camilla Ferreira; McCarthy, Cameron G; Szasz, Theodora; Goulopoulou, Styliani; Webb, R Clinton

    2015-04-01

    Fifty percent of trauma patients who present sepsis-like syndrome do not have bacterial infections. This condition is known as systemic inflammatory response syndrome (SIRS). A unifying factor of SIRS and sepsis is cardiovascular collapse. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns. Mitochondrial N-formyl peptides (F-MIT) are damage-associated molecular patterns that share similarities with bacterial N-formylated peptides and are potent immune system activators. The goal of this study was to investigate whether F-MIT trigger SIRS, including hypotension and vascular collapse via formyl peptide receptor (FPR) activation. We evaluated cardiovascular parameters in Wistar rats treated with FPR or histamine receptor antagonists and inhibitors of the nitric oxide pathway before and after F-MIT infusion. F-MIT, but not nonformylated peptides or mitochondrial DNA, induced severe hypotension via FPR activation and nitric oxide and histamine release. Moreover, F-MIT infusion induced hyperthermia, blood clotting, and increased vascular permeability. To evaluate the role of leukocytes in F-MIT-induced hypotension, neutrophil, basophil, or mast cells were depleted. Depletion of basophils, but not neutrophils or mast cells, abolished F-MIT-induced hypotension. Rats that underwent hemorrhagic shock increased plasma levels of mitochondrial formylated proteins associated with lung damage and antagonism of FPR ameliorated hemorrhagic shock-induced lung injury. Finally, F-MIT induced vasodilatation in isolated resistance arteries via FPR activation; however, F-MIT impaired endothelium-dependent relaxation in the presence of blood. These data suggest that F-MIT may be the link among trauma, SIRS, and cardiovascular collapse. Copyright © 2015 the American Physiological Society.

  19. Formyl Peptide Receptor Activation Elicits Endothelial Cell Contraction and Vascular Leakage.

    PubMed

    Wenceslau, Camilla F; McCarthy, Cameron G; Webb, R Clinton

    2016-01-01

    The major pathophysiological characteristic of systemic inflammatory response syndrome (SIRS) and sepsis is the loss of control of vascular tone and endothelial barrier dysfunction. These changes are attributed to pro-inflammatory mediators. It has been proposed that in patients and rats without infection, cell components from damaged tissue are the primary instigators of vascular damage. Mitochondria share several characteristics with bacteria, and when fragments of mitochondria are released into the circulation after injury, they are recognized by the innate immune system. N-Formyl peptides are common molecular signatures of bacteria and mitochondria and are known to play a role in the initiation of inflammation by activating the formyl peptide receptor (FPR). We have demonstrated that infusion of mitochondrial N-formyl peptides (F-MIT) leads to sepsis-like symptoms, including vascular leakage. We have also observed that F-MIT, via FPR activation, elicits changes in cytoskeleton-regulating proteins in endothelial cells. Therefore, we hypothesize that these FPR-mediated changes in cytoskeleton can cause endothelial cell contraction and, consequently vascular leakage. Here, we propose that endothelial FPR is a key contributor to impaired barrier function in SIRS and sepsis patients following trauma.

  20. Formyl Peptide Receptor Activation Elicits Endothelial Cell Contraction and Vascular Leakage

    PubMed Central

    Wenceslau, Camilla F.; McCarthy, Cameron G.; Webb, R. Clinton

    2016-01-01

    The major pathophysiological characteristic of systemic inflammatory response syndrome (SIRS) and sepsis is the loss of control of vascular tone and endothelial barrier dysfunction. These changes are attributed to pro-inflammatory mediators. It has been proposed that in patients and rats without infection, cell components from damaged tissue are the primary instigators of vascular damage. Mitochondria share several characteristics with bacteria, and when fragments of mitochondria are released into the circulation after injury, they are recognized by the innate immune system. N-Formyl peptides are common molecular signatures of bacteria and mitochondria and are known to play a role in the initiation of inflammation by activating the formyl peptide receptor (FPR). We have demonstrated that infusion of mitochondrial N-formyl peptides (F-MIT) leads to sepsis-like symptoms, including vascular leakage. We have also observed that F-MIT, via FPR activation, elicits changes in cytoskeleton-regulating proteins in endothelial cells. Therefore, we hypothesize that these FPR-mediated changes in cytoskeleton can cause endothelial cell contraction and, consequently vascular leakage. Here, we propose that endothelial FPR is a key contributor to impaired barrier function in SIRS and sepsis patients following trauma. PMID:27532003

  1. Inhibition by FK506 of formyl peptide-induced neutrophil activation and associated protein synthesis.

    PubMed

    Burnett, D; Adams, D H; Martin, T J; Liu, Q; Grant, R A; Stockley, R A; Lord, J M

    1994-09-15

    The macrolide FK506 inhibited, by up to 50%, neutrophil migration and the production of the superoxide radical in response to the formyl peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). The production of the superoxide radical in response to phorbol 12-myristate 13-acetate (PMA) was unaffected by FK506. The inhibition of neutrophil functions was accompanied by a partial reversal of FMLP-induced synthesis of cellular proteins, despite a rise in intracellular Ca2+. Neutrophils treated with FK506 demonstrated a small (average 23%) though significant decrease in formyl-peptide receptor numbers but receptor binding affinity was unaffected. The effects of FK506 on neutrophil activation appear to be analogous to those in T-lymphocytes. The incomplete inhibition, by FK506, of neutrophil responses suggests further that activation by FMLP is mediated via distinct multiple signalling pathways, including protein kinase activation and protein synthesis. The inability of FK506 to reduce FMLP-induced rises in cellular Ca2+ or PMA-induced activation of neutrophils suggests that its action is distal to Ca2+ mobilization and distinct from pathways relying on PKC activation. Thus the immunosuppressive effects of FK506 in vivo might be mediated through the inhibition of inflammatory cells other than lymphocytes and the drug therefore has therapeutic potential in a variety of inflammatory conditions. The drug also has potential in vitro for the characterization of signalling pathways from the plasma membrane to the nucleus.

  2. Structural and functional characterization of the human formyl peptide receptor ligand-binding region.

    PubMed Central

    Radel, S J; Genco, R J; De Nardin, E

    1994-01-01

    The formyl peptide (N-formyl-1-methionyl-1-leucyl-1-phenylalanine [FMLP]) receptor is involved in the activation of neutrophils and their subsequent response to chemotactic N-formylated peptides. Recently, we found that the first extracellular loop closest to the N-terminal end of the FMLP receptor exhibited the strongest ligand binding compared with that shown by other extracellular regions. By constructing amino acid substitutional variants of this domain, we have determined that residues Arg-84 and Lys-85 on this loop play major roles in ligand-binding activity. Furthermore, random rearrangement of the residues of this receptor region demonstrated that the position of these charged amino acids did not affect their involvement in ligand binding, although their presence was essential for this binding to occur. We propose that the portion of the first N-terminal extracellular loop of the FMLP receptor containing residues Arg-84 and Lys-85 contributes significantly to the active site in ligand-receptor binding. We further propose that this binding is not dependent on defined structure but rather that these charged moieties may function as important "contacts" in receptor-ligand interactions. Images PMID:8168934

  3. Chemotactic properties and absence of the formyl peptide receptor in ferret (Mustela putorius furo) neutrophils.

    PubMed

    Nakata, Makoto; Otsubo, Kouji; Kikuchi, Tomoko; Itou, Takuya; Sakai, Takeo

    2010-02-01

    This study describes a chemotaxis assay of ferret polymorphonuclear cells (PMNs). The optimal conditions for this chemotaxis assay were investigated for three chemoattractants: zymosan activated serum (ZAS), recombinant human interleukin-8 (rhIL-8) and N-formyl-Met-Leu- Phe (fMLF). In this study, ferret polymorphonuclear cells (PMNs) reacted to ZAS and rhIL-8, but not fMLF. The optimal concentration of ZAS and rhIL-8 were 5% and 100 ng/ml, respectively. The optimal incubation time of each reagent was 60 min. Due to the lack of response shown from fMLF, the existence of formyl peptide receptors (FPR) on ferret PMNs was investigated by evaluating FPR binding using flow cytometry. The receptor was not detected, implying that ferret neutrophils may lack FPR. This study confirms the fundamental experimental conditions for ferret PMNs chemotaxis and elucidates new findings concerning FPR in ferret neutrophils.

  4. Formyl peptide-induced chemotaxis of human polymorphonuclear leukocytes does not require either marked changes in cytosolic calcium or specific granule discharge. Role of formyl peptide receptor reexpression (or recycling).

    PubMed Central

    Perez, H D; Elfman, F; Marder, S; Lobo, E; Ives, H E

    1989-01-01

    We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane. PMID:2723068

  5. International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the Formyl Peptide Receptor (FPR) Family

    PubMed Central

    YE, RICHARD D.; BOULAY, FRANÇOIS; WANG, JI MING; DAHLGREN, CLAES; GERARD, CRAIG; PARMENTIER, MARC; SERHAN, CHARLES N.; MURPHY, PHILIP M.

    2009-01-01

    Formyl peptide receptors (FPRs) are a small group of seven-transmembrane domain, G protein-coupled receptors that are expressed mainly by mammalian phagocytic leukocytes and are known to be important in host defense and inflammation. The three human FPRs (FPR1, FPR2/ALX, and FPR3) share significant sequence homology and are encoded by clustered genes. Collectively, these receptors bind an extraordinarily numerous and structurally diverse group of agonistic ligands, including N-formyl and nonformyl peptides of different composition, that chemoattract and activate phagocytes. N-formyl peptides, which are encoded in nature only by bacterial and mitochondrial genes and result from obligatory initiation of bacterial and mitochondrial protein synthesis with N-formylmethionine, is the only ligand class common to all three human receptors. Surprisingly, the endogenous anti-inflammatory peptide annexin 1 and its N-terminal fragments also bind human FPR1 and FPR2/ALX, and the anti-inflammatory eicosanoid lipoxin A4 is an agonist at FPR2/ALX. In comparison, fewer agonists have been identified for FPR3, the third member in this receptor family. Structural and functional studies of the FPRs have produced important information for understanding the general pharmacological principles governing all leukocyte chemoattractant receptors. This article aims to provide an overview of the discovery and pharmacological characterization of FPRs, to introduce an International Union of Basic and Clinical Pharmacology (IUPHAR)-recommended nomenclature, and to discuss unmet challenges, including the mechanisms used by these receptors to bind diverse ligands and mediate different biological functions. PMID:19498085

  6. The sensing of bacteria: emerging principles for the detection of signal sequences by formyl peptide receptors.

    PubMed

    Bufe, Bernd; Zufall, Frank

    2016-06-01

    The ability to detect specific chemical signatures released by bacteria and other microorganisms is a fundamental feature of immune defense against pathogens. There is increasing evidence that chemodetection of such microorganism-associated molecular patterns (MAMPs) occurs at many places in the body including specific sets of chemosensory neurons in the mammalian nose. Formyl peptide receptors (FPRs) are a unique family of G protein-coupled receptors (GPCRs) that can detect the presence of bacteria and function as chemotactic receptors. Here, we highlight the recent discovery of a vast family of natural FPR agonists, the bacterial signal peptides (or signal sequences), thus providing new insight into the molecular mechanisms of bacterial sensing by human and mouse FPRs. Signal peptides in bacteria are formylated, N-terminal protein signatures required for directing the transfer of proteins through the plasma membrane. After their cleavage and release, signal peptides are available for FPR detection and thus provide a previously unrecognized MAMP. With over 170 000 predicted sequences, bacterial signal peptides represent one of the largest families of GPCR ligands and one of the most complex classes of natural activators of the innate immune system. By recognizing a conserved three-dimensional peptide motif, FPRs employ an unusual detection mechanism that combines structural promiscuity with high specificity and sensitivity, thus solving the problem of detecting thousands of distinct sequences yet maintaining selectivity. How signal peptides are released by bacteria and sensed by GPCRs and how these processes shape the responses of other cells and whole organisms represents an important topic for future research.

  7. Monocytes and neutrophils from tuberculosis patients are insensitive to anti-inflammatory effects triggered by the prototypic formyl peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP)

    PubMed Central

    BEIGIER-BOMPADRE, M; ALEMÁN, M; BARRIONUEVO, P; FRANCO, M C; RUBEL, C J; SASIAIN, M Del C; PALERMO, M S; ABBATE, E; ISTURIZ, M A

    2003-01-01

    Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis where formyl peptides, which are cleavage products of bacterial and mitochondrial proteins, are present. In this study, we demonstrated that interferon gamma (IFN)-γ and interleukin (IL)-10 induced the overexpression of the receptor for the Fc portion of IgG I (FcγRI) in monocytes from tuberculosis (TB) patients, showing that these cells respond to IFN-γ and IL-10 signals. We also demonstrated that lower doses of IL-10 render monocytes from TB patients less responsive to higher doses of the cytokine. Although the prototypic formyl peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is a well-known proinflammatory agonist, we have demonstrated previously that preincubation of monocytes with FMLP inhibited the up-regulation of FcγRI induced by IFN-γ or IL-10. This effect was not observed in monocytes from TB patientes. FMLP also induced the down-regulation of the expression of FcγRI in monocytes that had been activated already with IFN-γ. However, this effect of FMLP was not observed in monocytes from TB patients and supernatants from monocytes obtained from these patients were incapable of inducing the down-regulation of FcγRI. In contrast to normal donors, supernatants from FMLP-treated neutrophils from TB patients did not modify the basal level of expression of FcγRI in monocytes from normal donors. In conclusion, in this study we demonstrated the existence of two novel mechanisms that may contribute to the pathological effects generated by M. tuberculosis: the enhancement of FcγRI in response to IFN-γ and IL-10, and the unresponsiveness to the anti-inflammatory effects induced by formyl peptides. PMID:12869034

  8. Combining Elements from Two Antagonists of Formyl Peptide Receptor 2 Generates More Potent Peptidomimetic Antagonists.

    PubMed

    Skovbakke, Sarah Line; Holdfeldt, André; Nielsen, Christina; Hansen, Anna Mette; Perez-Gassol, Iris; Dahlgren, Claes; Forsman, Huamei; Franzyk, Henrik

    2017-08-24

    Structural optimization of a peptidomimetic antagonist of formyl peptide receptor 2 (FPR2) was explored by an approach involving combination of elements from the two most potent FPR2 antagonists described: a Rhodamine B-conjugated 10-residue gelsonin-derived peptide (i.e., PBP10, RhB-QRLFQVKGRR-OH) and the palmitoylated α-peptide/β-peptoid hybrid Pam-(Lys-βNspe)6-NH2. This generated an array of hybrid compounds from which a new subclass of receptor-selective antagonists was identified. The most potent representatives displayed activity in the low nanomolar range. The resulting stable and potent FPR2-selective antagonists (i.e., RhB-(Lys-βNphe)n-NH2; n = 4-6) are expected to become valuable tools in further elucidation of the physiological role of FPR2 in health and disease.

  9. Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1.

    PubMed

    Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik

    2015-08-01

    In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses.

  10. Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides

    PubMed Central

    John, C. D.; Sahni, V.; Mehet, D.; Morris, J. F.; Christian, H. C.; Perretti, M.; Flower, R. J.; Solito, E.; Buckingham, J. C.

    2007-01-01

    The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA11-188 and ANXA1Ac2-26) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1Ac2-26. However, lipoxin A4 (LXA4, 0.02-2μM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 μM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 μM) overcame the effects of dexamethasone, ANXA11-188, ANXA1Ac2-26, fMLF, and LXA4 on ACTH release, although at a lower concentration (50 μM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They

  11. Formyl Peptide Receptor Polymorphisms: 27 Most Possible Ways for Phagocyte Dysfunction.

    PubMed

    Skvortsov, S S; Gabdoulkhakova, A G

    2017-04-01

    Formyl peptide receptors (FPRs) expressed by mammalian myeloid cells are the important part of innate immunity. They belong to the seven-transmembrane domain class of receptors coupled to heterotrimeric GTP-binding proteins. Binding of the receptor with a wide spectrum of exogenous and endogenous ligands triggers such defensive phagocyte reactions as chemotaxis, secretory degranulation, and respiratory burst, keeping a balance of inflammatory and antiinflammatory processes in the organism. The association between single nucleotide polymorphisms in the gene of FPR1 receptor resulting in disruption of the receptor structure and the development of certain pathologies accompanied with inflammation, such as aggressive periodontitis, macular degeneration, and even gastric cancer (Maney, P., and Walters, J. D. (2009) J. Periodontol., 80, 1498-1505; Liang, X. Y., et al. (2014) Eye, 28, 1502-1510; Otani, T., et al. (2011) Biochem. Biophys. Res. Commun., 405, 356-361) has been shown. In this review, we matched the missense mutation of formyl-peptide receptors with their known functional domains and classified them according to their potential significance in pathology.

  12. T4-lysozyme fusion for the production of human formyl peptide receptors for structural determination.

    PubMed

    Wang, Xiaoqiang; Cui, Ying; Wang, Jiqian

    2014-03-01

    T4-lysozyme (T4L) fusion was introduced in the intracellular loop of a G protein-coupled receptor (GPCR) of human formyl peptide receptor 3 (FPR3), and the ability of T4L fusion to be used in the production of human FPR3 for structural determination was evaluated in this work. The T4L variant of human FPR3 termed FPR3-T4L was expressed in stable tetracycline-inducible HEK293 cells. A systematic detergent screening showed that fos-choline-14 was the optimal detergent to solubilize and subsequently purify FPR3-T4L from HEK293 cells. Immunoaffinity purification in combination with gel filtration was employed to purify the T4L-fused receptor to high homogeneity. The final yield of the human FPR3-T4L monomer from 2 g of cells was 0.2 mg. Circular dichroism spectroscopy indicated that the receptor adopted a correct secondary structure after purification, while ligand binding measurement indicated that the receptor was functional. Thus, the presence of T4L fusion did not evidently disturb the expression in HEK293 cells, proper folding, and functionality of human FPR3. Our study of evaluating T4L fusion for the recombinant production of human formyl peptide receptor would facilitate ongoing efforts in the structural characterization of GPCRs.

  13. Formyl peptide receptor as a novel therapeutic target for anxiety-related disorders.

    PubMed

    Gallo, Irene; Rattazzi, Lorenza; Piras, Giuseppa; Gobbetti, Thomas; Panza, Elisabetta; Perretti, Mauro; Dalley, Jeffrey W; D'Acquisto, Fulvio

    2014-01-01

    Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe-associated molecules and inform various cellular and sensorial mechanisms to the presence of pathogens in the host. Here we demonstrate that Fpr2/3-deficient mice show a distinct profile of behaviour characterised by reduced anxiety in the marble burying and light-dark box paradigms, increased exploratory behaviour in an open-field, together with superior performance on a novel object recognition test. Pharmacological blockade with a formyl peptide receptor antagonist, Boc2, in wild type mice reproduced most of the behavioural changes observed in the Fpr2/3(-/-) mice, including a significant improvement in novel object discrimination and reduced anxiety in a light/dark shuttle test. These effects were associated with reduced FPR signalling in the gut as shown by the significant reduction in the levels of p-p38. Collectively, these findings suggest that homeostatic FPR signalling exerts a modulatory effect on anxiety-like behaviours. These findings thus suggest that therapies targeting FPRs may be a novel approach to ameliorate behavioural abnormalities present in neuropsychiatric disorders at the cognitive-emotional interface.

  14. Multimodal formyl peptide receptor 1 targeted inflammation imaging probe: cFLFLF-MHI-DOTA.

    PubMed

    Li, Jie; Zhang, Yi; Chordia, Mahendra D; Wu, Hua; Shao, Li; Pan, Dongfeng

    2016-02-01

    Formyl peptide receptor 1 (FPR1) targeting multimodal probe cFLFLFK-MHI-DOTA for leukocyte based inflammation imaging is described. The compound consists of three domains, (a) cFLFLF peptide for FPR1 recognition and binding for activated leukocyte, (b) heptamethine cyanine dye (MHI) for near infrared fluorescence (NIRF) detection and imaging, and (c) metal chelator DOTA ligand that could form complex with a radiometal for nuclear (PET/SPECT) imaging or with a paramagnetic metal for MRI imaging. Detailed synthesis, characterization and in vitro evaluation are reported. The availability of dual mode inflammation imaging probe would allow in vivo gross level imaging of inflammation foci as well as ex vivo microscopic level cellular imaging for role played by innate immune cells in inflamed tissue. Copyright © 2016. Published by Elsevier Ltd.

  15. Design and synthesis of tryptophan containing dipeptide derivatives as formyl peptide receptor 1 antagonist.

    PubMed

    Hwang, Tsong-Long; Hung, Chih-Hao; Hsu, Ching-Yun; Huang, Yin-Ting; Tsai, Yu-Chi; Hsieh, Pei-Wen

    2013-06-14

    Our previous studies identified an Fmoc-(S,R)-tryptophan-containing dipeptide derivative, 1, which selectively inhibited neutrophil elastase release induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in human neutrophils. In an attempt to improve pharmacological activity, a series of tryptophan-containing dipeptides were synthesized and their pharmacological activities were investigated in human neutrophils. Of these, five compounds 3, 6, 19a, 24a, and 24b exhibited potent and dual inhibitory effects on FMLP-induced superoxide anion (O2˙(-)) generation and neutrophil elastase release in neutrophils with IC50 values of 0.23/0.60, 1.88/2.47, 1.87/3.60, 0.12/0.37, and 1.32/1.03 μM, respectively. Further studies indicated that inhibition of superoxide production in human neutrophils by these dipeptides was associated with the selective inhibition of formyl peptide receptor 1 (FPR1). Furthermore, the results of structure-activity relationship studies concluded that the fragment N-benzoyl-Trp-Phe-OMe (3) was most suitable as a core structure for interaction with FPR1, and may be approved as a lead for the development of new drugs in the treatment of neutrophilic inflammatory diseases. As some of the synthesized compounds exhibited separable conformational isomers, and showed diverse bioactivities, the conformation analysis of these compounds is also discussed herein.

  16. Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils

    PubMed Central

    1989-01-01

    Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F- actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G- actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F- actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N- formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand- receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively. PMID:2768337

  17. Formyl Peptide Receptors from Immune and Vomeronasal System Exhibit Distinct Agonist Properties*

    PubMed Central

    Bufe, Bernd; Schumann, Timo; Zufall, Frank

    2012-01-01

    The formyl peptide receptor (Fpr) family is well known for its contribution to immune defense against pathogens in human and rodent leukocytes. Recently, several structurally related members of these receptors were discovered in sensory neurons of the mouse vomeronasal organ (VNO), key detectors of pheromones and related semiochemicals. Although the biological role of vomeronasal Fprs is not yet clear, the known contribution of other Fprs to host immune defense suggested that they could contribute to vomeronasal pathogen sensing. Precise knowledge about the agonist properties of mouse Fprs is required to determine their function. We expressed all seven mouse and three human Fprs using an in vitro system and tested their activation with 32 selected compounds by conducting high throughput calcium measurements. We found an intriguing functional conservation between human and mouse immune Fprs that is most likely a consequence of closely similar biological constraints. By contrast, our data suggest a neofunctionalization of the vomeronasal Fprs. We show that the vomeronasal receptor mFpr-rs1 can be activated robustly by W-peptide and structural derivatives but not by other typical ligands of immune Fprs. mFpr-rs1 exhibits a stereo-selective preference for peptides containing d-amino acids. The same peptide motifs are contained in pathogenic microorganisms. Thus, the ligand profile of mFpr-rs1 is consistent with a role in vomeronasal pathogen sensing. PMID:22859307

  18. Distinct Signaling Cascades Elicited by Different Formyl Peptide Receptor 2 (FPR2) Agonists

    PubMed Central

    Cattaneo, Fabio; Parisi, Melania; Ammendola, Rosario

    2013-01-01

    The formyl peptide receptor 2 (FPR2) is a remarkably versatile transmembrane protein belonging to the G-protein coupled receptor (GPCR) family. FPR2 is activated by an array of ligands, which include structurally unrelated lipids and peptide/proteins agonists, resulting in different intracellular responses in a ligand-specific fashion. In addition to the anti-inflammatory lipid, lipoxin A4, several other endogenous agonists also bind FPR2, including serum amyloid A, glucocorticoid-induced annexin 1, urokinase and its receptor, suggesting that the activation of FPR2 may result in potent pro- or anti-inflammatory responses. Other endogenous ligands, also present in biological samples, include resolvins, amyloidogenic proteins, such as beta amyloid (Aβ)-42 and prion protein (Prp)106–126, the neuroprotective peptide, humanin, antibacterial peptides, annexin 1-derived peptides, chemokine variants, the neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP)-27, and mitochondrial peptides. Upon activation, intracellular domains of FPR2 mediate signaling to G-proteins, which trigger several agonist-dependent signal transduction pathways, including activation of phospholipase C (PLC), protein kinase C (PKC) isoforms, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, the mitogen-activated protein kinase (MAPK) pathway, p38MAPK, as well as the phosphorylation of cytosolic tyrosine kinases, tyrosine kinase receptor transactivation, phosphorylation and nuclear translocation of regulatory transcriptional factors, release of calcium and production of oxidants. FPR2 is an attractive therapeutic target, because of its involvement in a range of normal physiological processes and pathological diseases. Here, we review and discuss the most significant findings on the intracellular pathways and on the cross-communication between FPR2 and tyrosine kinase receptors triggered by different FPR2 agonists. PMID

  19. Antagonism of Human Formyl Peptide Receptor 1 with Natural Compounds and their Synthetic Derivatives

    PubMed Central

    Schepetkin, Igor A.; Khlebnikov, Andrei I.; Kirpotina, Liliya N.; Quinn, Mark T.

    2015-01-01

    Formyl peptide receptor 1 (FPR1) regulates a wide variety of neutrophil functional responses and plays an important role in inflammation and the pathogenesis of various diseases. To date, a variety of natural and synthetic molecules have been identified as FPR1 ligands. Here, we review current knowledge on natural products and natural product-inspired small-molecules reported to antagonize and/or inhibit the FPR1-mediated responses. Based on this literature, additional screening of selected commercially available natural compounds for their ability to inhibit fMLF-induced Ca2+ mobilization in human neutrophils and FPR1 transfected HL-60 cells, and pharmacophore modeling, natural products with potential as FPR1 antagonists are considered and discussed in this review. The identification and characterization of natural products that antagonize FPR1 activity may have potential for the development of novel therapeutics to limit or alter the outcome of inflammatory processes. PMID:26382576

  20. Antagonism of human formyl peptide receptor 1 with natural compounds and their synthetic derivatives.

    PubMed

    Schepetkin, Igor A; Khlebnikov, Andrei I; Kirpotina, Liliya N; Quinn, Mark T

    2016-08-01

    Formyl peptide receptor 1 (FPR1) regulates a wide variety of neutrophil functional responses and plays an important role in inflammation and the pathogenesis of various diseases. To date, a variety of natural and synthetic molecules have been identified as FPR1 ligands. Here, we review current knowledge on natural products and natural product-inspired small molecules reported to antagonize and/or inhibit the FPR1-mediated responses. Based on this literature, additional screening of selected commercially available natural compounds for their ability to inhibit fMLF-induced Ca(2+) mobilization in human neutrophils and FPR1 transfected HL-60 cells, and pharmacophore modeling, natural products with potential as FPR1 antagonists are considered and discussed in this review. The identification and characterization of natural products that antagonize FPR1 activity may have potential for the development of novel therapeutics to limit or alter the outcome of inflammatory processes. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis.

    PubMed

    Snapkov, Igor; Öqvist, Carl Otto; Figenschau, Yngve; Kogner, Per; Johnsen, John Inge; Sveinbjørnsson, Baldur

    2016-07-18

    Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor mainly expressed by the cells of myeloid origin, where it mediates the innate immune response to bacterial formylated peptides. High expression of FPR1 has been detected in various cancers but the function of FPR1 in tumorigenesis is poorly understood. Expression of FPR1 in neuroblastoma cell lines and primary tumors was studied using RT-PCR, western blotting, immunofluorescence and immunohistochemistry. Calcium mobilization assays and western blots with phospho-specific antibodies were used to assess the functional activity of FPR1 in neuroblastoma. The tumorigenic capacity of FPR1 was assessed by xenografting of neuroblastoma cells expressing inducible FPR1 shRNA, FPR1 cDNA or control shRNA in nude mice. FPR1 is expressed in neuroblastoma primary tumors and cell lines. High expression of FPR1 corresponds with high-risk disease and poor patient survival. Stimulation of FPR1 in neuroblastoma cells using fMLP, a selective FPR1 agonist, induced intracellular calcium mobilization and activation of MAPK/Erk, PI3K/Akt and P38-MAPK signal transduction pathways that were inhibited by using Cyclosporin H, a selective receptor antagonist for FPR1. shRNA knock-down of FPR1 in neuroblastoma cells conferred a delayed xenograft tumor development in nude mice, whereas an ectopic overexpression of FPR1 promoted augmented tumorigenesis in nude mice. Our data demonstrate that FPR1 is involved in neuroblastoma development and could represent a therapy option for the treatment of neuroblastoma.

  2. Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides.

    PubMed

    John, C D; Sahni, V; Mehet, D; Morris, J F; Christian, H C; Perretti, M; Flower, R J; Solito, E; Buckingham, J C

    2007-04-01

    The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely

  3. Enterococcus faecium stimulates human neutrophils via the formyl-peptide receptor 2.

    PubMed

    Bloes, Dominik Alexander; Otto, Michael; Peschel, Andreas; Kretschmer, Dorothee

    2012-01-01

    The human formyl-peptide receptor 2 (FPR2/ALX) senses phenol-soluble modulin (PSM) peptide toxins produced by pathogenic staphylococcal species and plays a crucial role in directing neutrophil influx during staphylococcal infection. However, it has remained unclear if FPR2 responds also to molecules from other bacterial pathogens. Here we analyzed a variety of gram-positive and gram-negative pathogens and found that apart from staphylococci only certain enterococcal strains have the capacity to stimulate FPR2/ALX. Most of the analyzed Enterococcus faecium but only sporadic Enterococcus faecalis strains released FPR2/ALX-stimulating molecules leading to neutrophil calcium ion fluxes, chemotaxis, and complement receptor upregulation. Among ten test strains vancomycin-resistant E. faecium had a significantly higher capacity to stimulate FPR2/ALX than vancomycin-susceptible strains, suggesting an association of strong FPR2/ALX activation with health-care associated strains. The enterococcal FPR2/ALX agonists were found to be peptides or proteins, which appear, however, to be unrelated to staphylococcal PSMs in sequence and physicochemical properties. Enterococci are among the most frequent invasive bacterial pathogens but the basis of enterococcal virulence and immune activation has remained incompletely understood. Our study indicates that previously unrecognized proteinaceous agonists contribute to Enterococcus-host interaction and underscores the importance of FPR2/ALX in host defense against major endogenous bacterial pathogens.

  4. Targeting formyl peptide receptor 1 of activated macrophages to monitor inflammation of experimental osteoarthritis in rat.

    PubMed

    Yang, Xinlin; Chordia, Mahendra D; Du, Xuejun; Graves, John L; Zhang, Yi; Park, Yong-Sang; Guo, Yongfei; Pan, Dongfeng; Cui, Quanjun

    2016-09-01

    Macrophages play a crucial role in the pathogenesis of osteoarthritis (OA). In this study, the feasibility of a formyl peptide receptor 1 (Fpr1)-targeting peptide probe cFLFLF-PEG-(64) Cu via positron emission tomography (PET) imaging was investigated for detection of macrophage activity during development of OA. Monoiodoacetate (MIA) was intraarticularly injected into the knee joint of Sprague-Dawley rats to induce OA. Five days later, cFLFLF-PEG-(64) Cu (∼7,400 kBq/rat) was injected into the tail vein and microPET/CT imaging was performed to assess the OA inflammation by detecting infiltration of macrophages by Fpr1 expression. In addition, a murine macrophage cell line RAW264.7 and two fluorescent probes cFLFLF-PEG-cyanine 7 (cFLFLF-PEG-Cy7) and cFLFLF-PEG-cyanine 5 (cFLFLF-PEG-Cy5) were used to define the binding specificity of the peptide to macrophages. It was found with the MIA model that the maximal standard uptake values (SUVmax ) for right (MIA treated) and left (control) knees were 17.96 ± 5.45 and 3.00 ± 1.40, respectively. Histological evaluation of cryomicrotome sections showed that Fpr1 expression, cFLFLF-PEG-Cy5 binding, and tartrate-resistant acid phosphatase activity were elevated in the injured synovial membranes. The in vitro experiments demonstrated that both fluorescent peptide probes could bind specifically to RAW264.7 cells, which was blocked by cFLFLF but not by the scramble peptide. The findings highlighted the use of cFLFLF-PEG-(64) Cu/PET as an effective method potentially applied for detection and treatment evaluation of OA. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1529-1538, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  5. Pachymodulin, a new functional formyl peptide receptor 2 peptidic ligand isolated from frog skin has Janus-like immunomodulatory capacities.

    PubMed

    Lacombe, Claire; Piesse, Christophe; Sagan, Sandrine; Combadière, Christophe; Rosenstein, Yvonne; Auvynet, Constance

    2015-02-12

    Recruitment of leukocytes is essential to fight infections or to heal injuries; however, excessive and/or prolonged responses favor the development of major inflammatory pathologies, such as cardiovascular or neurodegenerative diseases. Thus, it is of great interest to seek novel compounds that can regulate leukocyte recruitment depending on the degree of inflammation. We have isolated and characterized, by different chromatographic techniques, mass spectrometry, and Edman sequencing, a new hexapeptide (SSLSKL) from the Mexican frog Pachymedusa dacnicolor, which we named pachymodulin. In vitro, pachymodulin promotes the migration of leukocytes through the binding and activation of the human and mouse N-formyl peptide receptor 2 (huFPR2). In vivo, it exhibits opposite biological activities: under homeostatic conditions, pachymodulin induces the recruitment of leukocytes, whereas under inflammatory conditions, it inhibits this process. Therefore, pachymodulin represents an interesting template in the quest to design new immunomodulatory drugs in the therapy of immune-related diseases.

  6. Heterologously expressed formyl peptide receptor 2 (FPR2/ALX) does not respond to lipoxin A₄.

    PubMed

    Hanson, Julien; Ferreirós, Nerea; Pirotte, Bernard; Geisslinger, Gerd; Offermanns, Stefan

    2013-06-15

    Lipoxin A₄ (LXA₄) has been described as an anti-inflammatory mediator, which exerts its effects through the formyl peptide receptor FPR2, also known as ALX. However, there has been a controversy whether or not cells expressing FPR2/ALX, such as neutrophils, respond to LXA₄. We, therefore, systematically examined the ability of the human and murine forms of the receptor to respond to LXA₄. We show that both receptor orthologues responded to the FPR2/ALX peptide agonist WKYMVM when expressed heterologously. In contrast, LXA₄ from different sources neither increased [Ca²⁺](i) and extracellular-signal-regulated kinase (ERK) phosphorylation, nor did it induce a decrease in cAMP levels or a translocation of β-arrestin. Also, several LXA₄ analogs were found to be unable to signal through FPR2/ALX. We conclude that FPR2/ALX is not activated by LXA₄ and that the molecular mechanism by which LXA₄ functions still needs to be identified. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Computational Structure-activity Relationship Analysis of Small-Molecule Agonists for Human Formyl Peptide Receptors

    PubMed Central

    Khlebnikov, Andrei I.; Schepetkin, Igor A; Quinn, Mark T.

    2010-01-01

    N-formyl peptide receptors (FPR) are important in host defense. Because of the potential for FPRs as therapeutic targets, recent efforts have focused on identification of non-peptide agonists for two FPR subtypes, FPR1 and FPR2. Given that a number of specific small molecule agonists have recently been identified, we hypothesized that computational structure-activity relationship (SAR) analysis of these molecules could provide new information regarding molecular features required for activity. We used a training set of 71 compounds, including 10 FPR1-specific agonists, 36 FPR2-specific agonists, and 25 non-active analogs. A sequence of (1) one-way analysis of variance selection, (2) cluster analysis, (3) linear discriminant analysis, and (4) classification tree analysis led to the derivation of SAR rules with high (95.8%) accuracy for correct classification of compounds. These SAR rules revealed key features distinguishing FPR1 versus FPR2 agonists. To verify predictive ability, we evaluated a test set of 17 additional FPR agonists, and found that the majority of these agonists (>94%) were classified correctly as agonists. This study represents the first successful application of classification tree methodology based on atom pairs to SAR analysis of FPR agonists. Importantly, these SAR rules represent a relatively simple classification approach for virtual screening of FPR1/FPR2 agonists. PMID:20870313

  8. Cathepsin G-regulated release of formyl peptide receptor agonists modulate neutrophil effector functions.

    PubMed

    Woloszynek, Josh C; Hu, Ying; Pham, Christine T N

    2012-10-05

    Neutrophil serine proteases play an important role in inflammation by modulating neutrophil effector functions. We have previously shown that neutrophils deficient in the serine proteases cathepsin G and neutrophil elastase (CG/NE neutrophils) exhibit severe defects in chemokine CXCL2 release and reactive oxygen species (ROS) production when activated on immobilized immune complex. Exogenously added active CG rescues these defects, but the mechanism remains undefined. Using a protease-based proteomic approach, we found that, in vitro, the addition of exogenous CG to immune complex-stimulated CG/NE neutrophils led to a decrease in the level of cell-associated annexin A1 (AnxA1) and cathelin-related antimicrobial peptide (CRAMP), both known inflammatory mediators. We further confirmed that, in vivo, CG was required for the extracellular release of AnxA1 and CRAMP in a subcutaneous air pouch model. In vitro, CG efficiently cleaved AnxA1, releasing the active N-terminal peptide Ac2-26, and processed CRAMP in limited fashion. Ac2-26 and CRAMP peptides enhanced the release of CXCL2 by CG/NE neutrophils in a dose-dependent manner via formyl peptide receptor (FPR) stimulation. Blockade of FPRs by an antagonist, Boc2 (t-Boc-Phe-d-Leu-Phe-d-Leu-Phe), abrogates CXCL2 release, whereas addition of FPR agonists, fMLF and F2L, relieves Boc2 inhibition. Furthermore, the addition of active CG, but not inactive CG, also relieves Boc2 inhibition. These findings suggest that CG modulates neutrophil effector functions partly by controlling the release (and proteolysis) of FPR agonists. Unexpectedly, we found that mature CRAMP, but not Ac2-26, induced ROS production through an FPR-independent pathway.

  9. The Virulence Regulator Agr Controls the Staphylococcal Capacity to Activate Human Neutrophils via the Formyl Peptide Receptor 2

    PubMed Central

    Kretschmer, Dorothee; Nikola, Nele; Dürr, Manuela; Otto, Michael; Peschel, Andreas

    2012-01-01

    The Agr quorum-sensing system represents the master regulator for staphylococcal virulence factors and is known to have a strong impact on the release of pathogen-associated molecular pattern (PAMP) molecules. Among the various staphylococcal PAMPs, phenol-soluble modulin (PSM) peptides have attracted increasing interest because they are crucial for staphylococcal virulence and have neutrophil-recruiting properties. The latter depend on recognition of PSMs by the neutrophil formyl peptide receptor 2 (FPR2/ALX), for which PSMs are highly efficient agonists. We demonstrate that Agr inactivation in Staphylococcus aureus or S. epidermidis leads to strongly reduced neutrophil responses, which is in agreement with the previously reported strict control of PSM expression by Agr. Agr had a distinct and profound impact on activation of FPR2/ALX but not of the related FPR1 receptor that senses bacterial formylated peptides. S. epidermidis PSMs had similar FPR2/ALX-activating properties but differed in their dependence on N-terminal formylation compared to S. aureus PSMs. Moreover, S. aureus and S. epidermidis PSMs upregulated the neutrophil complement receptor CD11b via FPR2/ALX stimulation in an Agr-dependent fashion. Hence, Agr controls the capacity of staphylococcal pathogens to activate FPR2/ALX-dependent neutrophil responses, underscoring the crucial role of FPR2/ALX and PSMs in staphylococcus-host interaction. PMID:22067547

  10. The virulence regulator Agr controls the staphylococcal capacity to activate human neutrophils via the formyl peptide receptor 2.

    PubMed

    Kretschmer, Dorothee; Nikola, Nele; Dürr, Manuela; Otto, Michael; Peschel, Andreas

    2012-01-01

    The Agr quorum-sensing system represents the master regulator for staphylococcal virulence factors and is known to have a strong impact on the release of pathogen-associated molecular pattern (PAMP) molecules. Among the various staphylococcal PAMPs, phenol-soluble modulin (PSM) peptides have attracted increasing interest because they are crucial for staphylococcal virulence and have neutrophil-recruiting properties. The latter depend on recognition of PSMs by the neutrophil formyl peptide receptor 2 (FPR2/ALX), for which PSMs are highly efficient agonists. We demonstrate that Agr inactivation in Staphylococcus aureus or S. epidermidis leads to strongly reduced neutrophil responses, which is in agreement with the previously reported strict control of PSM expression by Agr. Agr had a distinct and profound impact on activation of FPR2/ALX but not of the related FPR1 receptor that senses bacterial formylated peptides. S. epidermidis PSMs had similar FPR2/ALX-activating properties but differed in their dependence on N-terminal formylation compared to S. aureus PSMs. Moreover, S. aureus and S. epidermidis PSMs upregulated the neutrophil complement receptor CD11b via FPR2/ALX stimulation in an Agr-dependent fashion. Hence, Agr controls the capacity of staphylococcal pathogens to activate FPR2/ALX-dependent neutrophil responses, underscoring the crucial role of FPR2/ALX and PSMs in staphylococcus-host interaction. Copyright © 2012 S. Karger AG, Basel.

  11. Upregulation of the N-formyl Peptide receptors in scleroderma fibroblasts fosters the switch to myofibroblasts.

    PubMed

    Rossi, Francesca Wanda; Napolitano, Filomena; Pesapane, Ada; Mascolo, Massimo; Staibano, Stefania; Matucci-Cerinic, Marco; Guiducci, Serena; Ragno, Pia; di Spigna, Gaetano; Postiglione, Loredana; Marone, Gianni; Montuori, Nunzia; de Paulis, Amato

    2015-06-01

    Systemic sclerosis (SSc) is characterized by chronic inflammation and fibrosis. N-Formyl peptide (fMLF) receptors (FPRs) are chemotactic receptors involved in inflammation. Three FPRs have been identified: FPR1, FPR2, and FPR3. We have examined, by RT-PCR, Western blot and immunohistochemistry, FPRs expression in skin fibroblasts from 10 normal subjects and 10 SSc patients, showing increased expression in SSc fibroblasts. Several functions of FPRs occur through the interaction with a region of the urokinase-type plasminogen activator receptor (uPAR88-92), able to interact with FPRs and to mediate urokinase (uPA) or fMLF-dependent cell migration. Soluble uPAR84-95 peptide can act as a direct ligand of FPRs. Furthermore, uPA or its aminoterminal fragment (ATF) can promote the exposure of the uPAR88-92 region. The WKYMVm peptide is a FPRs pan-agonist. We investigated the functional effects of these agonists on normal and SSc fibroblasts. ATF, uPAR84-95, and WKYMVm regulated adhesion, migration, and proliferation of normal fibroblasts. Despite FPR overexpression, the response of SSc fibroblasts to the same agonists was greatly reduced, except for the proliferative response to ATF. SSc fibroblasts showed increased α-smooth muscle actin expression and improved capability to induce wound closure. Indeed, they overexpressed a cleaved uPAR form, exposing the uPAR88-92 region, and vitronectin, both involved in fibrosis and in the fibroblast-to-myofibroblast transition. FPR stimulation promoted α-smooth muscle actin expression in normal fibroblasts as well as motility, matrix deposition, αvβ5 integrin expression, and radical oxygen species generation in normal and SSc fibroblasts. This study provides evidence that FPRs may play a role in fibrosis and in the fibroblast-to-myofibroblast transition. Copyright © 2015 by The American Association of Immunologists, Inc.

  12. Annexin A1 Induces Skeletal Muscle Cell Migration Acting through Formyl Peptide Receptors

    PubMed Central

    Bizzarro, Valentina; Belvedere, Raffaella; Dal Piaz, Fabrizio; Parente, Luca; Petrella, Antonello

    2012-01-01

    Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca2+-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca2+ perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca2+. These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the

  13. Promotion of formyl peptide receptor 1-mediated neutrophil chemotactic migration by antimicrobial peptides isolated from the centipede Scolopendra subspinipes mutilans.

    PubMed

    Park, Yoo Jung; Lee, Sung Kyun; Jung, Young Su; Lee, Mingyu; Lee, Ha Young; Kim, Sang Doo; Park, Joon Seong; Koo, JaeHyung; Hwang, Jae Sam; Bae, Yoe-Sik

    2016-09-01

    We investigated the effects of two antimicrobial peptides (AMPs) isolated from Scolopendra subspinipes mutilans on neutrophil activity. Stimulation of mouse neutrophils with the two AMPs elicited chemotactic migration of the cells in a pertussis toxin-sensitive manner. The two AMPs also stimulated activation of ERK and Akt, which contribute to chemotactic migration of neutrophils. We found that AMP-stimulated neutrophil chemotaxis was blocked by a formyl peptide receptor (FPR) 1 antagonist (cyclosporin H); moreover the two AMPs stimulated the chemotactic migration of FPR1-expressing RBL-2H3 cells but not of vector-expressing RBL-2H3 cells. We also found that the two AMPs stimulate neutrophil migration in vivo, and that this effect is blocked in FPR1-deficient mice. Taken together, our results suggest that the two AMPs stimulate neutrophils, leading to chemotactic migration through FPR1, and the two AMPs will be useful for the study of FPR1 signaling and neutrophil activation. [BMB Reports 2016; 49(9): 520-525].

  14. Promotion of formyl peptide receptor 1-mediated neutrophil chemotactic migration by antimicrobial peptides isolated from the centipede Scolopendra subspinipes mutilans

    PubMed Central

    Park, Yoo Jung; Lee, Sung Kyun; Jung, Young Su; Lee, Mingyu; Lee, Ha Young; Lee, Ha Young; Park, Joon Seong; Koo, JaeHyung; Koo, JaeHyung; Bae, Yoe-Sik

    2016-01-01

    We investigated the effects of two antimicrobial peptides (AMPs) isolated from Scolopendra subspinipes mutilans on neutrophil activity. Stimulation of mouse neutrophils with the two AMPs elicited chemotactic migration of the cells in a pertussis toxin-sensitive manner. The two AMPs also stimulated activation of ERK and Akt, which contribute to chemotactic migration of neutrophils. We found that AMP-stimulated neutrophil chemotaxis was blocked by a formyl peptide receptor (FPR) 1 antagonist (cyclosporin H); moreover the two AMPs stimulated the chemotactic migration of FPR1-expressing RBL-2H3 cells but not of vector-expressing RBL-2H3 cells. We also found that the two AMPs stimulate neutrophil migration in vivo, and that this effect is blocked in FPR1-deficient mice. Taken together, our results suggest that the two AMPs stimulate neutrophils, leading to chemotactic migration through FPR1, and the two AMPs will be useful for the study of FPR1 signaling and neutrophil activation. [BMB Reports 2016; 49(9): 520-525] PMID:27502013

  15. Walker 256 Tumor Growth Suppression by Crotoxin Involves Formyl Peptide Receptors and Lipoxin A4

    PubMed Central

    Brigatte, Patrícia; Faiad, Odair Jorge; Ferreira Nocelli, Roberta Cornélio; Landgraf, Richardt G.; Palma, Mario Sergio; Cury, Yara; Curi, Rui; Sampaio, Sandra Coccuzzo

    2016-01-01

    We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs. PMID:27190493

  16. Formyl Peptide Receptor 2 Plays a Deleterious Role During Influenza A Virus Infections.

    PubMed

    Tcherniuk, Sergey; Cenac, Nicolas; Comte, Marjorie; Frouard, Julie; Errazuriz-Cerda, Elisabeth; Galabov, Angel; Morange, Pierre-Emmanuel; Vergnolle, Nathalie; Si-Tahar, Mustapha; Alessi, Marie-Christine; Riteau, Béatrice

    2016-07-15

    The pathogenesis of influenza A virus (IAV) infections is a multifactorial process that includes the replication capacity of the virus and a harmful inflammatory response to infection. Formyl peptide receptor 2 (FPR2) emerges as a central receptor in inflammatory processes controlling resolution of acute inflammation. Its role in virus pathogenesis has not been investigated yet. We used pharmacologic approaches to investigate the role of FPR2 during IAV infection in vitro and in vivo. In vitro, FPR2 expressed on A549 cells was activated by IAV, which harbors its ligand, annexin A1, in its envelope. FPR2 activation by IAV promoted viral replication through an extracellular-regulated kinase (ERK)-dependent pathway. In vivo, activating FPR2 by administering the agonist WKYMVm-NH2 decreased survival and increased viral replication and inflammation after IAV infection. This effect was abolished by treating the mice with U0126, a specific ERK pathway inhibitor, showing that, in vivo, the deleterious role of FPR2 also occurs through an ERK-dependent pathway. In contrast, administration of the FPR2 antagonist WRW4 protected mice from lethal IAV infections. These data show that viral replication and IAV pathogenesis depend on FPR2 signaling and suggest that FPR2 may be a promising novel strategy to treat influenza. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  17. Formyl Peptide receptor 1 expression is associated with tumor progression and survival in gastric cancer.

    PubMed

    Cheng, Tsu-Yao; Wu, Ming-Shiang; Lin, Jaw-Town; Lin, Ming-Tsan; Shun, Chia-Tung; Hua, Kuo-Tai; Kuo, Min-Liang

    2014-05-01

    Formyl peptide receptor 1 (FPR1) as a regulator of innate inflammatory response has been implicated in tumor progression of gliomas. The purpose of the present study was to evaluate the prognostic significance and the ligand-receptor interaction of FPR1 in gastric cancer (GC). FPR1 was immunohistochemically-analyzed in tissue sections originating from 116 GC patients. Reverse transcription-polymerase chain reaction (RT-PCR) was used for the assessment of interaction between FPR1 and the FPR1 ligand annexin A1 (AnxA1) in GC cells. High FPR1 expression was significantly associated with stage IV disease, submucosal invasion, serosal invasion, and clinical outcome of GC. Multivariate analysis showed that high FPR1 expression was an independent risk factor of poor overall survival in GC patients. FPR1 expression increased significantly when AnxA1 overexpression was present in GC cells. A positive feedback regulation of FPR1 was involved in the AnxA1-FPR1 signal transduction. FPR1 expression may be used as a novel indicator to predict outcome in GC patients after gastrectomy.

  18. Identification of FAM3D as a new endogenous chemotaxis agonist for the formyl peptide receptors.

    PubMed

    Peng, Xinjian; Xu, Enquan; Liang, Weiwei; Pei, Xiaolei; Chen, Dixin; Zheng, Danfeng; Zhang, Yang; Zheng, Can; Wang, Pingzhang; She, Shaoping; Zhang, Yan; Ma, Jing; Mo, Xiaoning; Zhang, Yingmei; Ma, Dalong; Wang, Ying

    2016-05-01

    The family with sequence similarity 3 (FAM3) gene family is a cytokine-like gene family with four members FAM3A, FAM3B, FAM3C and FAM3D. In this study, we found that FAM3D strongly chemoattracted human peripheral blood neutrophils and monocytes. To identify the FAM3D receptor, we used chemotaxis, receptor internalization, Ca(2+) flux and radioligand-binding assays in FAM3D-stimulated HEK293 cells that transiently expressed formyl peptide receptor (FPR)1 or FPR2 to show that FAM3D was a high affinity ligand of these receptors, both of which were highly expressed on the surface of neutrophils, and monocytes and macrophages. After being injected into the mouse peritoneal cavity, FAM3D chemoattracted CD11b+ Ly6G+ neutrophils in a short time. In response to FAM3D stimulation, phosphorylated ERK1/2 and phosphorylated p38 MAPK family proteins were upregulated in the mouse neutrophils, and this increase was inhibited upon treatment with an inhibitor of FPR1 or FPR2. FAM3D has been reported to be constitutively expressed in the gastrointestinal tract. We found that FAM3D expression increased significantly during colitis induced by dextran sulfate sodium. Taken together, we propose that FAM3D plays a role in gastrointestinal homeostasis and inflammation through its receptors FPR1 and FPR2. © 2016. Published by The Company of Biologists Ltd.

  19. Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair

    PubMed Central

    Leoni, Giovanna; Alam, Ashfaqul; Neumann, Philipp-Alexander; Lambeth, J. David; Cheng, Guangjie; McCoy, James; Hilgarth, Roland S.; Kundu, Kousik; Murthy, Niren; Kusters, Dennis; Reutelingsperger, Chris; Perretti, Mauro; Parkos, Charles A.; Neish, Andrew S.; Nusrat, Asma

    2012-01-01

    N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1–/–IEC and AnxA1–/– mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair. PMID:23241962

  20. Nuclear localization of Formyl-Peptide Receptor 2 in human cancer cells.

    PubMed

    Cattaneo, Fabio; Parisi, Melania; Fioretti, Tiziana; Sarnataro, Daniela; Esposito, Gabriella; Ammendola, Rosario

    2016-08-01

    Current models of G protein-coupled receptors (GPCRs) signaling describe binding of external agonists to cell surface receptors which, in turn, trigger several biological responses. New paradigms indicate that GPCRs localize to and signal at the nucleus, thus regulating distinct signaling cascades. The formyl-peptide receptor FPR2 belongs to the GPCR super-family and is coupled to PTX-sensitive Gi proteins. We show by western blot analysis, immunofluorescence experiments and radioligand binding assays that FPR2 is expressed at nuclear level in CaLu-6 and AGS cells. Nuclear FPR2 is a functional receptor, since it participates in intra-nuclear signaling, as assessed by decreased G protein-FPR2 association and enhanced ERK2, c-Jun and c-Myc phosphorylation upon stimulation of intact nuclei with the FPR2 agonist, WKYMVm. We analyzed FPR2 sequence for the search of a nuclear localization sequence (NLS) and we found a stretch of basic aminoacids (227-KIHKK-231) in the third cytoplasmic loop of the receptor. We performed single (K230A) and multiple (H229A/K230A/K231A) mutagenesis of NLS. The constructs were individually overexpressed in HEK293 cells and immunofluorescence and western blot analysis showed that nuclear localization or translocation of FPR2 depends on the integrity of the H(229) and K(231) residues within the NLS.

  1. Production of a bioengineered G-protein coupled receptor of human formyl peptide receptor 3.

    PubMed

    Wang, Xiaoqiang; Zhang, Shuguang

    2011-01-01

    G-protein coupled receptors (GPCRs) participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3). FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S). After a systematic detergent screening, fos-choline-14 (FC-14) was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets.

  2. Physiological characterization of formyl peptide receptor expressing cells in the mouse vomeronasal organ

    PubMed Central

    Ackels, Tobias; von der Weid, Benoît; Rodriguez, Ivan; Spehr, Marc

    2014-01-01

    The mouse vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Based on largely monogenic expression of either type 1 or 2 vomeronasal receptors (V1Rs/V2Rs) or members of the formyl peptide receptor (FPR) family, the vomeronasal sensory epithelium harbors at least three neuronal subpopulations. While various neurophysiological properties of both V1R- and V2R-expressing neurons have been described using genetically engineered mouse models, the basic biophysical characteristics of the more recently identified FPR-expressing vomeronasal neurons have not been studied. Here, we employ a transgenic mouse strain that coexpresses an enhanced variant of yellow fluorescent protein together with FPR-rs3 allowing to identify and analyze FPR-rs3-expressing neurons in acute VNO tissue slices. Single neuron electrophysiological recordings allow comparative characterization of the biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. In this study, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal striking similarities in the basic (electro) physiological architecture of both transgene-expressing and non-expressing neurons, confirming the suitability of this genetically engineered mouse model for future studies addressing more specialized issues in vomeronasal FPR neurobiology. PMID:25484858

  3. Targeting formyl peptide receptor 2 reduces leukocyte-endothelial interactions in a murine model of stroke.

    PubMed

    Smith, Helen K; Gil, Cristiane Damas; Oliani, Sonia M; Gavins, Felicity N E

    2015-05-01

    Ischemia/reperfusion (I/R) injury following stroke can worsen patient outcome through excess inflammation. This study investigated the pharmacologic potential of targeting an endogenous anti-inflammatory circuit via formyl peptide receptor (FPR) 2/lipoxin receptor (ALX) (Fpr2/3 in mouse) in global cerebral I/R. Mice (C57BL/6 and Fpr2/3(-/-)) were subjected to bilateral common carotid artery occlusion, followed by reperfusion and treatment with FPR agonists: AnxA1Ac2-26 [Annexin A1 mimetic peptide (Ac-AMVSEFLKQAWFIENEEQEYVQTVK), 2.5 μg/kg] and 15-epimer-lipoxin A4 (15-epi-LXA4; FPR2/ALX specific, 12.5 and 100 ng/kg). Leukocyte-endothelial (L-E) interactions in the cerebral microvasculature were then quantified in vivo using intravital fluorescence microscopy. 15-epi-LXA4 administration at the start of reperfusion reduced L-E interactions after 40 min (which was sustained at 2 h with high-dose 15-epi-LXA4) to levels seen in sham-operated animals. AnxA1Ac2-26 treatment decreased leukocyte adhesion at 40 min and all L-E interactions at 2 h (up to 95%). Combined treatment with AnxA1Ac2-26 plus FPR antagonists t-Boc-FLFLF (250 ng/kg) or WRW4 (FPR2/ALX selective, 1.4 μg/kg) abrogated the effects of AnxA1Ac2-26 fully at 40 min. Antagonists were less effective at 2 h, which we demonstrate is likely because of their impact on early L-E interactions. Our findings indicate that FPR2/ALX activity elicits considerable control over vascular inflammatory responses during cerebral I/R and, therefore, provide evidence that targeting FPR2/ALX may be beneficial for patients who suffered from stroke. © FASEB.

  4. Binding of formyl peptides to Walker 256 carcinosarcoma cells and the chemotactic response of these cells.

    PubMed

    Rayner, D C; Orr, F W; Shiu, R P

    1985-05-01

    N-Formylmethionylleucylphenylalanine (fMLP) induces chemotaxis in leukocytes, the response being mediated by peptide binding to a receptor on the plasma membrane. In tumor cells, this peptide has been reported to induce cellular swelling and chemotaxis in vitro and to enhance the localization of circulating tumor cells in vivo. In the Boyden chamber, we evaluated the migratory responses of Walker carcinosarcoma 256 cells to varying concentrations of fMLP. Sigmoidal dose-response curves were obtained with the dose of chemotactic factor that elicits a half-maximal chemotactic response of 5.0 +/- 2.5 X 10(-8) M. Checkerboard analysis indicated that these responses were dependent upon a concentration gradient of fMLP with increases in migration of circa 2 to 2.5 times that of random movement. To examine the binding of fMLP, the tumor cells were incubated with 5 X 10(-9) M fML-[3H]P in Hanks' balanced salt solution. Specific binding (0.5 to 1% of total radioligand, to whole cells inhibited by 5 X 10(-6) M fMLP) approached equilibrium after 4 to 6 h at 4 degrees C and after 6 to 10 h at 22 degrees C. Autoradiographic studies demonstrated heterogeneous binding of the peptide by tumor cells and also showed its intracellular localization. In homogenates of Walker cells prepared in 0.1 M Tris HCl, pH 7.4, with 10 mM MgCl2 and bovine serum albumin (1 mg/ml), specific binding of approximately 0.5% of total fML-[3H]P reached equilibrium after 60 min at 4 degrees C. In whole cells and homogenates, binding was reversible by addition of unlabeled fMLP. In whole cells, displacement curves demonstrated a Kd of 1.9 +/- 0.1 X 10(-7) M, whereas in homogenates there was a background of low affinity (Kd greater than 10(-5) M) nonsaturable binding, but also a high-affinity component with Kd of 4.9 +/- 1.8 X 10(-8) M. Both chemotaxis and binding were inhibited by the oligopeptide, N-carbobenzoxy-L-phenylalanyl-L-methionine, which is a competitive inhibitor of formyl peptide

  5. Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus.

    PubMed

    Boer, J C; Domanska, U M; Timmer-Bosscha, H; Boer, I G J; de Haas, C J C; Joseph, J V; Kruyt, F A E; de Vries, E G E; den Dunnen, W F A; van Strijp, J A G; Walenkamp, A M E

    2013-02-19

    High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.

  6. The peptidomimetic Lau-(Lys-βNSpe)6-NH2 antagonizes formyl peptide receptor 2 expressed in mouse neutrophils.

    PubMed

    Skovbakke, Sarah Line; Winther, Malene; Gabl, Michael; Holdfeldt, André; Linden, Sara; Wang, Ji Ming; Dahlgren, Claes; Franzyk, Henrik; Forsman, Huamei

    2016-11-01

    The formyl peptide receptor (FPR) gene family has a complex evolutionary history and comprises eight murine members but only three human representatives. To enable translation of results obtained in mouse models of human diseases, more comprehensive knowledge of the pharmacological similarities/differences between the human and murine FPR family members is required. Compared to FPR1 and FPR2 expressed by human neutrophils, very little is known about agonist/antagonist recognition patterns for their murine orthologues, but now we have identified two potent and selective formylated peptide agonists (fMIFL and PSMα2) for Fpr1 and Fpr2, respectively. These peptides were used to determine the inhibition profile of a set of antagonists with known specificities for the two FPRs in relation to the corresponding murine receptors. Some of the most potent and selective antagonists for the human receptors proved to be devoid of effect on their murine orthologues as determined by their inability to inhibit superoxide release from murine neutrophils upon stimulation with receptor-specific agonists. The Boc-FLFLF peptide was found to be a selective antagonist for Fpr1, whereas the lipidated peptidomimetic Lau-(Lys-βNSpe)6-NH2 and the hexapeptide WRW4 were identified as Fpr2-selective antagonists. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

    PubMed

    Wagener, Brant M; Marjon, Nicole A; Prossnitz, Eric R

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

  8. Strain-specific Loss of Formyl Peptide Receptor 3 in the Murine Vomeronasal and Immune Systems.

    PubMed

    Stempel, Hendrik; Jung, Martin; Pérez-Gómez, Anabel; Leinders-Zufall, Trese; Zufall, Frank; Bufe, Bernd

    2016-04-29

    Formyl peptide receptor 3 (Fpr3, also known as Fpr-rs1) is a G protein-coupled receptor expressed in subsets of sensory neurons of the mouse vomeronasal organ, an olfactory substructure essential for social recognition. Fpr3 has been implicated in the sensing of infection-associated olfactory cues, but its expression pattern and function are incompletely understood. To facilitate visualization of Fpr3-expressing cells, we generated and validated two new anti-Fpr3 antibodies enabling us to analyze acute Fpr3 protein expression. Fpr3 is not only expressed in murine vomeronasal sensory neurons but also in bone marrow cells, the primary source for immune cell renewal, and in mature neutrophils. Consistent with the notion that Fpr3 functions as a pathogen sensor, Fpr3 expression in the immune system is up-regulated after stimulation with a bacterial endotoxin (lipopolysaccharide). These results strongly support a dual role for Fpr3 in both vomeronasal sensory neurons and immune cells. We also identify a large panel of mouse strains with severely altered expression and function of Fpr3, thus establishing the existence of natural Fpr3 knock-out strains. We attribute distinct Fpr3 expression in these strains to the presence or absence of a 12-nucleotide in-frame deletion (Fpr3Δ424-435). In vitro calcium imaging and immunofluorescence analyses demonstrate that the lack of four amino acids leads to an unstable, truncated, and non-functional receptor protein. The genome of at least 19 strains encodes a non-functional Fpr3 variant, whereas at least 13 other strains express an intact receptor. These results provide a foundation for understanding the in vivo function of Fpr3. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Antagonism of human formyl peptide receptor 1 (FPR1) by chromones and related isoflavones.

    PubMed

    Schepetkin, Igor A; Kirpotina, Liliya N; Khlebnikov, Andrei I; Cheng, Ni; Ye, Richard D; Quinn, Mark T

    2014-12-15

    Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. Because FPRs play an important role in the regulation of inflammatory reactions implicated in disease pathogenesis, FPR antagonists may represent novel therapeutics for modulating innate immunity. Previously, 4H-chromones were reported to be potent and competitive FPR1 antagonists. In the present studies, 96 additional chromone analogs, including related synthetic and natural isoflavones were evaluated for FPR1 antagonist activity. We identified a number of novel competitive FPR1 antagonists that inhibited fMLF-induced intracellular Ca2+ mobilization in FPR1-HL60 cells and effectively competed with WKYMVm-FITC for binding to FPR1 in FPR1-HL60 and FPR1-RBL cells. Compound 10 (6-hexyl-2-methyl-3-(1-methyl-1H-benzimidazol-2-yl)-4-oxo-4H-chromen-7-yl acetate) was found to be the most potent FPR1-specific antagonist, with binding affinity Ki∼100 nM. These chromones inhibited Ca2+ flux and chemotaxis in human neutrophils with nanomolar-micromolar IC50 values. In addition, the most potent novel FPR1 antagonists inhibited fMLF-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in FPR1-RBL cells. These antagonists were specific for FPR1 and did not inhibit WKYMVM/WKYMVm-induced intracellular Ca2+ mobilization in FPR2-HL60 cells, FPR3-HL60 cells, RBL cells transfected with murine Fpr1, or interleukin 8-induced Ca2+ flux in human neutrophils and RBL cells transfected with CXC chemokine receptor 1 (CXCR1). Moreover, pharmacophore modeling showed that the active chromones had a significantly higher degree of similarity with the pharmacophore template as compared to inactive analogs. Thus, the chromone/isoflavone scaffold represents a relevant backbone for development of novel FPR1 antagonists. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. A formyl peptide receptor agonist suppresses inflammation and bone damage in arthritis.

    PubMed

    Kao, W; Gu, R; Jia, Y; Wei, Xuemin; Fan, H; Harris, J; Zhang, Zhiyi; Quinn, J; Morand, E F; Yang, Y H

    2014-09-01

    Annexin A1 (AnxA1) is an endogenous anti-inflammatory protein and agonist of the formyl peptide receptor 2 (FPR2). However, the potential for therapeutic FPR ligands to modify immune-mediated disease has been little explored. We investigated the effects of a synthetic FPR agonist on joint disease in the K/BxN model of rheumatoid arthritis (RA) and RA fibroblast-like synoviocytes (FLS). Arthritis was induced by injection of K/BxN serum at day 0 and 2 in wild-type (WT) or AnxA1(-/-) mice and clinical and histopathological manifestations measured 8-11 days later. WT mice were given the FPR agonist compound 43 (Cpd43) (6 or 30 mg·kg(-1) i.p.) for 4 days. Effects of AnxA1 and Cpd43 on RANKL-induced osteoclastogenesis were assessed in RAW 264.7 cells and human RA FLS and macrophages. Treatment with Cpd43 before or after the onset of arthritis reduced clinical disease severity and attenuated synovial TNF-α and osteoclast-associated gene expression. Deletion of AnxA1 in mice exacerbated arthritis severity in the K/BxN model. In vitro, Cpd43 suppressed osteoclastogenesis and NFAT activity elicited by RANKL, and inhibited IL-6 secretion by mouse macrophages. In human RA joint-derived FLS and monocyte-derived macrophages, Cpd43 treatment inhibited IL-6 release, while blocking FPR2 or silencing AnxA1 increased this release. The FPR agonist Cpd43 reduced osteoclastogenesis and inflammation in a mouse model of RA and exhibited anti-inflammatory effects in relevant human cells. These data suggest that FPR ligands may represent novel therapeutic agents capable of ameliorating inflammation and bone damage in RA. © 2014 The British Pharmacological Society.

  11. Pleiotropic regulation of macrophage polarization and tumorigenesis by formyl peptide receptor-2.

    PubMed

    Li, Y; Cai, L; Wang, H; Wu, P; Gu, W; Chen, Y; Hao, H; Tang, K; Yi, P; Liu, M; Miao, S; Ye, D

    2011-09-08

    Cancer cells recruit monocytes, macrophages and other inflammatory cells by producing abundant chemoattractants and growth factors, such as macrophage colony-stimulating factor (M-CSF/CSF-1) and monocyte chemoattractant protein-1 (MCP-1/CCL2), to promote tumor growth and dissemination. An understanding of the mechanisms that target cancer cells and regulate tumor microenvironment is essential in designing anticancer therapies. Here, we showed that serum amyloid-A (SAA) and cathelicidin (LL-37) stimulated M-CSF and MCP-1 expression with or without lipopolysaccharide (LPS) administration; conversely, lipoxin-A(4) (LXA(4)) and annexin-A1 (ANXA1) inhibited LPS-induced M-CSF and MCP-1 production by human (HepG2) and mouse (H22) hepatocellular carcinoma cells (HCCs). The effects of LXA(4), ANXA1, SAA and LL-37 were dependent on the activation of their mutual cell-surface receptor formyl peptide receptor-2 (FPR2) and subsequent ROS-MAPK-NF-kB signalings. Furthermore, our results indicated that LPS switched macrophages into an IL-10(low)IL-12(high) M1 profile, whereas M-CSF+MCP-1 and FPR2 agonists skewed them into M2 (IL-10(high)IL-12(low)). In that respect, through modulating the phosphorylation of signal transducer and activator of transcription-3 (STAT3), LXA(4) and ANXA1 induced monocyte differentiation into M2a+M2c-like cells and showed antitumorigenetic activities, whereas SAA, LL-37 and M-CSF+MCP-1 led to M2b- or M2d-like polarization, which exacerbated HCC invasion in vitro and in vivo, respectively. Our results suggest that FPR2 has an appreciable pleiotropic regulator role in tumor immunoediting.

  12. Antagonism of Human Formyl Peptide Receptor 1 (FPR1) by Chromones and Related Isoflavones

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Cheng, Ni; Ye, Richard D.; Quinn, Mark T.

    2014-01-01

    Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. Because FPRs play an important role in the regulation of inflammatory reactions implicated in disease pathogenesis, FPR antagonists may represent novel therapeutics for modulating innate immunity. Previously, 4H-chromones were reported to be potent and competitive FPR1 antagonists. In the present studies, 96 additional chromone analogs, including related synthetic and natural isoflavones were evaluated for FPR1 antagonist activity. We identified a number of novel competitive FPR1 antagonists that inhibited fMLF-induced intracellular Ca2+ mobilization in FPR1-HL60 cells and effectively competed with WKYMVm-FITC for binding to FPR1 in FPR1-HL60 and FPR1-RBL cells. Compound 10 (6-hexyl-2-methyl-3-(1-methyl-1H-benzimidazol-2-yl)-4-oxo-4H-chromen-7-yl acetate) was found to be the most potent FPR1-specific antagonist, with binding affinity Ki~100 nM. These chromones inhibited Ca2+ flux and chemotaxis in human neutrophils with nanomolar-micromolar IC50 values. In addition, the most potent novel FPR1 antagonists inhibited fMLF-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in FPR1-RBL cells. These antagonists were specific for FPR1 and did not inhibit WKYMVM/WKYMVm-induced intracellular Ca2+ mobilization in FPR2-HL60 cells, FPR3-HL60 cells, RBL cells transfected with murine Fpr1, or interleukin 8-induced Ca2+ flux in human neutrophils and RBL cells transfected with CXC chemokine receptor 1 (CXCR1). Moreover, pharmacophore modeling showed that the active chromones had a significantly higher degree of similarity with the pharmacophore template as compared to inactive analogs. Thus, the chromone/isoflavone scaffold represents a relevant backbone for development of novel FPR1 antagonists. PMID:25450672

  13. Differential priming effects of proinflammatory cytokines on human neutrophil oxidative burst in response to bacterial N-formyl peptides.

    PubMed Central

    Elbim, C; Bailly, S; Chollet-Martin, S; Hakim, J; Gougerot-Pocidalo, M A

    1994-01-01

    Cytokines such as tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 8 (IL-8), IL-6, IL-1 alpha, and IL-1 beta produced during the immune and inflammatory responses to bacterial stimuli have been reported to interact with polymorphonuclear neutrophil (PMN) activities. However, contradictory findings on their direct and priming effects on the PMN oxidative burst, which is essential for bacterial killing, have been reported. We have used a flow cytometry method to study the effects of these cytokines on the oxidative burst of PMN in whole blood to avoid PMN activation related to isolation procedures. None of the cytokines tested directly activated the PMN oxidative burst, but they did have differential priming effects on the oxidative burst in response to bacterial N-formyl peptides. TNF, GM-CSF, and IL-8 strongly primed a subpopulation of PMN to produce H2O2 in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP), while IL-1 alpha, IL-1 beta, and IL-6 failed to do so. Furthermore, the addition of TNF, GM-CSF, or IL-8 to whole blood increased the capacity of a subpopulation of PMN to bind N-formyl peptides, a phenomenon that could account, at least in part, for the strong H2O2 production in response to FMLP after priming by the cytokines. The size of the primed hyperresponsive subpopulation was greater after priming with TNF or GM-CSF than after priming with IL-8. However, GM-CSF, TNF, and IL-8 at suboptimal concentrations cooperated in the induction of a subpopulation hyperresponsive to FMLP. These results show that, of the various proinflammatory cytokines tested, TNF, GM-CSF, and IL-8 strongly prime the PMN oxidative burst in response to bacterial peptides in whole blood and suggest that these cytokines may play a critical role in bacterial killing in vivo. PMID:8188340

  14. Identification of Novel Small-Molecule Agonists for Human Formyl Peptide Receptors and Pharmacophore Models of Their RecognitionS⃞

    PubMed Central

    Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Schepetkin, Igor A.; Ye, Richard D.; Rabiet, Marie-Josèphe; Jutila, Mark A.

    2010-01-01

    N-formyl peptide receptor (FPR1) and N-formyl peptide receptor-like 1 (FPRL1, now known as FPR2) are G protein-coupled receptors involved in host defense and sensing cellular dysfunction. Because of the potential for FPR1/FPR2 as a therapeutic target, our recent high-throughput screening efforts have focused on the identification of unique nonpeptide agonists of FPR1/FPR2. In the present studies, we screened a chemolibrary of drug-like molecules for their ability to induce intracellular calcium mobilization in RBL-2H3 cells transfected with human FPR1 or FPR2. Screening of these compounds resulted in the identification of novel and potent agonists that activated both FPR1 and FPR2, as well as compounds that were specific for either FPR1 or FPR2 with EC50 values in the low micromolar range. Specificity of the compounds was supported by analysis of calcium mobilization in HL-60 cells transfected with human FPR1 and FPR2. In addition, all but one agonist activated intracellular calcium flux and chemotaxis in human neutrophils, irrespective of agonist specificity for FPR1 or FPR2. Molecular modeling of the group of FPR1 and FPR2 agonists using field point methodology allowed us to create pharmacophore models for ligand binding sites and formulate requirements for these specific N-formyl peptide receptor agonists. These studies further demonstrate that agonists of FPR1/FPR2 include compounds with wide chemical diversity and that analysis of such compounds can enhance our understanding of their ligand/receptor interaction. PMID:19903830

  15. Identification of C-terminal Phosphorylation Sites of N-Formyl Peptide Receptor-1 (FPR1) in Human Blood Neutrophils*

    PubMed Central

    Maaty, Walid S.; Lord, Connie I.; Gripentrog, Jeannie M.; Riesselman, Marcia; Keren-Aviram, Gal; Liu, Ting; Dratz, Edward A.; Bothner, Brian; Jesaitis, Algirdas J.

    2013-01-01

    Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu312–Arg322 and Arg323–Lys350) and extracellular FPR1 peptide (Ile191–Arg201) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala323–Lys350 only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr325, Ser328, Thr329, Thr331, Ser332, Thr334, and Thr339. No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nm. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of

  16. Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils.

    PubMed

    Maaty, Walid S; Lord, Connie I; Gripentrog, Jeannie M; Riesselman, Marcia; Keren-Aviram, Gal; Liu, Ting; Dratz, Edward A; Bothner, Brian; Jesaitis, Algirdas J

    2013-09-20

    Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites

  17. Pharmacological profile of a bifunctional ligand of the formyl peptide receptor1 fused to the myc epitope.

    PubMed

    Charest-Morin, Xavier; Roy, Caroline; Fernandes, Maria J G; Marceau, François

    2015-03-01

    In human peripheral blood neutrophils or in myeloid PLB-985 cells differentiated towards a neutrophil-like phenotype, the peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleucyl-L-tyrosyl-L-leucyl-fluorescein isothiocyanate (f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC) binds to and activates formyl peptide receptor1 (FPR1) and is submitted to receptor-mediated endocytosis (microscopy, cytofluorometry). This peptide may be considered a C-terminally extended version of f-Met-Leu-Phe which carries a fluorescent cargo into cells. By analogy to other peptide hormones for which we have evaluated epitope-tagged agonists as carriers of antibody cargoes, we have designed and evaluated f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, C-terminally extended with the 10-residue myc tag. This peptide is as potent as f-Met-Leu-Phe to compete for f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC uptake by PLB-985 cells, but did not mediate (10-1000nM) the internalization of the fluorescent anti-myc monoclonal antibody 4A6 added to the extracellular fluid at ~7nM (microscopy). The nonfluorescent version of the antibody (28nM) acts as a pre-receptor antagonist of f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, but not of f-Met-Leu-Phe (superoxide release assay in differentiated PLB-985 cells). A further prolonged analog, f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly)5-myc, designed to decrease the possible steric hindrance between FPR1 and the bound anti-myc antibody, has little affinity for the receptor, precluding a direct assessment of this issue. Thus, the relatively low-affinity anti-myc antibody used at a high concentration functionally behaves as a selective pre-receptor antagonist of the agonist f-Nle-Leu-Phe-Nle-Tyr-Lys-myc. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors

    DTIC Science & Technology

    2005-01-01

    activated phagocytes. Therefore, we examined the chemotactic migration of platelets qualita- tively by videomicroscopy . Platelets in medium were al- lowed...significantly decreased M. Czapiga et al. /Experimental Hematology 33 (2005) 73–84 79Figure 3. Videomicroscopy of human platelets in response to formyl...selected platelets during videomicroscopy from the time of the addition of fMLF (104 M in 1 µL) or PBS. Movement between markers represents 10 frames

  19. Peptide length and folding state govern the capacity of staphylococcal β-type phenol-soluble modulins to activate human formyl-peptide receptors 1 or 2.

    PubMed

    Kretschmer, Dorothee; Rautenberg, Maren; Linke, Dirk; Peschel, Andreas

    2015-04-01

    Most staphylococci produce short α-type PSMs and about twice as long β-type PSMs that are potent leukocyte attractants and toxins. PSMs are usually secreted with the N-terminal formyl group but are only weak agonists for the leukocyte FPR1. Instead, the FPR1-related FPR2 senses PSMs efficiently and is crucial for leukocyte recruitment in infection. Which structural features distinguish FPR1 from FPR2 ligands has remained elusive. To analyze which peptide properties may govern the capacities of β-type PSMs to activate FPRs, full-length and truncated variants of such peptides from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis were synthesized. FPR2 activation was observed even for short N- or C-terminal β-type PSM variants once they were longer than 18 aa, and this activity increased with length. In contrast, the shortest tested peptides were potent FPR1 agonists, and this property declined with increasing peptide length. Whereas full-length β-type PSMs formed α-helices and exhibited no FPR1-specific activity, the truncated peptides had less-stable secondary structures, were weak agonists for FPR1, and required N-terminal formyl-methionine residues to be FPR2 agonists. Together, these data suggest that FPR1 and FPR2 have opposed ligand preferences. Short, flexible PSM structures may favor FPR1 but not FPR2 activation, whereas longer peptides with α-helical, amphipathic properties are strong FPR2 but only weak FPR1 agonists. These findings should help to unravel the ligand specificities of 2 critical human PRRs, and they may be important for new, anti-infective and anti-inflammatory strategies.

  20. Identification of oligomer proanthocyanidins (F2) isolated from grape seeds as a formyl peptide receptor 1 partial agonist.

    PubMed

    Yang, Jingyu; Wang, Qing; Zhao, Ruijun; Sun, Baoshan; Wang, Lihui; Hou, Yue; Li, Xiaoqin; Wu, Chunfu

    2013-04-01

    Formyl peptide receptor 1 (FPR1) plays an important role in the rapid progression of glioblastoma and has been considered as a molecular target for the treatment. Previously, we have shown that oligomer proanthocyanidins (F2, degree of polymerization 2-15), isolated from grape seeds, inhibited FPR1-mediated chemotaxis of U-87 glioblastoma cells. In the present study, we investigated the capacity of F2 to interact with FPR1. The cross attenuation of chemotaxis revealed that F2 shared FPR1 with formyl-methionyl-leucyl-phenylalanine (fMLF), which is a prototype agonist of FPR1. F2 was chemotactic for U-87 cells, and the chemotactic response was abolished when FPR1 gene was silenced or FPR1 was competitively occupied. We further show that F2 specifically blocked the binding of fluorescent agonist to FPR1. Interestingly, F2 exhibited the characteristic of a partial agonist for FPR1, as shown by its capacity to activate FPR1-mediated PI3K-PKC-MAPK pathways. Meanwhile, F2 also attenuated fMLF-triggered MAPK activation, suggesting that F2 could antagonize the effect of an agonist. Furthermore, F2 abolished the invasion of U-87 cells induced by fMLF. Thus, we have identified F2 as a novel, partial agonist for FPR1, which may be useful for glioblastoma therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates.

    PubMed

    Yang, Hui; Shi, Peng

    2010-12-01

    Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemosensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantly with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.

  2. Stimulation of human formyl peptide receptors by calpain inhibitors: homology modeling of receptors and ligand docking simulation.

    PubMed

    Fujita, Hisakazu; Kato, Takayuki; Watanabe, Norifumi; Takahashi, Tatsuji; Kitagawa, Seiichi

    2011-12-15

    Calpain inhibitors, including peptide aldehydes (N-acetyl-Leu-Leu-Nle-CHO and N-acetyl-Leu-Leu-Met-CHO) and α-mercapto-acrylic acid derivatives (PD150606 and PD151746), have been shown to stimulate phagocyte functions via activation of human formyl peptide receptor (hFPR) and/or hFPR-like 1 (hFPRL1). Using the homology modeling of the receptors and the ligand docking simulation, here we show that these calpain inhibitors could bind to the putative N-formyl-Met-Leu-Phe (fMLF) binding site on hFPR and/or hFPRL1. The studies with HEK-293 cells stably expressing hFPR or hFPRL1 showed that the concentrations of calpain inhibitors required to induce an increase in cytoplasmic free Ca(2+) ([Ca(2+)](i)) was much higher (>100 folds) than those of fMLF and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm). HEK-293 cells expressing hFPR or hFPRL1 with the mutated fMLF binding site never exhibited the [Ca(2+)](i) response to calpain inhibitors. When the optimal concentrations of each stimulus were used, pretreatment of cells with fMLF or WKYMVm abolished an increase in [Ca(2+)](i) induced by calpain inhibitors as well as the same stimulus, whereas pretreatment of cells with calpain inhibitors significantly suppressed, but never abolished, the [Ca(2+)](i) response induced by fMLF or WKYMVm, suggesting that the binding affinity of the inhibitors to the putative fMLF binding site may be lower than that of fMLF or WKYMVm. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Mitochondrial DAMPs from femoral reamings activate neutrophils via formyl peptide receptors and P44/42 MAP Kinase

    PubMed Central

    Hauser, Carl J.; Sursal, Tolga; Rodriguez, Edward K.; Appleton, Paul T.; Zhang, Qin; Itagaki, Kiyoshi

    2010-01-01

    Hypothesis Fractures and femoral reaming are associated with lung injury. The mechanisms linking fractures and inflammation are unclear; but tissue disruption might release mitochondria. Mitochondria are evolutionarily derived from bacteria and contain “Damage Associated Molecular Patterns” (DAMPs) like formylated peptides that can activate immunocytes. We therefore studied whether fracture reaming releases mitochondrial DAMPs (MTD) and how MTD act on immune cells. Methods Femur fracture reamings (FFx) from 10 patients were spun to remove bone particulates. Supernatants were assayed for mitochondrial DNA (mtDNA). Mitochondria were isolated from the residual reaming slurry, sonicated and spun at 12,000g. The resultant MTD were assayed for their ability to cause neutrophil (PMN) Ca2+ transient production, p44/42 MAPK phosphorylation, IL-8 release and matrix metalloproteinase-9 (MMP9) release with and without formyl peptide receptor-1 (FPR1) blockade. Rats were injected with MTD and whole lung assayed for p44/42 activation. Results mtDNA appears at many thousand fold normal plasma levels in FFx and at intermediate levels in patients’ plasma, suggesting release from fracture to plasma. FFx MTD caused brisk PMN Ca2+ flux, activated PMN p44/42 MAPK and caused PMN release of IL-8 and MMP9. Responses to MTD were inhibited by FPR1 blockade using Cyclosporin H and anti-FPR1. MTD injection caused P44/42 phosphorylation in rat lung. Conclusions FFx reaming releases mitochondria into the wound and circulation. MTD then activates PMN. Release of damage signals like MTD from FFx may underlie activation of the cytokine cascades known to be associated with facture fixation and lung injury. PMID:20736789

  4. Formation of truncated peptide by-products via sequence-specific formyl group transfer from Trp(For) residues to Nα in the course of Boc-SPPS.

    PubMed

    Azev, Viatcheslav N; Mustaeva, Leila G; Gorbunova, Elena Yu; Molchanov, Maksim V; Rodionov, Igor L

    2013-10-01

    (N(In))-Formyl protective group of tryptophan has been introduced as a base/nucleophile-labile protective group. It has long been known that a free Nα-amino group of the peptide can serve as a nucleophile: an irreversible formyl N(In)  → NH(2) transfer is consistently observed when deformylation is performed last on an otherwise deprotected peptide that possesses free Nα-amino group. Obviously, this particular side reaction should be expected any time free amino group is exposed to Trp(For), but, at the best of our knowledge, has never been reported in the course of Boc-SPPS. In the present communication, we describe a set of appropriately designed model experiments that permitted to detect the title side reaction both in solution and in solid-phase reactions. We observed intermolecular formyl group transfer with a model compound, Trp(For)-NH(2). Importantly, we also observed this migration on solid support with the rate roughly estimated to be up to 1% of residues per minute. We also observed that the formyl-group transfer reaction occurred in a sequence-dependent manner and was suppressed to a non-detectable level using 'in situ neutralization' technique. Because this side reaction is sequence dependent, there might be situations when the rate of the formation of Nα -formyl termination by-products is significant. In other cases, the Nα -For truncated by-products would not contaminate the final peptide significantly but still could be a source of microheterogeneity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

  5. Real-time detection of implant-associated neutrophil responses using a formyl peptide receptor-targeting NIR nanoprobe

    PubMed Central

    Zhou, Jun; Tsai, Yi-Ting; Weng, Hong; Tang, Ewin N; Nair, Ashwin; Davé, Digant P; Tang, Liping

    2012-01-01

    Neutrophils play an important role in implant-mediated inflammation and infection. Unfortunately, current methods which monitor neutrophil activity, including enzyme measurements and histological evaluation, require many animals and cannot be used to accurately depict the dynamic cellular responses. To understand the neutrophil interactions around implant-mediated inflammation and infection it is critical to develop methods which can monitor in vivo cellular activity in real time. In this study, formyl peptide receptor (FPR)-targeting near-infrared nanoprobes were fabricated. This was accomplished by conjugating near-infrared dye with specific peptides having a high affinity to the FPRs present on activated neutrophils. The ability of FPR-targeting nanoprobes to detect and quantify activated neutrophils was assessed both in vitro and in vivo. As expected, FPR-targeting nanoprobes preferentially accumulated on activated neutrophils in vitro. Following transplantation, FPR-targeting nanoprobes preferentially accumulated at the biomaterial implantation site. Equally important, a strong relationship was observed between the extent of fluorescence intensity in vivo and the number of recruited neutrophils at the implantation site. Furthermore, FPR-targeting nanoprobes may be used to detect and quantify the number of neutrophils responding to a catheter-associated infection. The results show that FPR-targeting nanoprobes may serve as a powerful tool to monitor and measure the extent of neutrophil responses to biomaterial implants in vivo. PMID:22619542

  6. N-formyl peptide receptors in human neutrophils display distinct membrane distribution and lateral mobility when labeled with agonist and antagonist

    PubMed Central

    1993-01-01

    Receptors for bacterial N-formyl peptides are instrumental for neutrophil chemotactic locomotion and activation at sites of infection. As regulatory mechanisms for signal transduction, both rapid coupling of the occupied receptor to cytoskeletal components, and receptor lateral redistribution, have been suggested (Jesaitis et al., 1986, 1989). To compare the distribution and lateral diffusion of the nonactivated and activated neutrophil N-formyl-peptide receptor, before internalization, we used a new fluorescent N-formyl-peptide receptor antagonist, tertbutyloxycarbonyl-Phe(D)-Leu-Phe(D)-Leu-Phe-OH (Boc- FLFLF, 0.1-1 microM), and the fluorescent receptor agonist formyl-Nle- Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK, 0.1-1 microM). Fluorescent Boc-FLFLF did not elicit an oxidative burst in the neutrophil at 37 degrees C, as assessed by chemiluminescence and reduction of p-nitroblue tetrazolium chloride, but competed efficiently both with formyl-methionyl-leucyl- phenylalanine (fMLF) and fnLLFnLYK. It was not internalized, as evidenced by confocal microscopy and acid elution of surface bound ligand. The lateral mobility characteristics of the neutrophil fMLF receptor were investigated with the technique of FRAP. The diffusion coefficient (D) was similar for antagonist- and agonist-labeled receptors (D approximately 5 x 10(-10) cm2/s), but the fraction of mobile receptors was significantly lower in agonist- compared to antagonist-labeled cells, approximately 40% in contrast to approximately 60%. This reduction in receptor mobile fraction was slightly counteracted, albeit not significantly, by dihydrocytochalasin B (dhcB, 5 microM). To block internalization of agonist-labeled receptors, receptor mobility measurements were done at 14 degrees C. At this temperature, confocal microscopy revealed clustering of receptors in response to agonist binding, compared to a more uniform receptor distribution in antagonist-labeled cells. The pattern of agonist- induced receptor clustering was

  7. In vivo and in vitro assessment of porcine neutrophil activation responses to chemoattractants: flow cytometric evidence for the selective absence of formyl peptide receptors.

    PubMed

    Fletcher, M P; Stahl, G L; Longhurst, J C

    1990-04-01

    Interest in the role that activated granulocytes play in C5a-induced myocardial ischemia prompted us to investigate and compare activation responses of pig and human neutrophils. The responses of Hypaque-Ficoll purified porcine (P-PMN) and human neutrophils (H-PMN) to stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C5a, phorbol myristate acetate (PMA), and calcium ionophore A23187 (A23187) were compared by flow cytometrically measured changes in the cells' forward (FWD-SC) (a measure of shape/volume change) and right angle (90 degrees-SC) light scatter (a measure of secretion), and in the distribution of the membrane potential sensitive fluorescent probe di-O-C (3). FMLP, C5a, and Zymosan-activated serum (ZAS stimulated chemotaxis and FMLP vs. PMA-stimulated adherence to plastic were also compared. Unstimulated P-PMN had lower FWD-SC and 90 degrees-SC than H-PMN (39.4 +/- 1.4 vs. 48.4 +/- 2.0 P less than 0.05, and 32.7 +/- 2.7 vs. 52.4 +/- 1.5 units, P less than 0.005, for FWD-SC and 90 degrees-SC of P-PMN vs. H-PMN, respectively). P-PMN selectively failed to increase their FWD-SC upon stimulation with FMLP (0.0 +/- 0.5% vs. 26.1 +/- 6.8%, P-PMN vs. H-PMN), or decrease their 90 degrees-SC when treated with cytochalasin B + FMLP (secretion) (2.4 +/- 0.1% vs. -35.8 +/- 4.6% change in 90 degrees-SC, P-PMN vs. H-PMN), while responding comparably to C5a, PMA, and A23187. P-PMN failed to depolarize in response to FMLP but responded similarly to H-PMN when activated by C5a, A23187, and PMA. P-PMN's chemotactic response to FMLP was selectively absent since the cells responded well to purified pig C5a. FMLP stimulated significant increases in H-PMN adherence to bovine serum albumin-coated plastic (44.1 +/- 6.7% vs. 12.6 +/- 3.7%, FMLP vs. buffer, P less than 0.025), but failed to increase adherence of P-PMN above baseline 0.68 +/- 0.20% vs. 2.12 +/- 1.90%, FMLP vs. buffer, P greater than 0.05. PMA (100 ng/ml) stimulated comparable increases in adherence in

  8. Involvement of formyl peptide receptors in the stimulatory effect of crotoxin on macrophages co-cultivated with tumour cells.

    PubMed

    Costa, E S; Faiad, O J; Landgraf, R G; Ferreira, A K; Brigatte, P; Curi, R; Cury, Y; Sampaio, S C

    2013-11-01

    Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 μg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1β increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Proteomic analysis of the palmitate-induced myotube secretome reveals involvement of the annexin A1-formyl peptide receptor 2 (FPR2) pathway in insulin resistance.

    PubMed

    Yoon, Jong Hyuk; Kim, Dayea; Jang, Jin-Hyeok; Ghim, Jaewang; Park, Soyeon; Song, Parkyong; Kwon, Yonghoon; Kim, Jaeyoon; Hwang, Daehee; Bae, Yoe-Sik; Suh, Pann-Ghill; Berggren, Per-Olof; Ryu, Sung Ho

    2015-04-01

    Elevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity.

  10. Proteomic Analysis of the Palmitate-induced Myotube Secretome Reveals Involvement of the Annexin A1-Formyl Peptide Receptor 2 (FPR2) Pathway in Insulin Resistance*

    PubMed Central

    Yoon, Jong Hyuk; Kim, Dayea; Jang, Jin-Hyeok; Ghim, Jaewang; Park, Soyeon; Song, Parkyong; Kwon, Yonghoon; Kim, Jaeyoon; Hwang, Daehee; Bae, Yoe-Sik; Suh, Pann-Ghill; Berggren, Per-Olof; Ryu, Sung Ho

    2015-01-01

    Elevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity. PMID:25616869

  11. Involvement of formyl peptide receptors in receptor for advanced glycation end products (RAGE) - and amyloid beta 1-42-induced signal transduction in glial cells

    PubMed Central

    2012-01-01

    Background Recent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role in the inflammatory response involved in neurodegenerative disorders such as Alzheimer’s disease (AD). Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-β 1–42 (Aβ1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains. Results We demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aβ1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy. Conclusions The results suggest that both formyl peptide receptors play an essential role in Aβ1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections. PMID:23164356

  12. Involvement of formyl peptide receptors in receptor for advanced glycation end products (RAGE)--and amyloid beta 1-42-induced signal transduction in glial cells.

    PubMed

    Slowik, Alexander; Merres, Julika; Elfgen, Anne; Jansen, Sandra; Mohr, Fabian; Wruck, Christoph J; Pufe, Thomas; Brandenburg, Lars-Ove

    2012-11-20

    Recent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role in the inflammatory response involved in neurodegenerative disorders such as Alzheimer's disease (AD).Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-β 1-42 (Aβ1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains. We demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aβ1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy. The results suggest that both formyl peptide receptors play an essential role in Aβ1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections.

  13. Oxidized low-density lipoprotein-induced foam cell formation is mediated by formyl peptide receptor 2.

    PubMed

    Lee, Ha Young; Oh, Eunseo; Kim, Sang Doo; Seo, Jeong Kon; Bae, Yoe-Sik

    2014-01-17

    The increased level of LDL and its modification into oxLDL has been regarded as an important risk factor for the development of cardiovascular diseases such as atherosclerosis. Although some scavenger receptors including CD36 and RAGE have been considered as target receptors for oxLDL, involvement of other receptors should be investigated for oxLDL-induced pathological responses. In this study, we found that oxLDL-induced foam cell formation was inhibited by formyl peptide receptor 2 (FPR2) antagonist WRW(4). oxLDL also stimulated calcium signaling and chemotactic migration in FPR2-expressing RBL-2H3 cells but not in vector-expressing RBL-2H3 cells. Moreover, oxLDL stimulated TNF-α production, which was also almost completely inhibited by FPR2 antagonist. Our findings therefore suggest that oxLDL stimulates macrophages, resulting in chemotactic migration, TNF-α production, and foam cell formation via FPR2 signaling, and thus likely contributes to atherogenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Small-molecule-biased formyl peptide receptor agonist compound 17b protects against myocardial ischaemia-reperfusion injury in mice

    PubMed Central

    Qin, Cheng Xue; May, Lauren T.; Li, Renming; Cao, Nga; Rosli, Sarah; Deo, Minh; Alexander, Amy E.; Horlock, Duncan; Bourke, Jane E.; Yang, Yuan H.; Stewart, Alastair G.; Kaye, David M.; Du, Xiao-Jun; Sexton, Patrick M.; Christopoulos, Arthur; Gao, Xiao-Ming; Ritchie, Rebecca H.

    2017-01-01

    Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI. PMID:28169296

  15. Small-molecule-biased formyl peptide receptor agonist compound 17b protects against myocardial ischaemia-reperfusion injury in mice.

    PubMed

    Qin, Cheng Xue; May, Lauren T; Li, Renming; Cao, Nga; Rosli, Sarah; Deo, Minh; Alexander, Amy E; Horlock, Duncan; Bourke, Jane E; Yang, Yuan H; Stewart, Alastair G; Kaye, David M; Du, Xiao-Jun; Sexton, Patrick M; Christopoulos, Arthur; Gao, Xiao-Ming; Ritchie, Rebecca H

    2017-02-07

    Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI.

  16. Annexin A1 contributes to pancreatic cancer cell phenotype, behaviour and metastatic potential independently of Formyl Peptide Receptor pathway

    PubMed Central

    Belvedere, Raffaella; Bizzarro, Valentina; Forte, Giovanni; Dal Piaz, Fabrizio; Parente, Luca; Petrella, Antonello

    2016-01-01

    Annexin A1 (ANXA1) is a Ca2+-binding protein over-expressed in pancreatic cancer (PC). We recently reported that extracellular ANXA1 mediates PC cell motility acting on Formyl Peptide Receptors (FPRs). Here, we describe other mechanisms by which intracellular ANXA1 could mediate PC progression. We obtained ANXA1 Knock-Out (KO) MIA PaCa-2 cells using the CRISPR/Cas9 genome editing technology. LC-MS/MS analysis showed altered expression of several proteins involved in cytoskeletal organization. As a result, ANXA1 KO MIA PaCa-2 partially lost their migratory and invasive capabilities with a mechanism that appeared independent of FPRs. The acquisition of a less aggressive phenotype has been further investigated in vivo. Wild type (WT), PGS (scrambled) and ANXA1 KO MIA PaCa-2 cells were engrafted orthotopically in SCID mice. No differences were found about PC primary mass, conversely liver metastatization appeared particularly reduced in ANXA1 KO MIA PaCa-2 engrafted mice. In summary, we show that intracellular ANXA1 is able to preserve the cytoskeleton integrity and to maintain a malignant phenotype in vitro. The protein has a relevant role in the metastatization process in vivo, as such it appears attractive and suitable as prognostic and therapeutic marker in PC progression. PMID:27412958

  17. Inhibition of formyl peptide receptor 1 reduces the efficacy of anticancer chemotherapy against carcinogen-induced breast cancer

    PubMed Central

    Baracco, Elisa E.; Pietrocola, Federico; Buqué, Aitziber; Bloy, Norma; Senovilla, Laura; Zitvogel, Laurence; Vacchelli, Erika; Kroemer, Guido

    2016-01-01

    ABSTRACT The loss-of-function mutation of formyl peptide receptor 1 (FPR1) has a negative impact on the progression-free and overall survival of breast cancer patients treated with anthracycline-based adjuvant chemotherapy. This effect may be attributed to the fact that chemotherapy-induced antitumor immunity requires FPR1 and that such anticancer immune responses are responsible for the long-term effects of chemotherapy. Here, we investigated the possible contribution of FPR1 to the efficacy of a combination of mitoxantrone (MTX) and cyclophosphamide (CTX) for the treatment of hormone-induced breast cancer. Breast cancer induced by a combination of medroxyprogesterone acetate (MPA) and 7,12-Dimethylbenz[a]anthracene (DMBA) could be successfully treated with MTX plus CTX in thus far that tumor growth was retarded and overall survival was extended (as compared to vehicle-only treated controls). However, the therapeutic efficacy of the combination therapy was completely abolished when FPR1 receptors were blocked by means of cyclosporin H (CsH). Future genetic studies on neoadjuvant chemotherapy-treated breast cancers are warranted to validate these findings at the clinical level. PMID:27471610

  18. Formyl peptide derived lipopeptides disclose differences between the receptors in mouse and men and call the pepducin concept in question

    PubMed Central

    Winther, Malene; Holdfeldt, André; Sundqvist, Martina; Rajabkhani, Zahra; Gabl, Michael; Bylund, Johan; Dahlgren, Claes

    2017-01-01

    A pepducin is a lipopeptide containing a peptide sequence that is identical to one of the intracellular domains of the G-protein coupled receptor (GPCR) assumed to be the target. Neutrophils express two closely related formyl peptide receptors belonging to the family of GPCRs; FPR1 and FPR2 in human and their respective orthologue Fpr1 and Fpr2 in mouse. By applying the pepducin concept, we have earlier identified FPR2 activating pepducins generated from the third intracellular loop of FPR2. The third intracellular loop of FPR2 differs in two amino acids from that of FPR1, seven from Fpr2 and three from Fpr1. Despite this, we found that pepducins generated from FPR1, FPR2, Fpr1 and Fpr2 all targeted FPR2 in human neutrophils and Fpr2 in mouse, but with different modulating outcomes. Whereas the FPR1/Fpr1 derived pepducins inhibited the FPR2 function in human neutrophils, they activated Fpr2 in mouse. The FPR2 derived pepducin activated FPR2/Fpr2, whereas the pepducin generated from Fpr2 inhibited both FPR2 and Fpr2. In summary, our data demonstrate that pepducins generated from the third intracellular loop of human FPR1/2 and mouse Fpr1/2, all targeted FPR2 in human and Fpr2 in mouse. With respect to the modulating outcomes, pepducin inhibitors identified for FPR2 are in fact activators for Fpr2 in mouse neutrophils. Our data thus questions the validity of pepducin concept regarding their receptor selectivity but supports the notion that FPR2/Fpr2 may recognize a lipopeptide molecular pattern, and highlight the differences in ligand recognition profile between FPR2 and its mouse orthologue Fpr2. PMID:28934373

  19. Development of potent antagonists for formyl peptide receptor 1 based on Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH.

    PubMed

    Hayashi, Ryo; Kitajima, Toshiki; Mizuguchi, Hikaru; Fujimoto, Miki; Yamaguchi, Aya; Koga, Shuichiro; Koga, Yuya; Osada, Satoshi; Kodama, Hiroaki

    2014-08-01

    While stimulation of formyl peptide receptors (FPRs) on the surface of human neutrophils induces several immune responses, under conditions of continuous activation of the receptor by agonists such as formyl-Met-Leu-Phe-OH (fMLP), neutrophil-dependent tissue damage ensues. Thus, FPR antagonists could be anticipated as drugs for FPR-related disease. In this study, Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH (Boc-FlFlF), one of several FPR subtype selective antagonists, was chosen and the positions at the Phe residues were optimized. We found that substitution with unnatural amino acids resulted in an improvement of two orders of magnitude. The most potent antagonist indicated FPR subtype selectivity at 1 μM. In addition to finding a potent antagonist, the structure-activity trends observed in this study should be valuable in designing a new type of FPR subtype selective antagonist. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. The N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) increases the lateral diffusion of complement receptor 1 (CR1/CD35) in human neutrophils; a causative role for oxidative metabolites?

    PubMed

    Rasmusson, B J; Carpentier, J L; Paccaud, J P; Magnusson, K E

    1996-10-01

    The effects of the N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) on the lateral mobility of the complement receptor type 1 (CR1/CD35) in glass-adherent human neutrophils were investigated, using fluorescence recovery after photobleaching (FRAP) and confocal microscopy (CSLM). It was found that addition of 0.1-1 microM fMLF increased the diffusion constant (D) of CR1/CD35 to 167-228% of controls. No effect was observed on the receptor distribution or the mobile fraction of receptors. The effect of fMLF on the lateral diffusion of CR1/CD35 could be totally inhibited by addition of pertussis toxon (PD, 250 ng/ml) or of the free radical scavenger enzymes superoxide dismutase (SOD, 2000 U/ml) and catalase (CAT, 200 U/ml), added together the results show that oxidative metabolites produced by neutrophils in response to fMLF can modulate CR1/CD35 diffusion, and indicate a regulatory role for oxygen radicals in phagocytosis.

  1. The Annexin I Sequence Gln9-Ala10-Trp11-Phe12 Is a Core Structure for Interaction with the Formyl Peptide Receptor 1*

    PubMed Central

    Movitz, Charlotta; Brive, Lars; Hellstrand, Kristoffer; Rabiet, Marie-Josèphe; Dahlgren, Claes

    2010-01-01

    The N-terminal part of the calcium-regulated and phospholipid-binding protein annexin AI contains peptide sequences with pro- and anti-inflammatory activities. We have earlier shown that a proinflammatory signal triggered by one of these peptides, Gln9–Lys25, is mediated by FPR1, a member of the formyl peptide receptor family expressed in human neutrophils. To determine the core structure in Gln9–Lys25, smaller peptides were generated, and their capacity to activate neutrophils was determined. A peptide spanning from amino acid Glu14 to Lys25 was inactive, whereas the activity was retained in the Gln9–Tyr20 peptide. Removal of amino acids from the C and N terminus of Gln9–Tyr20 revealed that the first amino acid (Gln9) was of the utmost importance for activity. The core structure that activated the neutrophil NADPH oxidase to release superoxide anions was Gln9-Ala10-Trp11-Phe12. This peptide also inhibited the activity induced by N-formyl-Met-Leu-Phe and WKYMVM. A structural model of the peptide agonist-FPR1 complex suggests that the transmembrane part of the binding pocket of the receptor binds optimally to a tetrapeptide. According to the model and the results presented, the N-terminal amino acid glutamine in Gln9–Phe12 is located close to the bottom of the binding cleft, leaving for steric reasons insufficient space to extend the peptide at the N terminus. The addition of amino acids at the C terminus will not affect binding. The model presented may be helpful in developing specific FPR1 ligands. PMID:20220135

  2. Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1

    PubMed Central

    Liu, Song; Omatsu, Tatsushi; Janka-Junttila, Mirkka; Casolaro, Vincenzo; Reinecker, Hans-Christian; Parent, Carole A.; Fasano, Alessio

    2015-01-01

    Background Gliadin, the immunogenic component within gluten and trigger of celiac disease, is known to induce the production of Interleukin-8, a potent neutrophil-activating and chemoattractant chemokine. We sought to study the involvement of neutrophils in the early immunological changes following gliadin exposure. Methods Utilizing immunofluorescence microscopy and flow cytometry, the redistribution of major tight junction protein, Zonula occludens (ZO)-1, and neutrophil recruitment were assessed in duodenal tissues of gliadin-gavaged C57BL/6 wild-type and Lys-GFP reporter mice, respectively. Intravital microscopy with Lys-GFP mice allowed monitoring of neutrophil recruitment in response to luminal gliadin exposure in real time. In vitro chemotaxis assays were used to study murine and human neutrophil chemotaxis to gliadin, synthetic alpha-gliadin peptides and the neutrophil chemoattractant, fMet-Leu-Phe, in the presence or absence of a specific inhibitor of the fMet-Leu-Phe receptor-1 (FPR1), cyclosporine H. An irrelevant protein, zein, served as a control. Results Redistribution of ZO-1 and an influx of CD11b+Lys6G+ cells in the lamina propria of the small intestine were observed upon oral gavage of gliadin. In vivo intravital microscopy revealed a slowing down of GFP+ cells within the vessels and influx in the mucosal tissue within 2 hours after challenge. In vitro chemotaxis assays showed that gliadin strongly induced neutrophil migration, similar to fMet-Leu-Phe. We identified thirteen synthetic gliadin peptide motifs that induced cell migration. Blocking of FPR1 completely abrogated the fMet-Leu-Phe-, gliadin- and synthetic peptide-induced migration. Conclusions Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process. PMID:26378785

  3. Inflammation and N-formyl peptide receptors mediate the angiogenic activity of human vitreous humour in proliferative diabetic retinopathy.

    PubMed

    Rezzola, Sara; Corsini, Michela; Chiodelli, Paola; Cancarini, Anna; Nawaz, Imtiaz M; Coltrini, Daniela; Mitola, Stefania; Ronca, Roberto; Belleri, Mirella; Lista, Liliana; Rusciano, Dario; De Rosa, Mario; Pavone, Vincenzo; Semeraro, Francesco; Presta, Marco

    2017-04-01

    Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses. Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous. PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45(+) cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay. This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR

  4. Differential neutrophil chemotactic response towards IL-8 and bacterial N-formyl peptides in term newborn infants

    PubMed Central

    Stålhammar, Maria E.; Douhan Håkansson, Lena; Jonzon, Anders; Sindelar, Richard

    2017-01-01

    Background A prerequisite for an effective innate immunity is the migrative ability of neutrophils to respond to inflammatory and infectious agents such as the intermediate interleukin (IL)-8 and the end-target formyl-methionyl-leucyl-phenylalanine (fMLP) chemoattractants. The aim was to study the chemotactic capacity of neutrophils from newborn infants and adults in response to IL-8 and the bacterial peptide fMLP. Methods In the under-agarose cell migration assay, isolated leukocytes from healthy adults and from cord blood of healthy term newborn infants were studied with dose responses towards IL-8 and fMLP. The same number of leukocytes (1 × 105 cells), with the same distribution of neutrophils and monocytes, were analyzed in neonates and adults. Chemotaxis was distinguished from randomly migrating neutrophils, and the neutrophil pattern of migration, i.e. the migration distance and the number of migrating neutrophils per distance, was evaluated. Results In comparison to adults, fewer neutrophils from newborn infants migrated towards IL-8 and for a shorter distance (P < .01, respectively). The number of neutrophils migrating to different gradients of fMLP, the distance they migrated, and the correlation between the number and the distance were the same for neonates and adults. Random migration did not differ in any instance. Conclusion Chemotaxis of neutrophils from newborn infants was as co-ordinated as neutrophils from adults in response to fMLP, whereas the response to IL-8 was reduced. The differential response of neutrophils from neonates to intermediate and end-target chemoattractants could indicate a reduced infectious response. PMID:27690722

  5. Bioactive secondary metabolites of a marine Bacillus sp. inhibit superoxide generation and elastase release in human neutrophils by blocking formyl peptide receptor 1.

    PubMed

    Yang, Shun-Chin; Lin, Chwan-Fwu; Chang, Wen-Yi; Kuo, Jimmy; Huang, Yin-Ting; Chung, Pei-Jen; Hwang, Tsong-Long

    2013-06-03

    It is well known that overwhelming neutrophil activation is closely related to acute and chronic inflammatory injuries. Formyl peptide receptor 1 (FPR1) plays an important role in activation of neutrophils and may represent a potent therapeutic target in inflammatory diseases. In the present study, we demonstrated that IA-LBI07-1 (IA), an extract of bioactive secondary metabolites from a marine Bacillus sp., has anti-inflammatory effects in human neutrophils. IA significantly inhibited superoxide generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated neutrophils, but failed to suppress the cell responses activated by non-FPR1 agonists. IA did not alter superoxide production and elastase activity in cell-free systems. IA also attenuated the downstream signaling from FPR1, such as the Ca2+, MAP kinases and AKT pathways. In addition, IA inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analogue of FMLP, to FPR1 in human neutrophils and FPR1-transfected HEK293 cells. Taken together, these results show that the anti-inflammatory effects of IA in human neutrophils are through the inhibition of FPR1. Also, our data suggest that IA may have therapeutic potential to decrease tissue damage induced by human neutrophils.

  6. Further Studies on 2-Arylacetamide Pyridazin-3(2H)-ones: Design, Synthesis and Evaluation of 4,6-Disubstituted Analogues as Formyl Peptide Receptors (FPRs) Agonists

    PubMed Central

    Giovannoni, Maria Paola; Schepetkin, Igor A.; Cilibrizzi, Agostino; Crocetti, Letizia; Khlebnikov, Andrei I.; Dahlgren, Claes; Graziano, Alessia; Piaz, Vittorio Dal; Kirpotina, Liliya N.; Zerbinati, Serena; Vergelli, Claudia; Quinn, Mark T.

    2013-01-01

    Formyl peptide receptors (FPRs) play an essential role in the regulation of endogenous inflammation and immunity. In the present studies, a large series of pyridazin-3(2H)-one derivatives bearing an arylacetamide chain at position 2 was synthesized and tested for FPR agonist activity. The pyridazin-3(2H)-one ring was confirmed to be an appropriate scaffold to support FPR agonist activity, and its modification at the 4 and 6 positions led to the identification of additional active agonists, which induced intracellular Ca2+ flux in HL-60 cells transfected with either FPR1, FPR2, or FPR3. Seven formyl peptide receptor 1 (FPR1)-specific and several mixed FPR1/FPR2 dual agonists were identified with low micromolar EC50 values. Furthermore, these agonists also activated human neutrophils, inducing intracellular Ca2+ flux and chemotaxis. Finally, molecular docking studies indicated that the most potent pyridazin-3(2H)-ones overlapped in their best docking poses with fMLF and WKYMVM peptides in the FPR1 and FPR2 ligand binding sites, respectively. Thus, pyridazinone-based compounds represent potential lead compounds for further development of selective and/or potent FPR agonists. PMID:23685570

  7. Human formyl peptide receptor ligand binding domain(s). Studies using an improved mutagenesis/expression vector reveal a novel mechanism for the regulation of receptor occupancy.

    PubMed

    Perez, H D; Vilander, L; Andrews, W H; Holmes, R

    1994-09-09

    Recently, we reported the domain requirements for the binding of formyl peptide to its specific receptor. Based on experiments using receptor chimeras, we also postulated an importance for the amino-terminal domain of the receptor in ligand binding (Perez, H. D., Holmes, R., Vilander, L., Adams, R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295). We have begun to perform a detailed analysis of the regions within the formyl peptide receptor involved in ligand binding. To address the importance of the receptor amino-terminal domain, we substituted (or inserted) hydrophilic sequences within the amino-terminal domain, expressed the receptors, and determined their ability to bind ligand. A stretch of nine amino acids next to the initial methionine was identified as crucial for receptor occupancy. A peptide containing such a sequence specifically completed binding of the ligand to the receptor. Alanine screen mutagenesis of the second extracellular domain also identified amino acids involved in ligand binding as well as a disulfide bond (Cys98 to Cys176) crucial for maintaining the binding pocket. These studies provide evidence for a novel mechanism involved in regulation of receptor occupancy. Binding of the ligand induces conformational changes in the receptor that result in the apposition of the amino-terminal domain over the ligand, providing a lid to the binding pocket.

  8. Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

    PubMed

    Honda, S; Campbell, J J; Andrew, D P; Engelhardt, B; Butcher, B A; Warnock, R A; Ye, R D; Butcher, E C

    1994-04-15

    Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

  9. Airway lipoxin A4/formyl peptide receptor 2-lipoxin receptor levels in pediatric patients with severe asthma.

    PubMed

    Gagliardo, Rosalia; Gras, Delphine; La Grutta, Stefania; Chanez, Pascal; Di Sano, Caterina; Albano, Giusy D; Vachier, Isabelle; Montalbano, Angela M; Anzalone, Giulia; Bonanno, Anna; Riccobono, Loredana; Gjomarkaj, Mark; Profita, Mirella

    2016-06-01

    Lipoxins are biologically active eicosanoids with anti-inflammatory properties. Lipoxin A4 (LXA4) signaling blocks asthmatic responses in human and experimental model systems. There is evidence that patients with respiratory diseases, including severe asthma (SA), display defective generation of lipoxin signals despite glucocorticoid therapy. We investigated airway levels of formyl peptide receptor 2-lipoxin receptor (FPR2/ALXR), LXA4, and its counterregulatory compound, leukotriene B4 (LTB4), in patients with childhood asthma. We addressed the potential interplay of the LXA4-FPR2/ALXR axis and glucocorticoids in the resolution of inflammation. We examined LXA4 and LTB4 concentrations in induced sputum supernatants from children with intermittent asthma (IA), children with SA, and healthy control (HC) children. In addition, we investigated FPR2/ALXR expression in induced sputum cells obtained from the study groups. Finally, we evaluated in vitro the molecular interaction between LXA4 and glucocorticoid receptor-based mechanisms. We found that children with SA have decreased LXA4 concentrations in induced sputum supernatants in comparison with children with IA. In contrast to decreases in LXA4 concentrations, LTB4 concentrations were increased in children with asthma independent of severity. LXA4 concentrations negatively correlated with LTB4 concentrations and with exacerbation numbers in children with SA. FPR2/ALXR expression was reduced in induced sputum cells of children with SA compared with that seen in HC subjects and children with IA. Finally, we describe in vitro the existence of crosstalk between LXA4 and glucocorticoid receptor at the cytosolic level mediated by G protein-coupled FPR2/ALXR in peripheral blood granulocytes isolated from HC subjects, children with IA, and children with SA. Our findings provide evidence for defective LXA4 generation and FPR2/ALXR expression that, associated with increased LTB4, might be involved in a reduction in the

  10. Serum amyloid A1 isoforms display different efficacy at Toll-like receptor 2 and formyl peptide receptor 2.

    PubMed

    Chen, Mingjie; Zhou, Huibing; Cheng, Ni; Qian, Feng; Ye, Richard D

    2014-12-01

    Serum amyloid A (SAA) is a major acute-phase protein and a precursor of amyloid A, the deposit of which leads to amyloidosis. Different alleles exist in SAA1, a predominant form of the human SAA gene family. Emerging evidence has shown correlations between these alleles and diseases including familiar Mediterranean fever and amyloidosis. However, it remains unclear how the proteins encoded by these SAA1 alleles act differently. Here we report the characterization of proteins encoded by SAA1.1, SAA1.3, and SAA1.5, in comparison to that encoded by SAA2.2, for their preference of the SAA receptors including formyl peptide receptor 2 (FPR2) and Toll-like receptor 2 (TLR2). SAA1.1 was more efficacious than SAA1.3 and SAA1.5 but equally efficacious to SAA2.2 in calcium mobilization and chemotaxis assays, which measure the activation of the G protein-coupled FPR2. In agreement with this, SAA1.1 and SAA2.2 induced more robust phosphorylation of ERK than SAA1.3 and SAA1.5. Only small differences were observed between the SAA1 proteins tested and SAA2.2 in TLR2-dependent NF-κB luciferase reporter assay. In comparison, SAA1.3 was most effective in stimulating ERK and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from C57BL/10ScN (Tlr4lps-del) mice, we examined the SAA isoforms for their induction of selected pro- and anti-inflammatory cytokines. SAA1.3 was most potent in the induction of TNFα and IL-1rn, whereas SAA1.5 induced robust IL-10 expression. These results show differences of the SAA1 isoforms in their selectivity for the SAA receptors, which may affect their roles in modulating inflammation and immunity. Copyright © 2014 Elsevier GmbH. All rights reserved.

  11. The formyl peptide receptor 1 exerts a tumor suppressor function in human gastric cancer by inhibiting angiogenesis.

    PubMed

    Prevete, N; Liotti, F; Visciano, C; Marone, G; Melillo, R M; de Paulis, A

    2015-07-01

    N-formyl peptide receptors (FPR1, FPR2 and FPR3) are involved in innate immunity, inflammation and cancer. FPR expression, initially described in immune cells, was later observed in non-hematopoietic cell populations and tissues. Several studies suggested a role for FPRs in the progression of various tumor histotypes, including gastric cancer (GC), for which a positive association with a specific FPR1 polymorphism has recently been described. We previously showed that FPRs are expressed on gastric epithelium and are required for wound repair and restitution of barrier integrity. Here we assess the role of FPRs in GC. We characterized the functions of FPRs in GC epithelial cells (MKN28, AGS and MKN45) cultured in vitro by assessing migration, proliferation, resistance to apoptosis and activation of the epithelial-to-mesenchymal transition. Activation of each FPR induced the epithelial-to-mesenchymal transition, proliferation, resistance to apoptosis and migration of GC cells in culture. Blocking compounds or RNA interference of each FPR reverted these effects. We also defined the in vivo tumorigenic potential of GC epithelial cells silenced for FPRs by xenograft experiments in immunocompromised mice. Interestingly, FPR1 silencing in GC cells (shFPR1) significantly enhanced xenograft growth with respect to shCTR, shFPR2 and shFPR3 xenografts, because of augmented vessel density and cell proliferation. Accordingly, HIF-1α and VEGF mRNA levels were higher in shFPR1 xenografts than in controls. Moreover, the in vitro production of proangiogenic factors in response to FPR2/3 agonists (WKYMVm, LL-37, uPA, uPAR84-95, AnxA1) or to other proinflammatory mediators (IL-1α) was higher in shFPR1 GC cells than in shCTR, shFPR2 and shFPR3 cells, suggesting that FPR1 functions as an inhibitor of CG angiogenesis. Thus, we propose that FPR1 stimulation may represent a novel therapeutic approach to counteract tumor angiogenesis.

  12. Simultaneous mobilization of Mac-1 (CD11b/CD18) and formyl peptide chemoattractant receptors in human neutrophils.

    PubMed

    Graves, V; Gabig, T; McCarthy, L; Strour, E F; Leemhuis, T; English, D

    1992-08-01

    Mobilization of a distinct subset of specific granules provides a physiologically important mechanism to recruit Mac-1 (CD11b/CD18) from an intracellular pool to the external surface of the neutrophil plasma membrane, where the functionally active heterodimer mediates several adherence-dependent processes that are crucial for adequate host defense and cellular inflammatory responses. We observed similar characteristics for translocation of Mac-1 and neutrophil formyl peptide receptors (FPR) and hypothesize that the readily accessible pools of both Mac-1 and FPR are colocalized within this specific granule subset. Plasma membrane levels of both FPR (assessed with 3H-FMLP) and Mac-1 (assessed by fluorescence-activated cell sorter analysis of fluorescein isothiocyanate [FITC]-Mo-1-labeled cells) were markedly downregulated in cells prepared at low temperature from blood cooled to 4 degrees C immediately after removal from the circulation. Levels of both FPR and Mac-1 remained low on cells held at 4 degrees C. Upon warming, spontaneous upregulation of Mac-1 and FPR occurred with similar kinetics and temperature dependency. Translocation of both Mac-1 and FPR was markedly potentiated by exposure of cells to either fluoride ion (which has been shown by others to specifically elicit exocytosis of gelatinase-rich and vitamin B-12 binding protein-poor granules) or granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that markedly potentiates the neutrophils' host defense capabilities. Levels of both FPR and Mac-1 on F-- or GM-CSF-treated neutrophils exceeded those present on cells incubated at 37 degrees C for extended time intervals, indicating that stimulated translocation may involve mobilization of an additional granule subset. Scatchard analysis showed that only low-affinity FPR were translocated during spontaneous and stimulus-dependent upregulation. To directly compare FPR levels on the surface of cells displaying varying levels of Mac-1 within a

  13. Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1

    PubMed Central

    Douthwaite, Julie A; Sridharan, Sudharsan; Huntington, Catherine; Hammersley, Jayne; Marwood, Rose; Hakulinen, Jonna K; Ek, Margareta; Sjögren, Tove; Rider, David; Privezentzev, Cyril; Seaman, Jonathan C; Cariuk, Peter; Knights, Vikki; Young, Joyce; Wilkinson, Trevor; Sleeman, Matthew; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2015-01-01

    Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs. PMID:25484051

  14. Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1.

    PubMed

    Douthwaite, Julie A; Sridharan, Sudharsan; Huntington, Catherine; Hammersley, Jayne; Marwood, Rose; Hakulinen, Jonna K; Ek, Margareta; Sjögren, Tove; Rider, David; Privezentzev, Cyril; Seaman, Jonathan C; Cariuk, Peter; Knights, Vikki; Young, Joyce; Wilkinson, Trevor; Sleeman, Matthew; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2015-01-01

    Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.

  15. Functional N-Formyl Peptide Receptor 2 (FPR2) Antagonists Based on Ureidopropanamide Scaffold Have Potential to Protect Against Inflammation-associated Oxidative Stress.

    PubMed

    Stama, Madia Letizia; Lacivita, Enza; Kirpotina, Liliya N; Niso, Mauro; Perrone, Roberto; Schepetkin, Igor A; Quinn, Mark T; Leopoldo, Marcello

    2017-09-18

    Formyl peptide receptor-2 (FPR2) is a G protein-coupled receptor belonging to the N-formyl peptide receptor (FPR) family that plays critical roles in peripheral and brain inflammatory responses. FPR2 has been proposed as a target for the development of drugs that could facilitate the resolution of chronic inflammatory reactions by enhancing endogenous anti-inflammation systems. Starting from the structure of the FPR2 agonists (R)- and (S)-4 and 2, we designed a new series of ureidopropanamide derivatives with the goal of converting functional activity from agonism to antagonism and to develop new FPR2 antagonists. Although none of the compounds behaved as antagonist, some of the compounds were able to induce receptor desensitization, thus functionally behaving as antagonists. Evaluation of these compounds in an in vitro model of neuroinflammation showed that they reduced reactive oxygen species (ROS) production in mouse microglial N9 cells after stimulation with lipopolysaccharide (LPS). These FPR2 ligands may protect cells from damage due to inflammation-associated oxidative stress. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Novel 3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]propanamides as selective agonists of human formyl-peptide receptor 2.

    PubMed

    Lacivita, Enza; Schepetkin, Igor A; Stama, Madia L; Kirpotina, Liliya N; Colabufo, Nicola A; Perrone, Roberto; Khlebnikov, Andrei I; Quinn, Mark T; Leopoldo, Marcello

    2015-07-15

    N-Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. We here report the synthesis and biological evaluation of six pairs of chiral ureidopropanamido derivatives as potent and selective formyl peptide receptor-2 (FPR2) agonists that were designed starting from our lead agonist (S)-3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]propanamide ((S)-9a). The new compounds were obtained in overall yields considerably higher than (S)-9a. Several of the new compounds showed agonist properties comparable to that of (S)-9a along with higher selectivity over FPR1. Molecular modeling was used to define chiral recognition by FPR2. In vitro metabolic stability of selected compounds was also assessed to obtain preliminary insight on drug-like properties of this class of compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. 6-Methyl-2,4-Disubstituted Pyridazin-3(2H)-ones: A Novel Class of Small-Molecule Agonists for Formyl Peptide Receptors

    PubMed Central

    Cilibrizzi, Agostino; Quinn, Mark T.; Kirpotina, Liliya N.; Schepetkin, Igor A; Holderness, Jeff; Ye, Richard D.; Rabiet, Marie-Josephe; Biancalani, Claudio; Cesari, Nicoletta; Graziano, Alessia; Vergelli, Claudia; Pieretti, Stefano; Piaz, Vittorio Dal; Giovannoni, Maria Paola

    2010-01-01

    Following a ligand-based drug design approach, a potent mixed formyl peptide receptor 1 (FPR1) and formyl peptide receptor-like 1 (FPRL1) agonist (14a) and a potent and specific FPRL1 agonist (14x) were identified. These compounds belong to a large series of pyridazin-3(2H)-one derivatives substituted with a methyl group at position 6 and a methoxy benzyl at position 4. At position 2, an acetamide side chain is essential for activity. Likewise, the presence of lipophilic and/or electronegative substituents in the position para to the aryl group at the end of the chain plays a critical role for activity. Affinity for FPR1 receptors was evaluated by measuring intracellular calcium flux in HL-60 cells transfected with FPR1, FPRL1, and FPRL2. Agonists were able to activate intracellular calcium mobilization and chemotaxis in human neutrophils. The most potent chemotactic agent (EC50 = 0.6 μM) was the mixed FPR/FPRL1 agonist 14h. PMID:19639995

  18. Novel 3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]propanamides as Selective Agonists of Human Formyl-Peptide Receptor 2

    PubMed Central

    Lacivita, Enza; Schepetkin, Igor A.; Stama, Madia L.; Kirpotina, Liliya N.; Colabufo, Nicola A.; Perrone, Roberto; Khlebnikov, Andrei I.; Quinn, Mark T.; Leopoldo, Marcello

    2014-01-01

    N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. We here report the synthesis and biological evaluation of six pairs of chiral ureidopropanamido derivatives as potent and selective formyl peptide receptor-2 (FPR2) agonists, that were designed starting from our lead agonist (S )-3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N -[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]propanamide ((S)-9a). The new compounds were obtained in overall yields considerably higher than (S)-9a. Various of the new compounds showed agonist properties comparable to that of (S)-9a along with higher selectivity over FPR1. Molecular modeling was used to define chiral recognition by FPR2. In vitro metabolic stability of selected compounds was also assessed to obtain preliminary insight on drug-like properties of this class of compounds. PMID:25549897

  19. Influence of ionization on the conformational preferences of peptide models. Ramachandran surfaces of N-formyl-glycine amide and N-formyl-alanine amide radical cations.

    PubMed

    Gil, Adrià; Sodupe, Mariona; Bertran, Juan

    2009-09-01

    Ramachandran maps of neutral and ionized HCO-Gly-NH2 and HCO-Ala-NH2 peptide models have been built at the B3LYP/6-31++G(d,p) level of calculation. Direct optimizations using B3LYP and the recently developed MPWB1K functional have also been carried out, as well as single-point calculations at the CCSD(T) level of theory with the 6-311++G(2df,2p) basis set. Results indicate that for both peptide models ionization can cause drastic changes in the shape of the PES in such a way that highly disallowed regions in neutral PES become low-energy regions in the radical cation surface. The structures localized in such regions, epsilonL+* and epsilonD+* are highly stabilized due to the formation of 2-centre-3-electron interactions between the two carbonyl oxygens. Inclusion of solvent effects by the conductor-like polarizable continuum model (CPCM) shows that the solute-solvent interaction energy plays an important role in determining the stability order. Copyright 2008 Wiley Periodicals, Inc.

  20. Tumour necrosis factor (TNF)-α primes murine neutrophils when triggered via formyl peptide receptor-related sequence 2, the murine orthologue of human formyl peptide receptor-like 1, through a process involving the type I TNF receptor and subcellular granule mobilization

    PubMed Central

    Önnheim, Karin; Bylund, Johan; Boulay, Francois; Dahlgren, Claes; Forsman, Huamei

    2008-01-01

    Neutrophil granulocytes play an important role in innate host defence against microbial invasions and they are also the key effector cells in mediating host tissue damage. These functions often rely on the production of reactive oxygen species (ROS) from the membrane-bound NADPH-oxidase system. The magnitude of ROS production varies depending on the state of the cells, i.e. resting or primed. Many priming agents as well as potent NADPH-oxidase activators have been identified and characterized for human neutrophils. The cytokine tumour necrosis factor (TNF)-α is one prominent example of a priming agent and the synthetic hexapeptide WKYMVm is an agonist that triggers an activation of the NADPH-oxidase of human neutrophils through two members of the formyl peptide family of receptors, formyl peptide receptor (FPR) and FPR-like 1 (FPRL1). This peptide also activates murine neutrophils but the precise receptor involved has not been previously characterized. We show in this study that WKYMVm activates stably transfected HL60 cells expressing murine formyl peptide receptor-related sequence 2 (Fpr-rs2) and that activation of murine neutrophils with WKYMVm is blocked by an FPRL1-specific antagonist. WKYMVm is thus an agonist for Fpr-rs2 and we suggest that this receptor is in fact the mouse orthologue of FPRL1. In addition, we show that the WKYMVm response in murine neutrophils can be primed by TNF-α and this priming process involves mobilization of subcellular granules. The results obtained using neutrophils derived from TNF receptor type I (TNFRI)-deficient animals suggest that TNF-α exerts its priming effect via the TNFRI. PMID:18710405

  1. V101L of human formyl peptide receptor 1 (FPR1) increases receptor affinity and augments the antagonism mediated by cyclosporins

    PubMed Central

    Zhou, Caihong; Zhou, Yan; Wang, Jia; Feng, Yang; Wang, Haonan; Xue, Jinglun; Chen, Yani; Ye, Richard D.; Wang, Ming-Wei

    2013-01-01

    Genetic variation plays a major role in drug response variability. CsA (cyclosporin A), a widely used immunosuppressive agent, is a specific antagonist for FPR1 (formyl peptide receptor 1), which is an important G-protein-coupled chemoattractant receptor in the innate immune system. In order to study the variable responses of cyclosporins to different FPR1 mutants, we investigated the distribution of human FPR1 haplotypes among 209 healthy Han Chinese subjects. The haplotype pattern in Han Chinese were characterized on the basis of five SNPs (single nucleotide polymorphisms), including rs5030878 (p.T11I), rs2070745 (p.V101L), rs5030880 (p.R190W), rs1042229 (p.N192K) and rs867228 (p.A346E). Receptor binding affinity of cyclosporins to FPR1 haplotypes was assessed using N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys–FITC in CHO-Gα16 cells stably transfected with cDNAs encoding the top 12 FPR1 haplotypes in the Han Chinese. Variants of FPR1 carrying a single amino acid substitution of leucine for valine at position 101 (p.Leu101) displayed significantly higher pKi values for CsA and CsH (cyclosporin H), indicative of an improved receptor affinity. The polymorphism of FPR1 p.Leu101 also enhanced the inhibitory effects of cyclosporins on fMLF (N-formyl-methionyl-leucyl-phenylalanine)-induced activities, including calcium mobilization, cell chemotaxis and MAPK (mitogen-activated protein kinase) phosphorylation. These results point to a possible complication for clinical use of CsA in patients carrying the p.Leu101 allele of FPR1. PMID:23373827

  2. V101L of human formyl peptide receptor 1 (FPR1) increases receptor affinity and augments the antagonism mediated by cyclosporins.

    PubMed

    Zhou, Caihong; Zhou, Yan; Wang, Jia; Feng, Yang; Wang, Haonan; Xue, Jinglun; Chen, Yani; Ye, Richard D; Wang, Ming-Wei

    2013-04-15

    Genetic variation plays a major role in drug response variability. CsA (cyclosporin A), a widely used immunosuppressive agent, is a specific antagonist for FPR1 (formyl peptide receptor 1), which is an important G-protein-coupled chemoattractant receptor in the innate immune system. In order to study the variable responses of cyclosporins to different FPR1 mutants, we investigated the distribution of human FPR1 haplotypes among 209 healthy Han Chinese subjects. The haplotype pattern in Han Chinese were characterized on the basis of five SNPs (single nucleotide polymorphisms), including rs5030878 (p.T11I), rs2070745 (p.V101L), rs5030880 (p.R190W), rs1042229 (p.N192K) and rs867228 (p.A346E). Receptor binding affinity of cyclosporins to FPR1 haplotypes was assessed using N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-FITC in CHO-G(α16) cells stably transfected with cDNAs encoding the top 12 FPR1 haplotypes in the Han Chinese. Variants of FPR1 carrying a single amino acid substitution of leucine for valine at position 101 (p.Leu(101)) displayed significantly higher pK(i) values for CsA and CsH (cyclosporin H), indicative of an improved receptor affinity. The polymorphism of FPR1 p.Leu(101) also enhanced the inhibitory effects of cyclosporins on fMLF (N-formyl-methionyl-leucyl-phenylalanine)-induced activities, including calcium mobilization, cell chemotaxis and MAPK (mitogen-activated protein kinase) phosphorylation. These results point to a possible complication for clinical use of CsA in patients carrying the p.Leu(101) allele of FPR1.

  3. Gastrin-Releasing Peptide/Neuromedin B Receptor Antagonists PD176252, PD168368, and Related Analogs Are Potent Agonists of Human Formyl-Peptide ReceptorsS⃞

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Jutila, Mark A.

    2011-01-01

    N-Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) involved in host defense and sensing cellular dysfunction. Thus, FPRs represent important therapeutic targets. In the present studies, we screened 32 ligands (agonists and antagonists) of unrelated GPCRs for their ability to induce intracellular Ca2+ mobilization in human neutrophils and HL-60 cells transfected with human FPR1, FPR2, or FPR3. Screening of these compounds demonstrated that antagonists of gastrin-releasing peptide/neuromedin B receptors (BB1/BB2) PD168368 [(S)-a-methyl-a-[[[(4-nitrophenyl)amino]carbonyl]amino]-N-[[1-(2-pyridinyl) cyclohexyl]methyl]-1H-indole-3-propanamide] and PD176252 [(S)-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]-a-methyl-a-[[-(4-nitrophenyl)amino]carbonyl]amino-1H-indole-3-propanamide] were potent mixed FPR1/FPR2 agonists, with nanomolar EC50 values. Cholecystokinin-1 receptor agonist A-71623 [Boc-Trp-Lys(ε-N-2-methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH2] was also a mixed FPR1/FPR2 agonist, but with a micromolar EC50. Screening of 56 Trp- and Phe-based PD176252/PD168368 analogs and 41 related nonpeptide/nonpeptoid analogs revealed 22 additional FPR agonists. Most were potent mixed FPR1/FPR2/FPR3 agonists with nanomolar EC50 values for FPR2, making them among the most potent nonpeptide FPR2 agonists reported to date. In addition, these agonists were also potent chemoattractants for murine and human neutrophils and activated reactive oxygen species production in human neutrophils. Molecular modeling of the selected agonists using field point methods allowed us to modify our previously reported pharmacophore model for the FPR2 ligand binding site. This model suggests the existence of three hydrophobic/aromatic subpockets and several binding poses of FPR2 agonists in the transmembrane region of this receptor. These studies demonstrate that FPR agonists could include ligands of unrelated GPCR and that analysis of such compounds can enhance our

  4. A novel receptor cross-talk between the ATP receptor P2Y2 and formyl peptide receptors reactivates desensitized neutrophils to produce superoxide.

    PubMed

    Önnheim, Karin; Christenson, Karin; Gabl, Michael; Burbiel, Joachim C; Müller, Christa E; Oprea, Tudor I; Bylund, Johan; Dahlgren, Claes; Forsman, Huamei

    2014-04-15

    Neutrophils express several G-protein coupled receptors (GPCRs) and they cross regulate each other. We described a novel cross-talk mechanism in neutrophils, by which signals generated by the receptor for ATP (P2Y2) reactivate desensitized formyl peptide receptors (FPRs) so that these ligand-bound inactive FPRs resume signaling. At the signaling level, the cross-talk was unidirectional, i.e., P2Y2 ligation reactivated FPR, but not vice versa and was sensitive to the phosphatase inhibitor calyculinA. Further, we show that the cross talk between P2Y2 and FPR bypassed cytosolic Ca(2+) transients and did not rely on the actin cytoskeleton. In summary, our data demonstrate a novel cross-talk mechanism that results in reactivation of desensitized FPRs and, an amplification of the neutrophil response to ATP. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Synthesis, HPLC Enantioresolution, and X-ray Analysis of a New Series of C5-methyl Pyridazines as N-Formyl Peptide Receptor (FPR) Agonists

    PubMed Central

    Cilibrizzi, Agostino; Crocetti, Letizia; Giovannoni, Maria Paola; Graziano, Alessia; Vergelli, Claudia; Bartolucci, Gianluca; Soldani, Giacomo; Quinn, Mark T.; Schepetkin, Igor A.; Faggi, Cristina

    2015-01-01

    The synthesis of three racemates and the corresponding non chiral analogues of a C5-methyl pyridazine series is described here, as well as the isolation of pure enantiomers and their absolute configuration assignment. In order to obtain optically active compounds, direct chromatographic methods of separation by HPLC-UV were investigated using four chiral stationary phases (CSPs: Lux Amylose-2®, Lux Cellulose-1®, Lux Cellulose-2® and Lux Cellulose-3®). The best resolution was achieved using amylose tris(5-chloro-2-methylphenylcarbamate) (Lux Amylose-2®), and single enantiomers were isolated on a semipreparative scale with high enantiomeric excess, suitable for biological assays. The absolute configuration of optically active compounds was unequivocally established by X-ray crystallographic analysis and comparative chiral HPLC-UV profile. All compounds of the series were tested for formyl peptide receptor (FPR) agonist activity, and four were found to be active, with EC50 values in the micromolar range. PMID:23744588

  6. Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor.

    PubMed

    Werner, Kristin; Kälble, Solveig; Wolter, Sabine; Schneider, Erich H; Buschauer, Armin; Neumann, Detlef; Seifert, Roland

    2015-10-01

    The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

  7. Inhibition of neutrophil migration in mice by mouse formyl peptide receptors 1 and 2 dual agonist: indication of cross-desensitization in vivo

    PubMed Central

    Sogawa, Yoshitaka; Ohyama, Takao; Maeda, Hiroaki; Hirahara, Kazuki

    2011-01-01

    It has been reported that the stimulation of neutrophils with N-formyl-Met-Leu-Phe (fMLF), an agonist for formyl peptide receptor (Fpr) 1, renders cells unresponsive to other chemoattractants in vitro. This is known as cross-desensitization, but its functional relevance in neutrophil migration in vivo has not been investigated. Here, we show that precedent stimulation of mouse neutrophils with compound 43, a non-peptidyl agonist for mouse Fpr1 and Fpr2, rendered the cells unresponsive to a second stimulation with C5a, leukotriene B4, or keratinocyte-derived cytokine (KC) in calcium mobilization and chemotaxis assays in vitro. The expression of chemokine (C-X-C motif) receptor 2 (CXCR2) on the surface of neutrophils was concomitantly diminished by stimulating the cells with the compound. Moreover, oral administration of the compound to mice before they were exposed to lipopolysaccharide (LPS) aerosol resulted in a dose-dependent reduction in the neutrophil count in bronchoalveolar lavage fluid. The expression of CXCR2 on blood neutrophils was also reduced in the compound-administered mice. The recipient mice that underwent adoptive transfer of fluorescence-labelled neutrophils that had been incubated with the compound showed a substantial decrease in neutrophil counts in bronchoalveolar lavage fluid after they were exposed to LPS, when compared with the control mice to which vehicle-treated neutrophils had been transferred. These results are consistent with the idea that the agonist for Fpr1 and Fpr2 induced cross-desensitization in neutrophils and attenuated neutrophil migration into the airways. Our results also revealed the unpredicted effect of an Fpr1 and Fpr2 dual agonist, which may act as a functional antagonist for multiple chemoattractant receptors in vivo. PMID:21039475

  8. Lack of formyl peptide receptor 1 and 2 leads to more severe inflammation and higher mortality in mice with of pneumococcal meningitis

    PubMed Central

    Oldekamp, Sandra; Pscheidl, Sebastian; Kress, Eugenia; Soehnlein, Oliver; Jansen, Sandra; Pufe, Thomas; Wang, Ji Ming; Tauber, Simone C; Brandenburg, Lars-Ove

    2014-01-01

    Bacterial meningitis is, despite progress in research and the development of new treatment strategies, still a cause of severe neuronal sequelae. The brain is protected from penetrating pathogens by both the blood–brain barrier and the innate immune system. The invading pathogens are recognized by pattern recognition receptors including the G-protein coupled formyl peptide receptors (FPRs), which are expressed by immune cells of the central nervous system. The expression of FPRs is up-regulated during bacterial meningitis, but the consequence on the progression of inflammation and impact on mortality are far from clear. Therefore, we used mFPR1 and mFPR2-deficient mice to investigate the effects on inflammation, bacterial growth and mortality in a mouse model of pneumococcal meningitis. Our results revealed increased bacterial burden, increased neutrophil infiltration and higher mortality in mFPR1/2-deficient mice in comparison to wild-type mice. The mFPR1- or mFPR2-deficient mice also showed significantly increased glial cell density, whereas the immune responses including the expression of anti-inflammatory cytokines and antimicrobial peptides were decreased in bacterial meningitis. Taken together, the results suggest that FPR1 and FPR2 play an important role in the innate immune responses against Streptococcus pneumoniae within the central nervous system and the lack of the receptors leads to a dysregulation of the inflammatory response compared with wild-type mice. PMID:24863484

  9. Lack of formyl peptide receptor 1 and 2 leads to more severe inflammation and higher mortality in mice with of pneumococcal meningitis.

    PubMed

    Oldekamp, Sandra; Pscheidl, Sebastian; Kress, Eugenia; Soehnlein, Oliver; Jansen, Sandra; Pufe, Thomas; Wang, Ji Ming; Tauber, Simone C; Brandenburg, Lars-Ove

    2014-11-01

    Bacterial meningitis is, despite progress in research and the development of new treatment strategies, still a cause of severe neuronal sequelae. The brain is protected from penetrating pathogens by both the blood-brain barrier and the innate immune system. The invading pathogens are recognized by pattern recognition receptors including the G-protein coupled formyl peptide receptors (FPRs), which are expressed by immune cells of the central nervous system. The expression of FPRs is up-regulated during bacterial meningitis, but the consequence on the progression of inflammation and impact on mortality are far from clear. Therefore, we used mFPR1 and mFPR2-deficient mice to investigate the effects on inflammation, bacterial growth and mortality in a mouse model of pneumococcal meningitis. Our results revealed increased bacterial burden, increased neutrophil infiltration and higher mortality in mFPR1/2-deficient mice in comparison to wild-type mice. The mFPR1- or mFPR2-deficient mice also showed significantly increased glial cell density, whereas the immune responses including the expression of anti-inflammatory cytokines and antimicrobial peptides were decreased in bacterial meningitis. Taken together, the results suggest that FPR1 and FPR2 play an important role in the innate immune responses against Streptococcus pneumoniae within the central nervous system and the lack of the receptors leads to a dysregulation of the inflammatory response compared with wild-type mice. © 2014 John Wiley & Sons Ltd.

  10. The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.

    PubMed

    Skovbakke, Sarah Line; Heegaard, Peter M H; Larsen, Camilla J; Franzyk, Henrik; Forsman, Huamei; Dahlgren, Claes

    2015-01-15

    Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Leucine Leucine-37 Uses Formyl Peptide Receptor–Like 1 to Activate Signal Transduction Pathways, Stimulate Oncogenic Gene Expression, and Enhance the Invasiveness of Ovarian Cancer Cells

    PubMed Central

    Coffelt, Seth B.; Tomchuck, Suzanne L.; Zwezdaryk, Kevin J.; Danka, Elizabeth S.; Scandurro, Aline B.

    2009-01-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor–like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein–coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37–induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37–stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37–treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1. PMID:19491199

  12. Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells.

    PubMed

    Coffelt, Seth B; Tomchuck, Suzanne L; Zwezdaryk, Kevin J; Danka, Elizabeth S; Scandurro, Aline B

    2009-06-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1.

  13. Interplay between signaling via the formyl peptide receptor (FPR) and chemokine receptor 3 (CCR3) in human eosinophils.

    PubMed

    Svensson, Lena; Redvall, Elin; Johnsson, Marianne; Stenfeldt, Anna-Lena; Dahlgren, Claes; Wennerås, Christine

    2009-08-01

    Eosinophils express the chemoattractant receptors CCR3 and FPR. CCR3 binds several agonists such as eotaxin-1, -2, and -3 and RANTES, whereas the FPR binds the formylated tripeptide fMLP and a host of other ligands. The aim of this study was to investigate if there is interplay between these two receptors regarding the elicitation of migration and respiratory burst in human blood-derived eosinophils. Inhibition of the FPR with the antagonists CyH and boc-MLP abrogated the migration of eosinophils toward all of the CCR3 agonists. Similar results were seen when the FPR was desensitized with its cognate ligand, fMLP. In contrast, the respiratory burst triggered by eotaxin-1 was not inhibited by CyH. Thus, signals evoked via the FPR caused unidirectional down-regulation of CCR3-mediated chemotaxis but not respiratory burst in human eosinophils. The underlying mechanism was neither reduced ability of the CCR3 ligand eotaxin-1 to bind to CCR3 nor down-regulation of CCR3 from the cell surface. Finally, confocal microscopy and adFRET analysis ruled out homo- or heterodimer formation between FPR and/or CCR3 as an explanation for the reduction in chemotaxis via CCR3. Pharmacologic inhibition of signal transduction molecules showed that the release of free oxygen radicals in response to eotaxin-1 compared with fMLP is relatively more dependent on the p38 MAPK pathway.

  14. FAM19A4 is a novel cytokine ligand of formyl peptide receptor 1 (FPR1) and is able to promote the migration and phagocytosis of macrophages

    PubMed Central

    Wang, Wenyan; Li, Ting; Wang, Xiaolin; Yuan, Wanxiong; Cheng, Yingying; Zhang, Heyu; Xu, Enquan; Zhang, Yingmei; Shi, Shuang; Ma, Dalong; Han, Wenling

    2015-01-01

    FAM19A4 is an abbreviation for family with sequence similarity 19 (chemokine (C–C motif)-like) member A4, which is a secretory protein expressed in low levels in normal tissues. The biological functions of FAM19A4 remain to be determined, and its potential receptor(s) is unclarified. In this study, we demonstrated that FAM19A4 was a classical secretory protein and we verified for the first time that its mature protein is composed of 95 amino acids. We found that the expression of this novel cytokine was upregulated in lipopolysaccharide (LPS)-stimulated monocytes and macrophages and was typically in polarized M1. FAM19A4 shows chemotactic activities on macrophages and enhances the macrophage phagocytosis of zymosan both in vitro and in vivo with noticeable increases of the phosphorylation of protein kinase B (Akt). FAM19A4 can also increase the release of reactive oxygen species (ROS) upon zymosan stimulation. Furthermore, based on receptor internalization, radio ligand binding assays and receptor blockage, we demonstrated for the first time that FAM19A4 is a novel ligand of formyl peptide receptor 1 (FPR1). The above data indicate that upon inflammatory stimulation, monocyte/macrophage-derived FAM19A4 may play a crucial role in the migration and activation of macrophages during pathogenic infections. PMID:25109685

  15. Annexin A1 released from apoptotic cells acts through formyl peptide receptors to dampen inflammatory monocyte activation via JAK/STAT/SOCS signalling

    PubMed Central

    Pupjalis, Danute; Goetsch, Julia; Kottas, Diane J; Gerke, Volker; Rescher, Ursula

    2011-01-01

    The immunosuppressive effects of apoptotic cells involve inhibition of pro-inflammatory cytokine release and establishment of an anti-inflammatory cytokine profile, thus limiting the degree of inflammation and promoting resolution. We report here that this is in part mediated by the release of the anti-inflammatory mediator annexin A1 from apoptotic cells and the functional activation of annexin A1 receptors of the formyl peptide receptor (FPR) family on target cells. Supernatants from apoptotic neutrophils or the annexin A1 peptidomimetic Ac2-26 significantly reduced IL-6 signalling and the release of TNF-α from endotoxin-challenged monocytes. Ac2-26 activated STAT3 in a JAK-dependent manner, resulting in upregulated SOCS3 levels, and depletion of SOCS3 reversed the Ac2-26-mediated inhibition of IL-6 signalling. This identifies annexin A1 as part of the anti-inflammatory pattern of apoptotic cells and links the activation of FPRs to established signalling pathways triggering anti-inflammatory responses. PMID:21254404

  16. Evaluation of cell-free expression system for the production of soluble and functional human GPCR N-formyl peptide receptors.

    PubMed

    Wang, Xiaoqiang; Wang, Jiqian; Ge, Baosheng

    2013-11-01

    Human N-formyl peptide receptors (FPRs) belong to the G protein-coupled receptors (GPCRs) superfamily, the most frequently addressed drug targets in the pharmaceutical industry, and are considered to play important roles in innate immunity and host defense mechanisms. Although still a highly challenging task, the availability of soluble and functional GPCRs including FPRs in milligram quantities is essential to spur the advancement of protein-based structural and functional studies for drug discovery. In this report, the applicability of E. coli extracts-based cell-free expression system to producing soluble and active human FPRs and hence to FPRs protein-based research was evaluated, during which human FPR3 was selected as our prototype receptor. To better solubilize the freshly expressed human FPR3, a panel of different detergents (mostly nonionic detergents) were selected and evaluated in the cell-free system devoid of natural membrane. After one-step immunoaffinity purification, the secondary structure and biological function of purified FPR3 were characterized. A final yield of 0.6 mg functional human FPR3 per ml reaction volume was obtained. The demonstrated proper folding and functionality of the cell-free produced human FPR3 opens a new avenue for the fast and efficient generation of human FPRs (and even other GPCRs) for structural and functional analysis.

  17. Cutting Edge: LL-37-Mediated Formyl Peptide Receptor-2 Signaling in Follicular Dendritic Cells Contributes to B Cell Activation in Peyer's Patch Germinal Centers.

    PubMed

    Kim, Sae-Hae; Kim, Yu Na; Jang, Yong-Suk

    2017-01-15

    Peyer's patches (PPs) are the major mucosal immune-inductive site, and germinal centers (GCs) in PPs determine the quality of the Abs produced. PP GCs are continuously induced by the gut microbiota, and their maintenance contributes to the induction of strong IgA responses to Ags. In this study, we investigated the role of formyl peptide receptor (FPR)-mediated signaling in the maintenance of PP GCs, because FPRs recognize the microbiota and initiate an innate immune response by chemotaxis. We found that follicular dendritic cells (FDCs), a key organizer of B cell follicles and GCs in mucosal immunity, express Fpr2. Additionally, Fpr2-mediated signaling in PP FDCs promoted Cxcl13 and B cell activating factor expression, as well as B cell proliferation and activation. Therefore, we suggest that Fpr2-mediated signaling in FDCs plays a key role in GC maintenance in PPs and results in an Ag-specific IgA response in the gut mucosal immune compartment. Copyright © 2017 by The American Association of Immunologists, Inc.

  18. Targeting the annexin 1-formyl peptide receptor 2/ALX pathway affords protection against bacterial LPS-induced pathologic changes in the murine adrenal cortex.

    PubMed

    Buss, Nicholas A P S; Gavins, Felicity N E; Cover, Patricia O; Terron, Andrea; Buckingham, Julia C

    2015-07-01

    Hypothalamo-pituitary-adrenocortical dysfunction contributes to morbidity and mortality in a high proportion of patients with sepsis. Here, we provide new insights into the underlying adrenal pathology. Using a murine model of endotoxemia (LPS injection), we demonstrate that adrenal insufficiency is triggered early in the disease. LPS induced a local inflammatory response in the adrenal gland within 4 hours of administration, coupled with increased expression of mRNAs for annexin A1 (AnxA1) and the formyl peptide receptors [(Fprs) 1, 2, and 3], a loss of lipid droplets in cortical cells (index of availability of cholesterol, the substrate for steroidogenesis), and a failure to mount a steroidogenic response to ACTH. Deletion of AnxA1 or Fpr2/3 in mice prevented lipid droplet loss, but not leukocyte infiltration. LPS increased adrenal myeloid differentiation primary response gene 88 and TLR2 mRNA expression, but not lymphocyte antigen 96 or TLR4. By contrast, neutrophil depletion prevented leukocyte infiltration and increased AnxA1, Fpr1, and Fpr3 mRNAs but had no impact on lipid droplet loss. Our novel data demonstrate that AnxA1 and Fpr2 have a critical role in the manifestation of adrenal insufficiency in this model, through regulation of cholesterol ester storage, suggesting that pharmacologic interventions targeting the AnxA1/FPR/ALX pathway may provide a new approach for the maintenance of adrenal steroidogenesis in sepsis. © FASEB.

  19. Inhibition of U-87 human glioblastoma cell proliferation and formyl peptide receptor function by oligomer procyanidins (F2) isolated from grape seeds.

    PubMed

    Zhang, Feng-Jiao; Yang, Jing-Yu; Mou, Yan-Hua; Sun, Bao-Shan; Ping, Yi-Fang; Wang, Ji-Ming; Bian, Xiu-Wu; Wu, Chun-Fu

    2009-05-15

    Gliomas are the most common and lethal tumor type in the brain. The present study investigated the effect of oligomer procyanidins (F2) (F2, degree of polymerization 2-15), a natural fraction isolated from grape seeds on the biological behavior of glioblastoma cells. We found that F2 significantly inhibited the glioblastoma growth, with little cytotoxicity on normal cells, induced G2/M arrest and decreased mitochondrial membrane potential in U-87 cells. It also induced a non-apoptotic cell death phenotype resembling paraptosis in U-87 cells. In addition, it was found for the first time that F2 in non-cytotoxic concentrations selectively inhibited U-87 cell chemotaxis mediated by a G-protein coupled receptor formyl peptide receptor FPR, which is implicated in tumor cell invasion and metastasis. Further experiments indicated that F2 inhibited fMLF-induced U-87 cell calcium mobilization and MAP kinases ERK1/2 phosphorylation. Moreover, F2 attenuated the glioblastoma FPR expression, a new molecular target for glioma therapeutics, which has been shown to play important roles in glioma cells chemotaxis, proliferation and angiogenesis in addition to its promotion to tumor progression, but did not affect FPR mRNA expression in U-87 cells. Taken together, our results suggest that F2 may be a promising candidate for the development of novel anti-tumor therapeutics.

  20. Bacterial N-formyl Peptides Reduce PMA- and E.coli-induced Neutrophil Respiratory Burst in Term Neonates and Adults.

    PubMed

    Stålhammar, Maria E; Douhan Håkansson, Lena; Sindelar, Richard

    2017-02-15

    Neutrophil migration and respiratory burst is the prerequisite for efficient first line defense against invading microorganisms. However, migration and respiratory burst can be compromised in adults and especially in newborn infants, where sustained neutrophil accumulation, uncontrolled burst and reduced scavenging of ROS might cause inadvertent tissue damage due to uncontrolled inflammation. The aim of this study was to investigate the modulatory effect of the chemoattractants formyl-methionyl-leucyl-phenylalanine (fMLP) and IL-8 on respiratory burst in neutrophils from term newborn infants and adults. Whole blood from the umbilical cord of 17 healthy term newborn infants delivered by caesarean section and from 17 healthy adults as reference was preincubated with fMLP or IL-8 and stimulated with PMA or E.coli bacteria. Respiratory burst was quantified by flow cytometry analysis of dihydrorhodamine 123 fluorescence. fMLP reduced the PMA-induced respiratory burst of neutrophils from newborn infants and adults by 12% and 21% respectively (p<0.05). E.coli-induced burst was also reduced by fMLP in neutrophils from newborn infants (10%; p<0.01) and adults (6%; p<0.05). No such changes were observed with IL-8. Similar respiratory burst in response to single stimulus with PMA or E.coli were observed in both newborn infants and adults. fMLP reduced PMA- and E.coli-induced respiratory burst of neutrophils in whole blood from term newborn infants as well as in adults. The reduced respiratory burst by fMLP might be a mechanism to reduce the detrimental effects of uncontrolled inflammation during neutrophil migration. This article is protected by copyright. All rights reserved.

  1. Deficiency of formyl peptide receptor 1 and 2 is associated with increased inflammation and enhanced liver injury after LPS-stimulation.

    PubMed

    Giebeler, Arne; Streetz, Konrad L; Soehnlein, Oliver; Neumann, Ulf; Wang, Ji Ming; Brandenburg, Lars-Ove

    2014-01-01

    Formyl peptide-receptor 1 and 2 (FPR1 and FPR2) in mice were identified as receptors with contrary affinity for the PAMP fMLF. Formyl-methionyl-leucyl-phenylalanine is either part of the bacterial membrane and is secreted by the mitochondria of eukaryotic ceslls during apoptosis. Furthermore FPR1 and 2 are described as highly relevant factors for the chemotaxis of immune cells. Their role during the acute liver injury has not been investigated yet. Constitutive knockout mice for FPR1 (mFPR1-/-), FPR2 (mFPR2-/-) and wild type (WT) mice were challenged with LPS i.p. for 3 h and 6 h. Liver and serum were sampled for further analysis. Liver transaminases were elevated in all mice 3 h and 6 h post LPS stimulation. Gene expression analysis displayed a reduced expression of the pro-inflammatory cytokines IL-6 and CXCL1 after 3 h in the mFPR1-/- compared to wild type and mFPR2-/- mice. After 6 h, IL-6, TNF-α and CXCL1 were significantly higher in mice lacking mFPR1 or 2. Consistent to these findings the numbers of CD11b+ and Ly6G+ immune cells were altered in the livers. The analysis of TLR2 and TLR4 revealed time and genotype specific changes in theirs gene expression. Additionally, the liver in mFPR1- and mFPR2-deficient mice seem to be more susceptible to apoptosis by showing a significant higher number of TUNEL+-cells in the liver than WT-mice and displayed less Ki67-positive nuclei in the liver. The results suggest a prominent role of FPRs in the regulation of the hepatic inflammatory response after LPS induced liver injury. Deletion of mFPR1 or mFPR2 leads to deregulation of the inflammatory response compared to WT mice, associated with more severe liver injury represented by higher levels of transaminases, apoptotic cells and a reduced regenerative capacity.

  2. WKYMVm-induced activation of formyl peptide receptor 2 stimulates ischemic neovasculogenesis by promoting homing of endothelial colony-forming cells.

    PubMed

    Heo, Soon Chul; Kwon, Yang Woo; Jang, Il Ho; Jeong, Geun Ok; Yoon, Jung Won; Kim, Chi Dae; Kwon, Sang Mo; Bae, Yoe-Sik; Kim, Jae Ho

    2014-03-01

    Endothelial colony-forming cells (ECFCs) are recruited to the sites of ischemic injury in order to contribute to neovascularization and repair of injured tissues. However, therapeutic potential of ECFCs is limited due to low homing and engraftment efficiency of transplanted ECFCs. The G-protein-coupled formyl peptide receptor (FPR) 2 has been implicated in regulation of inflammation and angiogenesis, while the role of FPR2 in homing and engraftment of ECFCs and neovascularization in ischemic tissues has not been fully defined. This study was undertaken to investigate the effects of WKYMVm, a selective FPR2 agonist isolated by screening synthetic peptide libraries, on homing ability of ECFCs and vascular regeneration of ischemic tissues. WKYMVm stimulated chemotactic migration, angiogenesis, and proliferation ability of human ECFCs in vitro. Small interfering RNA-mediated silencing of FPR2, but not FPR3, abrogated WKYMVm-induced migration and angiogenesis of ECFCs. Intramuscular injection of WKYMVm resulted in attenuation of severe hind limb ischemia and promoted neovascularization in ischemic limb. ECFCs transplanted via tail vein into nude mice were incorporated into capillary vessels in the ischemic hind limb, resulting in augmented neovascularization and improved ischemic limb salvage. Intramuscular injection of WKYMVm promoted homing of exogenously administered ECFCs to the ischemic limb and ECFC-mediated vascular regeneration. Silencing of FPR2 expression in ECFCs resulted in abrogation of WKYMVm-induced in vivo homing of exogenously transplanted ECFCs to the ischemic limb, neovascularization, and ischemic limb salvage. These results suggest that WKYMVm promotes repair of ischemic tissues by stimulating homing of ECFCs and neovascularization via a FPR2-dependent mechanism. © AlphaMed Press.

  3. Formyl-Peptide Receptor 2/3/Lipoxin A4 Receptor Regulates Neutrophil-Platelet Aggregation and Attenuates Cerebral Inflammation: Impact for Therapy in Cardiovascular Disease.

    PubMed

    Vital, Shantel A; Becker, Felix; Holloway, Paul M; Russell, Janice; Perretti, Mauro; Granger, D Neil; Gavins, Felicity N E

    2016-05-31

    Platelet activation at sites of vascular injury is essential for hemostasis, but it is also a major pathomechanism underlying ischemic injury. Because anti-inflammatory therapies limit thrombosis and antithrombotic therapies reduce vascular inflammation, we tested the therapeutic potential of 2 proresolving endogenous mediators, annexin A1 N-terminal derived peptide (AnxA1Ac2-26) and aspirin-triggered lipoxin A4 (15-epi-lipoxin A4), on the cerebral microcirculation after ischemia/reperfusion injury. Furthermore, we tested whether the lipoxin A4 receptor formyl-peptide receptor 2/3 (Fpr2/3; ortholog to human FPR2/lipoxin A4 receptor) evoked neuroprotective functions after cerebral ischemia/reperfusion injury. Using intravital microscopy, we found that cerebral ischemia/reperfusion injury was accompanied by neutrophil and platelet activation and neutrophil-platelet aggregate formation within cerebral microvessels. Moreover, aspirin-triggered lipoxin A4 activation of neutrophil Fpr2/3 regulated neutrophil-platelet aggregate formation in the brain and inhibited the reactivity of the cerebral microvasculature. The same results were obtained with AnxA1Ac2-26 administration. Blocking Fpr2/lipoxin A4 receptor with the antagonist Boc2 reversed this effect, and treatments were ineffective in Fpr2/3 knockout mice, which displayed an exacerbated disease severity, evidenced by increased infarct area, blood-brain barrier dysfunction, increased neurological score, and elevated levels of cytokines. Furthermore, aspirin treatment significantly reduced cerebral leukocyte recruitment and increased endogenous levels of aspirin-triggered lipoxin A4, effects again mediated by Fpr2/3. Fpr2/lipoxin A4 receptor is a therapeutic target for initiating endogenous proresolving, anti-inflammatory pathways after cerebral ischemia/reperfusion injury. © 2016 American Heart Association, Inc.

  4. Spasmogenic activity of chemotactic N-formylated oligopeptides: identity of structure--function relationships for chemotactic and spasmogenic activities.

    PubMed

    Marasco, W A; Fantone, J C; Ward, P A

    1982-12-01

    The chemotactic N-formylated oligopeptides are potent spasmogenic agents for guinea pig ileum. Structure-activity studies with various N-formylated peptides suggest the presence of a specific receptor that resembles in specificity the formyl peptide receptor on leukocytes. A competitive antagonist of the formyl peptide receptor on leukocytes also inhibits formyl peptide-induced ileum contraction, whereas the antihistamine diphenhydramine is without effect. The contractile response caused by the synthetic N-formylated peptides differs from those induced by acetylcholine, histamine, and substance P. In particular, a latent period after treatment with the N-formyl peptides is seen before the onset of the response, and a sustained contractile response is not maintained. In addition, tachyphylaxis does occur, but complete recovery of activity is seen after a 20- to 30-min rest period. These observations suggest broad biological roles of prokaryotic signal peptides from bacteria as acute inflammatory mediators.

  5. Annexin 1 exerts anti-nociceptive effects after peripheral inflammatory pain through formyl-peptide-receptor-like 1 in rat dorsal root ganglion.

    PubMed

    Pei, L; Zhang, J; Zhao, F; Su, T; Wei, H; Tian, J; Li, M; Shi, J

    2011-12-01

    Annexin 1 (ANXA1) has analgesic effects in inflammatory pain. We aimed to investigate the anti-nociceptive role of ANXA1, at the dorsal root ganglion (DRG) level, through an interaction with formyl-peptide-receptor-like 1 (FPR2/ALX). Inflammatory pain was evoked by injecting complete Freund's adjuvant (CFA, 50 μl) into the hindpaw of male Sprague-Dawley rats. The distribution of ANXA1 and FPR2/ALX in L4/5 DRGs was evaluated by immunofluorescence. The expression of ANXA1 was measured by western blot. The involvement of FPR2/ALX in the anti-nociception of ANXA1 was investigated by thermal (irradiant heat) and mechanical (von Frey filament) pain tests with intrathecal (i.t.) ANXA1-derived peptide (Anxa1(2-26)), FPR2/ALX agonist 5(S)-6(R)-7-trihydroxyheptanoic-acid-methyl-ester (BML-111), and antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe (Boc1). ANXA1 and FPR2/ALX localized in the satellite glial cells and neurones in L4/5 DRGs. CFA treatment (n=20) increased ANXA1 expression in L4/5 DRGs within 7 days (P<0.01). I.T. Anxa1(2-26) (20 and 100 µg µl(-1)) and BML-111 (10 and 100 nmol) reduced CFA-induced thermal and mechanical nociception within 48 h (n=40) (P<0.05). However, i.t. Boc1 10 µg intensified inflammatory pain (P<0.05) and reversed the anti-nociceptive effect of Anxa1(2-26) (n=25) (P<0.05). Moreover, ANXA1 expression increased in L4/5 DRGs after i.t. Anxa1(2-26) (20 µg µl(-1)) (P<0.05) and BML-111 (10 nmol) (P<0.01) but decreased after i.t. Boc1 (10 and 100 µg) alone (P<0.01) or Boc1 (10 µg) co-injection with Anxa1(2-26) (20 µg µl(-1)) (P<0.05). Endogenous ANXA1 expression at the DRG level is involved in CFA-induced inflammatory pain, and i.t. ANXA1 20 µg µl(-1) produces its anti-nociceptive effect through FPR2/ALX.

  6. The Role of Water in Activation Mechanism of Human N-Formyl Peptide Receptor 1 (FPR1) Based on Molecular Dynamics Simulations

    PubMed Central

    Yuan, Shuguang; Ghoshdastider, Umesh; Trzaskowski, Bartosz; Latek, Dorota; Debinski, Aleksander; Pulawski, Wojciech; Wu, Rongliang; Gerke, Volker; Filipek, Slawomir

    2012-01-01

    The Formyl Peptide Receptor 1 (FPR1) is an important chemotaxis receptor involved in various aspects of host defense and inflammatory processes. We constructed a model of FPR1 using as a novel template the chemokine receptor CXCR4 from the same branch of the phylogenetic tree of G-protein-coupled receptors. The previously employed template of rhodopsin contained a bulge at the extracellular part of TM2 which directly influenced binding of ligands. We also conducted molecular dynamics (MD) simulations of FPR1 in the apo form as well as in a form complexed with the agonist fMLF and the antagonist tBocMLF in the model membrane. During all MD simulation of the fMLF-FPR1 complex a water molecule transiently bridged the hydrogen bond between W2546.48 and N1083.35 in the middle of the receptor. We also observed a change in the cytoplasmic part of FPR1 of a rotamer of the Y3017.53 residue (tyrosine rotamer switch). This effect facilitated movement of more water molecules toward the receptor center. Such rotamer of Y3017.53 was not observed in any crystal structures of GPCRs which can suggest that this state is temporarily formed to pass the water molecules during the activation process. The presence of a distance between agonist and residues R2015.38 and R2055.42 on helix TM5 may suggest that the activation of FPR1 is similar to the activation of β-adrenergic receptors since their agonists are separated from serine residues on helix TM5. The removal of water molecules bridging these interactions in FPR1 can result in shrinking of the binding site during activation similarly to the shrinking observed in β-ARs. The number of GPCR crystal structures with agonists is still scarce so the designing of new ligands with agonistic properties is hampered, therefore homology modeling and docking can provide suitable models. Additionally, the MD simulations can be beneficial to outline the mechanisms of receptor activation and the agonist/antagonist sensing. PMID:23189124

  7. [Formylation of porphyrin platinum complexes].

    PubMed

    Rumiantseva, V D; Konovalenko, L I; Nagaeva, E A; Mironov, A F

    2005-01-01

    The formylation reaction of platinum complexes of beta-unsubstituted porphyrins was studied. The interaction of deuteroporphyrin IX derivatives with the Vilsmeyer reagent led to the selective formylation of their macrocycles in the beta position. The resulting formyl derivatives of the porphyrins are of interest for fluorescent immunoassay.

  8. T21/DP107, A synthetic leucine zipper-like domain of the HIV-1 envelope gp41, attracts and activates human phagocytes by using G-protein-coupled formyl peptide receptors.

    PubMed

    Su, S B; Gao, J l; Gong, W h; Dunlop, N M; Murphy, P M; Oppenheim, J J; Wang, J M

    1999-05-15

    A leucine zipper-like domain, T21/DP107, located in the amino terminus of the ectodomain of gp41, is crucial to the formation of fusogenic configuration of the HIV-1 envelope protein gp41. We report that the synthetic T21/DP107 segment is a potent stimulant of migration and calcium mobilization in human monocytes and neutrophils. The activity of T21/DP107 on phagocytes was pertussis toxin-sensitive, suggesting this peptide uses Gi-coupled seven-transmembrane receptor(s). Since the bacterial chemotactic peptide fMLP partially desensitized the calcium-mobilizing activity of T21/DP107 in phagocytes, we postulated that T21/DP107 might preferentially use a lower affinity fMLP receptor. By using cells transfected to express cloned prototype chemotactic N-formyl peptide receptor (FPR) or its variant, FPR-like 1 (FPRL1), we demonstrate that T21/DP107 activates both receptors but has a much higher efficacy for FPRL1. In addition, T21/DP107 at nM concentrations induced migration of FPRL1-transfected human embryonic kidney 293 cells. In contrast, fMLP did not induce significant chemotaxis of the same cells at a concentration as high as 50 microM. Although a lipid metabolite, lipoxin A4, was a high-affinity ligand for FPRL1, it was not reported to induce Ca2+ mobilization or chemotaxis in FPRL1-transfected cells. Therefore, T21/DP107 is a first chemotactic peptide agonist identified thus far for FPRL1. Our results suggest that this peptide domain of the HIV-1 gp41 may have the potential to activate host innate immune response by interacting with FPR and FPRL1 on phagocytes.

  9. Serum amyloid A1α induces paracrine IL-8/CXCL8 via TLR2 and directly synergizes with this chemokine via CXCR2 and formyl peptide receptor 2 to recruit neutrophils.

    PubMed

    De Buck, Mieke; Berghmans, Nele; Pörtner, Noëmie; Vanbrabant, Lotte; Cockx, Maaike; Struyf, Sofie; Opdenakker, Ghislain; Proost, Paul; Van Damme, Jo; Gouwy, Mieke

    2015-12-01

    Cell migration depends on the ability of leukocytes to sense an external gradient of chemotactic proteins produced during inflammation. These proteins include chemokines, complement factors, and some acute phase proteins, such as serum amyloid A. Serum amyloid A chemoattracts neutrophils, monocytes, and T lymphocytes via its G protein-coupled receptor formyl peptide receptor 2. We demonstrate that serum amyloid A1α more potently chemoattracts neutrophils in vivo than in vitro. In contrast to CD14(+) monocytes, no rapid (within 2 h) induction of interleukin-8/CXC chemokine ligand 8 or macrophage-inflammatory protein-1α/CC chemokine ligand 3 was observed in purified human neutrophils after stimulation of the cells with serum amyloid A1α or lipopolysaccharide. Moreover, interleukin-8/CXC chemokine ligand 8 induction in monocytes by serum amyloid A1α was mediated by toll-like receptor 2 and was inhibited by association of serum amyloid A1α with high density lipoprotein. This indicates that the potent chemotactic response of neutrophils toward intraperitoneally injected serum amyloid A1α is indirectly enhanced by rapid induction of chemokines in peritoneal cells, synergizing in a paracrine manner with serum amyloid A1α. We observed direct synergy between IL-8/CXC chemokine ligand 8 and serum amyloid A1α, but not lipopolysaccharide, in chemotaxis and shape change assays with neutrophils. Furthermore, the selective CXC chemokine receptor 2 and formyl peptide receptor 2 antagonists, SB225002 and WRW4, respectively, blocked the synergy between IL-8/CXC chemokine ligand 8 and serum amyloid A1α in neutrophil chemotaxis in vitro, indicating that for synergy their corresponding G protein-coupled receptors are required. Additionally, SB225002 significantly inhibited serum amyloid A1α-mediated peritoneal neutrophil influx. Taken together, endogenous (e.g., IL-1β) and exogenous (e.g., lipopolysaccharide) inflammatory mediators induce primary chemoattractants such as

  10. The Lipidated Peptidomimetic Lau-((S)-Aoc)-(Lys-βNphe)6-NH2 Is a Novel Formyl Peptide Receptor 2 Agonist That Activates Both Human and Mouse Neutrophil NADPH Oxidase.

    PubMed

    Holdfeldt, André; Skovbakke, Sarah Line; Winther, Malene; Gabl, Michael; Nielsen, Christina; Perez-Gassol, Iris; Larsen, Camilla Josephine; Wang, Ji Ming; Karlsson, Anna; Dahlgren, Claes; Forsman, Huamei; Franzyk, Henrik

    2016-09-16

    Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists for this receptor display potent activity for the human receptor (FPR2) but low activity for the mouse receptor orthologue (Fpr2), rendering them inapplicable in murine models of human disease. Here we describe a novel FPR2 agonist, the proteolytically stable α-peptide/β-peptoid hybrid Lau-((S)-Aoc)-(Lys-βNphe)6-NH2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca(2+) concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated with lauric acid (C12 fatty acid), which is linked to a peptide/peptoid repeat ((Lys-βNphe)6-NH2). Both the fatty acid moiety and the (S)-Aoc residue were required for FPR2/Fpr2 activation. This type of proteolytically stable FPR2-specific peptidomimetics may serve as valuable tools for future analysis of FPR2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Helicobacter pylori HP(2-20) induces eosinophil activation and accumulation in superficial gastric mucosa and stimulates VEGF-alpha and TGF-beta release by interacting with formyl-peptide receptors.

    PubMed

    Prevete, N; Rossi, F W; Rivellese, F; Lamacchia, D; Pelosi, C; Lobasso, A; Necchi, V; Solcia, E; Fiocca, R; Ceppa, P; Staibano, S; Mascolo, M; D'Argenio, G; Romano, M; Ricci, V; Marone, G; De Paulis, A

    2013-01-01

    Eosinophils participate in the immune response against Helicobacter pylori, but little is known about their role in the gastritis associated to the infection. We recently demonstrated that the Hp(2-20) peptide derived from H. pylori accelerates wound healing of gastric mucosa by interacting with N-formyl peptide receptors (FPRs) expressed on gastric epithelial cells. The aim of the present study was to investigate whether eosinophils play a role in the repair of gastric mucosa tissue during H. pylori infection. Immuno-histochemistry and transmission electron microscopy were used to detect eosinophils in gastric mucosal biopsies. Eosinophil re-distribution occurred in the gastric mucosa of H. pylori-infected patients: their density did not change in the deep mucosal layer, whereas it increased in the superficial lamina propria just below the foveolar epithelium; eosinophils entered the epithelium itself as well as the lumen of foveolae located close to the area harboring bacteria, which in turn were also engulfed by eosinophils. The H. pylori-derived peptide Hp(2-20) stimulated eosinophil migration through the engagement of FPR2 and FPR3, and also induced production of VEGF-A and TGF-beta, two key mediators of tissue remodelling. We also demonstrate that Hp(2-20) in vivo induced eosinophil infiltration in rat gastric mucosa after injury brought about by indomethacin. This study suggests that eosinophil infiltrate could modulate the capacity of gastric mucosa to maintain or recover its integrity thereby shedding light on the role of eosinophils in H. pylori infection.

  12. Aspirin and (or) omega-3 polyunsaturated fatty acids protect against corticohippocampal neurodegeneration and downregulate lipoxin A4 production and formyl peptide receptor-like 1 expression in pentylenetetrazole-kindled rats.

    PubMed

    Abd-Elghafour, Basma A; El-Sayed, Norhan M; Ahmed, Amal A M; Zaitone, Sawsan A; Moustafa, Yasser M

    2016-11-10

    There is evidence for a relationship between inflammation and seizures because epilepsy can be caused by or result in inflammation. This study aimed to investigate the effect of aspirin and (or) omega-3 polyunsaturated fatty acids (PUFAs) on seizure activity and neurodegeneration in pentylenetetrazole (PTZ)-kindled rats focusing on their effect on corticohippocampal production of lipoxin A4 (LXA4) and expression of formyl peptide receptor-like 1 (FPRL1) receptors. Male rats were injected with PTZ (35 mg/kg, i.p.) 3 times per week for a total of 15 doses. Rats were treated daily with aspirin (20 mg/kg, i.p.), omega-3 PUFAs (85 mg/kg, p.o.), or a combination of them for 35 days. Both LXA4 level and expression of FPRL1 receptor in the cortices and hippocampi of rats' brains were greater in PTZ-kindled rats compared to a saline control group. Cotreatment with aspirin and (or) omega-3 PUFAs reduced convulsive behaviour; reduced levels of LXA4, interleukin-1β, and nuclear factor-κB; and showed a lower percentage of corticohippocampal degenerative cells compared to PTZ-kindled rats. The combination of the 2 therapeutic agents did not provide significant improvement in comparison with the monotherapies. These findings suggest the use of aspirin or omega-3 PUFAs may delay the development of seizures and provide neuroprotection in a clinical setting.

  13. Reactivation of Gαi-coupled formyl peptide receptors is inhibited by Gαq-selective inhibitors when induced by signals generated by the platelet-activating factor receptor.

    PubMed

    Holdfeldt, André; Dahlstrand Rudin, Agnes; Gabl, Michael; Rajabkhani, Zahra; König, Gabriele M; Kostenis, Evi; Dahlgren, Claes; Forsman, Huamei

    2017-09-01

    Formyl peptide receptor (FPR)-desensitized neutrophils display increased production/release of superoxide (O2(-)) when activated by platelet-activating factor (PAF), a priming of the response achieved through a unique receptor crosstalk mechanism. The aim of this study was to determine the effect of an inhibitor selective for small, heterotrimeric G proteins belonging to the Gαq subclass on that receptor crosstalk. We show that signals generated by FPRs and the PAF receptor (PAFR) induce activation of the neutrophil O2(-), producing NADPH-oxidase, and that response was sensitive to Gαq inhibition in cells activated by PAF, but no inhibition was obtained in cells activated by FPR agonists. Signaling in naive neutrophils is terminated fairly rapidly, and the receptors become homologously desensitized. The downstream sensitivity to Gαq inhibition in desensitized cells displaying increased production/release of O2(-) through the PAFR receptor crosstalk mechanism also comprised the reactivation of the FPRs, and the activation signals were redirected from the PAFR to the desensitized/reactivated FPRs. The Gαq-dependent activation signals generated by the PAFRs activate the Gαi-coupled FPRs, a receptor crosstalk that represents a novel pathway by which G protein-coupled receptors can be regulated and signaling can be turned on and off. © Society for Leukocyte Biology.

  14. Synthesis, enantioresolution, and activity profile of chiral 6-methyl-2,4-disubs-tituted pyridazin-3(2H)-ones as potent N-formyl peptide receptor agonists

    PubMed Central

    Cilibrizzi, Agostino; Schepetkin, Igor A.; Bartolucci, Gianluca; Crocetti, Letizia; Piaz, Vittorio Dal; Giovannoni, Maria Paola; Graziano, Alessia; Kirpotina, Liliya N.; Quinn, Mark T.; Vergelli, Claudia

    2012-01-01

    A series of chiral pyridazin-3(2H)-ones was synthesized, separated as pure enantiomers, and evaluated for N-formyl peptide receptor (FPR) agonist activity. Characterization of the purified enantiomers using combined chiral HPLC and chiroptical studies (circular dichroism, allowed unambiguous assignment of the absolute configuration for each pair of enantiomers). Evaluation of the ability of racemic mixtures and purified enantiomers to stimulate intracellular Ca2+ flux in FPR-transfected HL-60 cells and human neutrophils and to induce β-arrestin recruitment in FPR-transfected CHO-K1 cells showed that many enantiomers were potent agonists, inducing responses in the sub-micromolar to nanomolar range. Furthermore, FPRs exhibited enantiomer selectivity, generally preferring the R-(−)-forms over the S-(+)-enantiomers. Finally, we found that elongation of the carbon chain in the chiral center of the active compounds generally increased biological activity. Thus, these studies provide important new information regarding molecular features involved in FPR ligand preference and report the identification of a novel series of FPR agonists. PMID:22607879

  15. Derivative of wheat germ agglutinin specifically inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis by blocking re-expression (or recycling) of receptors

    SciTech Connect

    Perez, H.D.; Elfman, F.; Lobo, E.; Sklar, L.; Chenoweth, D.; Hooper, C.

    1986-03-01

    The mechanism of action of a derivative of wheat germ agglutinin (WGA-D) which specifically and irreversibly inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte (PMN) chemotaxis was examined. At a concentration that completely inhibited PMN chemotaxis, WGA-D had no effect on either the uptake or release of (/sup 3/H)-FMLP by PMN. Similarly, WGA-D did not affect either the short-term binding to, or internalization by, PMN of a fluoresceinated FMLP analog. WGA-D did interfere, however, with the re-expression (or recycling) of FMLP receptors by PMN that had been preincubated with 1 ..mu..M FMLP for 10 min at 4/sup 0/C. This effect was specific for WGA-D, because it was not observed when concanavalin A was used. Scatchard plot analysis of FMLP binding to PMN after receptor re-expression demonstrated that WGA-D-treated PMN had a significant diminution in the number of high affinity receptors. WGA-D-mediated inhibition of FMLP receptor re-expression was associated with inhibition of FMLP-induced PMN chemotaxis, but had no effect on either FMLP-induced PMN superoxide anion generation or degranulation. Studies using (/sup 12/%I)-WGA-D demonstrated that PMN did not internalize WGA-D spontaneously. The data indicate that WGA-D perhaps by binding to the FMLP receptor, inhibits FMLP-induced PMN chemotaxis by blocking the re-expression (or recycling) of a population of receptors required for continuous migration.

  16. Inhibition of phospholipase D activation by CYL-26z in formyl peptide-stimulated neutrophils involves the blockade of RhoA activation.

    PubMed

    Kuan, Yu-Hsiang; Lin, Ruey-Hseng; Tsao, Lo-Ti; Chen, Yeh-Long; Tzeng, Cherng-Chyi; Wang, Jih-Pyang

    2005-09-15

    5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity, which was assessed by the production of phosphatidylethanol (PEt) in the presence of ethanol, in rat neutrophils (IC50 1.2+/-0.2 microM). CYL-26z caused a slight but significant attenuation of the global protein tyrosine phosphorylation stimulated by fMLP only at concentrations of CYL-26z up to 30 microM. CYL-26z blocked the membrane recruitment of protein kinase C-alpha (PKC-alpha) at concentrations of CYL-26z > or =3 microM, but failed to affect the membrane association of PKC-betaI and -betaII. The translocation of RhoA to the membrane was attenuated by CYL-26z (IC50 3.8+/-0.8 microM) in fMLP-stimulated neutrophils, whereas CYL-26z caused no significant inhibition of the membrane recruitment of ADP-ribosylation factor (Arf). CYL-26z inhibited the activation of RhoA and dissociation of the RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex in fMLP-stimulated neutrophils (IC50 1.8+/-1.0 microM and 1.8+/-0.9 microM, respectively). In a cell-free system, CYL-26z effectively attenuated the membrane association of RhoA in response to GTPgammaS (IC50 1.3+/-0.5 microM). In contrast, the GTPgammaS-stimulated translocation of Arf to membrane was suppressed only at concentrations of CYL-26z up to 30 microM. CYL-26z inhibited the fMLP-stimulated membrane expression of CD11b, CD45 and CD63, and the release of lysozyme and beta-glucuronidase. These results indicate that CYL-26z inhibited the fMLP-stimulated PLD activity, mainly through the blockade of RhoA activation, and degranulation in rat neutrophils.

  17. A derivative of wheat germ agglutinin specifically inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis by blocking re-expression (or recycling) of receptors.

    PubMed

    Perez, H D; Elfman, F; Lobo, E; Sklar, L; Chenoweth, D; Hooper, C

    1986-03-01

    We examined the mechanism of action of a derivative of wheat germ agglutinin (WGA-D) which specifically and irreversibly inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte (PMN) chemotaxis. At a concentration that completely inhibited PMN chemotaxis, WGA-D had no effect on either the uptake or release of [3H]-FMLP by PMN. Similarly, WGA-D did not affect either the short-term binding to, or internalization by, PMN of a fluoresceinated FMLP analog. WGA-D did interfere, however, with the re-expression (or recycling) of FMLP receptors by PMN that had been preincubated with 1 microM FMLP for 10 min at 4 degrees C. This effect was specific for WGA-D, because it was not observed when concanavalin A was used. Scatchard plot analysis of FMLP binding to PMN after receptor re-expression demonstrated that WGA-D-treated PMN had a significant diminution in the number of high affinity receptors. WGA-D-mediated inhibition of FMLP receptor re-expression was associated with inhibition of FMLP-induced PMN chemotaxis, but had no effect on either FMLP-induced PMN superoxide anion generation or degranulation. Studies using [125I]-WGA-D demonstrated that PMN did not internalize WGA-D spontaneously. PMN did internalize [125I]-WGA-D, however, when stimulated with FMLP. Internalization of WGA-D by FMLP-stimulated PMN was rapid, dependent on the concentration of FMLP, and specific. Internalization of [125I]-WGA-D by PMN did not occur when highly purified human C5a, instead of FMLP, was used as a stimulus. Subcellular fractionation studies demonstrated that [125I]-WGA-D and [3H]-FMLP were co-internalized by PMN, and segregated to a compartment co-migrating with Golgi markers. Western blot analysis, using PMN plasma membranes, demonstrated that WGA-D bound to a single membrane glycoprotein that migrated with an apparent m.w. of 62,000. The data indicate that WGA-D, perhaps by binding to the FMLP receptor, inhibits FMLP-induced PMN chemotaxis by blocking

  18. 3-(1H-Indol-3-yl)-2-[3-(4-nitrophenyl)ureido]propanamide Enantiomers With Human Formyl-Peptide Receptor Agonist Activity: Molecular Modeling of Chiral Recognition by FPR2

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Leopoldo, Marcello; Lucente, Ermelinda; Lacivita, Enza; De Giorgio, Paola; Quinn, Mark T.

    2012-01-01

    N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. Recent studies indicated that FPRs have stereo-selective preference for chiral ligands. Here, we investigated the structure-activity relationship of 24 chiral ureidopropanamides, including previously reported compounds PD168368/PD176252 and their close analogs, and used molecular modeling to define chiral recognition by FPR2. Unlike previously reported 6-methyl-2,4-disubstituted pyridazin-3(2H)-ones, whose R-forms preferentially activated FPR1/FPR2, we found that four S-enantiomers in the seven ureidopropanamide pairs tested preferentially activated intracellular Ca2+ flux in FPR2-transfected cells, while the R-counterpart was more active in two enantiomer pairs. Thus, active enantiomers of FPR2 agonists can be in either R- or S- configurations, depending on the molecular scaffold and specific substituents at the chiral center. Using molecular modeling approaches, including field point methodology, homology modeling, and docking studies, we propose a model that can explain stereoselective activity of chiral FPR2 agonists. Importantly, our docking studies of FPR2 chiral agonists correlated well with the FPR2 pharmacophore model derived previously. We conclude that the ability of FPR2 to discriminate between the enantiomers is the consequence of the arrangement of the three asymmetric hydrophobic subpockets at the main orthosteric FPR2 binding site with specific orientation of charged regions in the subpockets. PMID:23219934

  19. 3-(1H-indol-3-yl)-2-[3-(4-nitrophenyl)ureido]propanamide enantiomers with human formyl-peptide receptor agonist activity: molecular modeling of chiral recognition by FPR2.

    PubMed

    Schepetkin, Igor A; Kirpotina, Liliya N; Khlebnikov, Andrei I; Leopoldo, Marcello; Lucente, Ermelinda; Lacivita, Enza; De Giorgio, Paola; Quinn, Mark T

    2013-02-01

    N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. Recent studies indicated that FPRs have stereo-selective preference for chiral ligands. Here, we investigated the structure-activity relationship of 24 chiral ureidopropanamides, including previously reported compounds PD168368/PD176252 and their close analogs, and used molecular modeling to define chiral recognition by FPR2. Unlike previously reported 6-methyl-2,4-disubstituted pyridazin-3(2H)-ones, whose R-forms preferentially activated FPR1/FPR2, we found that four S-enantiomers in the seven ureidopropanamide pairs tested preferentially activated intracellular Ca(2+) flux in FPR2-transfected cells, while the R-counterpart was more active in two enantiomer pairs. Thus, active enantiomers of FPR2 agonists can be in either R- or S-configurations, depending on the molecular scaffold and specific substituents at the chiral center. Using molecular modeling approaches, including field point methodology, homology modeling, and docking studies, we propose a model that can explain stereoselective activity of chiral FPR2 agonists. Importantly, our docking studies of FPR2 chiral agonists correlated well with the FPR2 pharmacophore model derived previously. We conclude that the ability of FPR2 to discriminate between the enantiomers is the consequence of the arrangement of the three asymmetric hydrophobic subpockets at the main orthosteric FPR2 binding site with specific orientation of charged regions in the subpockets.

  20. Signal transduction by the formyl peptide receptor. Studies using chimeric receptors and site-directed mutagenesis define a novel domain for interaction with G-proteins.

    PubMed

    Amatruda, T T; Dragas-Graonic, S; Holmes, R; Perez, H D

    1995-11-24

    The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q. Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16. These results suggest that

  1. The antimicrobial peptide cecropin A induces caspase-independent cell death in human promyelocytic leukemia cells.

    PubMed

    Cerón, José María; Contreras-Moreno, Judit; Puertollano, Elena; de Cienfuegos, Gerardo Álvarez; Puertollano, María A; de Pablo, Manuel A

    2010-08-01

    Most antimicrobial peptides have been shown to have antitumoral activity. Cecropin A, a linear 37-residue antimicrobial polypeptide produced by the cecropia moth, has exhibited cytotoxicity in various human cancer cell lines and inhibitory effects on tumor growth. In this study, we investigated the apoptosis induced by cecropin A in the promyelocytic cell line HL-60. Treatment of cells with cecropin A was characterized by loss of viability in a dose-dependent manner, lactate dehydrogenase (LDH) leakage, and modest attenuation of lysosomal integrity measured by neutral red assay. An increase of reactive oxygen species (ROS) generation, DNA fragmentation, and phosphatidylserine externalization were quantified following cecropin A exposure at a concentration of 30 microM, whereas cecropin A-induced apoptosis was independent of caspase family members, because the activity of caspase-8 and -9 were irrelevant. Nevertheless, caspase-3 activity showed a significant increase at concentrations of 20-40 microM, but a considerable reduction at 50 microM. Flow cytometry analysis revealed a dissipation of the mitochondrial transmembrane potential (Deltapsi(m)), and the accumulation of cells at sub-G1 phase measured by FACS analysis of propidium iodide (PI) stained nuclei suggested induction of apoptosis. Morphological changes measured by Hoechst 33342 or acridine orange/ethidium bromide staining showed nuclear condensation, corroborating the apoptotic action of cecropin A. Overall, these data indicate that cecropin A is able to induce apoptosis in HL-60 cells through a signaling mechanism mediated by ROS, but independently of caspase activation.

  2. Radiosynthesis and in vivo Evaluation of Carbon-11 (2S)-3-(1H-Indol-3-yl)-2-{[(4-methoxyphenyl)carbamoyl]amino}-N-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl}propanamide: An Attempt to Visualize Brain Formyl Peptide Receptors in Mouse Models of Neuroinflammation.

    PubMed

    Lacivita, Enza; Stama, Madia Letizia; Maeda, Jun; Fujinaga, Masayuki; Hatori, Akiko; Zhang, Ming-Rong; Colabufo, Nicola A; Perrone, Roberto; Higuchi, Makoto; Suhara, Tetsuya; Leopoldo, Marcello

    2016-07-01

    Here, we describe the very first attempt to visualize in vivo formyl peptide receptors (FPRs) in mouse brain by positron emission tomography (PET). FPRs are expressed in microglial cells where they mediate chemotactic activity of β-amyloid peptide in Alzheimer disease and, thus, are involved in neuroinflammatory processes. To this purpose, we have selected (2S)-3-(1H-Indol-3-yl)-2-{[(4-methoxyphenyl)carbamoyl]amino}-N-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl}propanamide ((S)-1), that we have previously identified as a potent non-peptidic FPR agonist. (S)-[(11) C]-1 has been prepared in high radiochemical yield. (S)-[(11) C]-1 showed very low penetration of blood-brain barrier and, thus, was unable to accumulate into the brain. In addition, (S)-[(11) C]-1 was not able to label FPRs receptors in brain slices of PS19 and APP23 mice, two animal models of Alzheimer disease. Although (S)-[(11) C]-1 was not suitable to visualize FPRs in the brain, this study provides useful information for the design and characterization of future potential PET radioligands for visualization of brain FPRs by PET. © 2016 Wiley-VHCA AG, Zürich.

  3. Standard Thermodynamic Functions of Tripeptides N-Formyl-l-methionyl-l-leucyl-l-phenylalaninol and N-Formyl-l-methionyl-l-leucyl-l-phenylalanine Methyl Ester

    PubMed Central

    2015-01-01

    The heat capacities of tripeptides N-formyl-l-methionyl-l-leucyl-l-phenylalaninol (N-f-MLF-OH) and N-formyl-l-methionyl-l-leucyl-l-phenylalanine methyl ester (N-f-MLF-OMe) were measured by precision adiabatic vacuum calorimetry over the temperature range from T = (6 to 350) K. The tripeptides were stable over this temperature range, and no phase change, transformation, association, or thermal decomposition was observed. The standard thermodynamic functions: molar heat capacity Cp,m, enthalpy H(T) – H(0), entropy S(T), and Gibbs energy G(T) – H(0) of peptides were calculated over the range from T = (0 to 350) K. The low-temperature (T ≤ 50 K) heat capacities dependencies were analyzed using the Debye’s and the multifractal theories. The standard entropies of formation of peptides at T = 298.15 K were calculated. PMID:24803685

  4. The American cockroach peptide periplanetasin-2 blocks Clostridium Difficile toxin A-induced cell damage and inflammation in the gut.

    PubMed

    Hong, Ji; Zhang, Peng; Yoon, I Na; Hwang, Jae Sam; Kang, Jin Ku; Kim, Ho

    2017-02-07

    Clostridium difficile, which causes pseudomembranous colitis, releases toxin A and toxin B. These toxins are considered to be the main causative agents for the disease pathogenesis, and their expression is associated with a marked increase of apoptosis in mucosal epithelial cells. Colonic epithelial cells are believed to form a physical barrier between the lumen and the submucosa, and abnormally increased mucosal epithelial cell apoptosis is considered to be an initial step in gut inflammation responses. Therefore, one approach to treating pseudomembranous colitis would be to develop agents that block the mucosal epithelial cell apoptosis caused by toxin A, thus restoring barrier function and curing inflammatory responses in the gut. We recently isolated an antimicrobial peptide, Periplanetasin-2 (Peri-2, YPCKLNLKLGKVPFH) from the American cockroach, whose extracts have shown great potential for clinical use. Here, we assessed whether Peri-2 could inhibit the cell toxicities and inflammation caused by C. difficile toxin A. Indeed, in human colonocyte HT29 cells, Peri-2 inhibited the toxin A-induced decrease in cell proliferation and ameliorated the cell apoptosis induced by this toxin. Moreover, in the toxin A-induced mouse enteritis model, Peri-2 blocked the mucosal disruption and inflammatory response caused by toxin A. These results suggest that the American cockroach peptide, Peri-2, could be a possible drug candidate for addressing the pseudomembranous colitis caused by C. difficile toxin A.

  5. Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA-induced human neutrophil activation and migration.

    PubMed

    Heineke, Marieke H; van der Steen, Lydia P E; Korthouwer, Rianne M; Hage, J Joris; Langedijk, Johannes P M; Benschop, Joris J; Bakema, Jantine E; Slootstra, Jerry W; van Egmond, Marjolein

    2017-07-24

    The cross-linking of the IgA Fc receptor (FcαRI) by IgA induces release of the chemoattractant LTB4, thereby recruiting neutrophils in a positive feedback loop. IgA autoantibodies of patients with autoimmune blistering skin diseases therefore induce massive recruitment of neutrophils, resulting in severe tissue damage. To interfere with neutrophil mobilization and reduce disease morbidity, we developed a panel of specific peptides mimicking either IgA or FcαRI sequences. CLIPS technology was used to stabilize three-dimensional structures and to increase peptides' half-life. IgA and FcαRI peptides reduced phagocytosis of IgA-coated beads, as well as IgA-induced ROS production and neutrophil migration in in vitro and ex vivo (human skin) experiments. Since topical application would be the preferential route of administration, Cetomacrogol cream containing an IgA CLIPS peptide was developed. In the presence of a skin permeation enhancer, peptides in this cream were shown to penetrate the skin, while not diffusing systemically. Finally, epitope mapping was used to discover sequences important for binding between IgA and FcαRI. In conclusion, a cream containing IgA or FcαRI peptide mimetics, which block IgA-induced neutrophil activation and migration in the skin may have therapeutic potential for patients with IgA-mediated blistering skin diseases. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The Spectroscopy of the Formyl Radical.

    NASA Astrophysics Data System (ADS)

    Adamson, George William

    Fluorescence-excitation and stimulated emission pumping (SEP) spectroscopies were used to study the properties of photolytically generated, gas phase, formyl radical (HCO). This work focused on the ground doublet electronic state, between 4000-11 200cm^{-1} of energy, and the second excited doublet electronic state, between 38 590-38 750cm^{-1 } of energy. From the fluorescence-excitation spectrum the rotational, centrifugal distortion, and spin -rotational constants were determined for the vibrationless level of the second excited doublet electronic state. From the stimulated emission pumping spectrum vibrational term values, rotational constants, spin-rotational constants, and rotational linewidths were determined for ground electronic state vibrational levels--both above and below the dissociation limit for formyl radical dissociating to hydrogen atom and carbon monoxide. The systematics of the dissociation lifetimes, inferred from the observed rotational linewidths, for rovibrational levels of the ground electronic state were studied. The dissociating lifetimes were found to be strongly correlated with bending vibrational excitation and the amount of angular momentum about the minimum inertial axis of formyl radical. The observed dissociation lifetimes indicates the need for an upward adjustment of the currently accepted dissociation limit and barrier height. (Copies available exclusively from MIT Libraries, Rm. 14-0951, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253 -1690.).

  7. Formyl peptides and ATP stimulate Ca2+ and Na+ inward currents through non-selective cation channels via G-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells. Involvement of Ca2+ and Na+ in the activation of beta-glucuronidase release and superoxide production.

    PubMed

    Krautwurst, D; Seifert, R; Hescheler, J; Schultz, G

    1992-12-15

    In human neutrophils, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces increases in the intracellular free Ca2+ concentration ([Ca2+]i) with subsequent activation of beta-glucuronidase release and superoxide (O2-) production. Results from several laboratories suggest that the increase in [Ca2+]i is due to activation of non-selective cation (NSC) channels. We studied the biophysical characteristics, pharmacological modulation and functional role of NSC channels in dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells. fMLP increased [Ca2+]i by release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. fMLP also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365). Under whole-cell voltage-clamp conditions, fMLP and ATP (a purinoceptor agonist) activated inward currents characterized by a linear current-voltage relationship and a reversal potential near 0 mV. NSC channels were substantially more permeable to Na+ than to Ca2+. SK&F 96365 inhibited fMLP- and ATP-stimulated currents with a half-maximal effect at about 3 microM. Pertussis toxin prevented stimulation by fMLP of NSC currents and reduced ATP-stimulated currents by about 80%. Intracellular application of the stable GDP analogue, guanosine 5'-O-[2-thio]diphosphate, completely blocked stimulation by agonists of NSC currents. In excised inside-out patches, single channel openings with an amplitude of 0.24 pA were observed in the presence of fMLP and the GTP analogue, guanosine 5'-O-[3-thio]triphosphate. The bath solution contained neither Ca2+ nor ATP. The current/voltage relationship was linear with a conductance of 4-5 pS and reversed at about 0 mV. fMLP-induced beta-glucuronidase release and O2- production were substantially reduced by replacement of extracellular CaCl2 or NaCl by ethylenebis(oxyethylenenitrilo)tetra-acetic acid and

  8. The opioid peptide dynorphin A induces leukocyte responses via integrin Mac-1 (αMβ2, CD11b/CD18).

    PubMed

    Podolnikova, Nataly P; Brothwell, Julie A; Ugarova, Tatiana P

    2015-06-03

    Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (αMβ2, CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor. In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages. Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

  9. A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene.

    PubMed

    Cha, Yeseul; Lee, Sang Hoon; Jang, Su Kil; Guo, Haiyu; Ban, Young-Hwan; Park, Dongsun; Jang, Gwi Yeong; Yeon, Sungho; Lee, Jeong-Yong; Choi, Ehn-Kyoung; Joo, Seong Soo; Jeong, Heon-Sang; Kim, Yun-Bae

    2017-01-01

    This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight. Male rats were orally administered with SP-NN (50 or 300mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine.

  10. Acid-labile formylation of amino terminal proline of human immunodeficiency virus type 1 p24(gag) was found by proteomics using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

    PubMed

    Fuchigami, Takashi; Misumi, Shogo; Takamune, Nobutoki; Takahashi, Ichiro; Takama, Michiho; Shoji, Shozo

    2002-05-10

    HIV-1(LAV-1) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified human immunodeficiency virus type 1 (HIV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained, and the stained spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-five proteins were identified as the proteins inside the virion and the acid-labile formyl group of an amino terminal proline residue of HIV-1(LAV-1) p24(gag) was determined by MALDI-TOF MS before and after weak-acid treatments (0.6 N hydrochloric acid) and confirmed by post-source decay (PSD) of the N-formylated N-terminal tryptic peptide (N-formylated Pro(1)-Arg(18)). The role of formylation has been unclear so far, but it is surmised that the acid-labile formylation of HIV-1(LAV-1) p24(gag) may play a critical role in the formation of the HIV-1 core for conferring HIV-1 infectivity.

  11. Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

    PubMed Central

    Lewandowski, Thomas; Huang, Jianzhong; Fan, Frank; Rogers, Shannon; Gentry, Daniel; Holland, Reannon; DeMarsh, Peter; Zalacain, Magdalena

    2013-01-01

    Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. PMID:23571548

  12. Antiangiogenic activity of 2-formyl-8-hydroxy-quinolinium chloride.

    PubMed

    Lam, K-H; Lee, K K-H; Kok, S H-L; Wong, R S-M; Lau, F-Y; Cheng, G Y-M; Wong, W-Y; Tong, S-W; Chan, K-W; Chan, R Y-K; Tang, J C-O; Cheng, C-H; Hau, D K-P; Bian, Z-X; Gambari, R; Chui, C-H

    2016-05-01

    Tumour growth is closely related to the development of new blood vessels to supply oxygen and nutrients to cancer cells. Without the neovascular formation, tumour volumes cannot increase and undergo metastasis. Antiangiogenesis is one of the most promising approaches for antitumour therapy. The exploration of new antiangiogenic agents would be helpful in antitumour therapy. Quinoline is an aromatic nitrogen compound characterized by a double-ring structure which exhibits a benzene ring fused to pyridine at two adjacent carbon atoms. The high stability of quinoline makes it preferable in a variety of therapeutic and pharmaceutical applications, including antitumour treatment. This work is to examine the potential antiangiogenic activity of the synthetic compound 2-Formyl-8-hydroxy-quinolinium chloride. We found that 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of human umbilical vein endothelial cells in vitro. Using the diethylnitrosamine-induced hepatocarcinogenesis model, 2-Formyl-8-hydroxy-quinolinium chloride showed strong antiangiogenic activity. Furthermore, 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of large Hep3B xenografted tumour from the nude mice. We assume that 2-Formyl-8-hydroxy-quinolinium chloride could be a potential antiangiogenic and antitumour agent and it is worthwhile to further study its underlying working mechanism. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Arabidopsis 10-formyl tetrahydrofolate deformylases are essential for photorespiration.

    PubMed

    Collakova, Eva; Goyer, Aymeric; Naponelli, Valeria; Krassovskaya, Inga; Gregory, Jesse F; Hanson, Andrew D; Shachar-Hill, Yair

    2008-07-01

    In prokaryotes, PurU (10-formyl tetrahydrofolate [THF] deformylase) metabolizes 10-formyl THF to formate and THF for purine and Gly biosyntheses. The Arabidopsis thaliana genome contains two putative purU genes, At4g17360 and At5g47435. Knocking out these genes simultaneously results in plants that are smaller and paler than the wild type. These double knockout (dKO) mutant plants show a 70-fold increase in Gly levels and accumulate elevated levels of 5- and 10-formyl THF. Embryo development in dKO mutants arrests between heart and early bent cotyledon stages. Mature seeds are shriveled, accumulate low amounts of lipids, and fail to germinate. However, the dKO mutant is only conditionally lethal and is rescued by growth under nonphotorespiratory conditions. In addition, culturing dKO siliques in the presence of sucrose restores normal embryo development and seed viability, suggesting that the seed and embryo development phenotypes are a result of a maternal effect. Our findings are consistent with the involvement of At4g17360 and At5g47435 proteins in photorespiration, which is to prevent excessive accumulation of 5-formyl THF, a potent inhibitor of the Gly decarboxylase/Ser hydroxymethyltransferase complex. Supporting this role, deletion of the At2g38660 gene that encodes the bifunctional 5,10-methylene THF dehydrogenase/5,10-methenyl THF cyclohydrolase that acts upstream of 5-formyl THF formation restored the wild-type phenotype in dKO plants.

  14. Deprotection of the indole (N(ind))-formyl (For) group on tryptophan employing a new reagent, N,N'-dimethylethylendiamine (DMEDA) in an aqueous solution.

    PubMed

    Odagami, Takenao; Tsuda, Yuko; Kogami, Yuji; Kouji, Hiroyuki; Okada, Yoshio

    2009-02-01

    The deprotection of the indole (N(ind))-formyl (For) group on Trp was achieved in a 95% yield using N,N'-dimethylethylendiamine (DMEDA) (1.5, 2.0, 3.0 eq) in water at room temperature. A new reagent was successfully applied to the deprotection of a model peptide, H-Phe-Trp(N(ind)-For)-Lys-Tyr-OH, to give H-Phe-Trp-Lys-Tyr-OH in a 91% yield.

  15. Structural, ac conductivity and dielectric properties of 3-formyl chromone

    NASA Astrophysics Data System (ADS)

    Ali, H. A. M.

    2017-07-01

    The structure for the powder of 3-formyl chromone was examined by X-ray diffraction technique in the 2θ° range ( 4° - 60° . The configuration of Al/3-formyl chromone/Al samples was designed. The electrical and dielectric properties were studied as a function of frequency (42- 5 × 106 Hz) and temperature (298-408K). The ac conductivity data of bulk of 3-formyl chromone varies as a power law with the frequency at different temperatures. The predominant mechanism for ac conduction was deduced. The ac conductivity shows a thermally activated process at different frequencies. The dielectric constant and dielectric loss were determined using the capacitance and dissipation factor measurements at different temperatures. The dielectric loss shows a peak of relaxation time that shifted to higher frequency with an increase in the temperature. The activation energy of the relaxation process was estimated.

  16. Ferromagnetic behavior of formyl-group-carrying stable thioaminyl radicals.

    PubMed

    Miura, Yozo; Nakamura, Shogo; Teki, Yoshio

    2003-10-17

    Four formyl-group-carrying thioaminyl radicals were generated, and one radical could be isolated as radical crystals. Magnetic susceptibility measurements of the isolated radical showed a ferromagnetic regular linear-chain interaction of 2J/k(B) = 3.2 K, which was explained in terms of the X-ray crystallographic results.

  17. Glycolaldehyde Formation via the Dimerization of the Formyl Radical

    NASA Astrophysics Data System (ADS)

    Woods, Paul M.; Slater, Ben; Raza, Zamaan; Viti, Serena; Brown, Wendy A.; Burke, Daren J.

    2013-11-01

    Glycolaldehyde, the simplest monosaccharide sugar, has recently been detected in low- and high-mass star-forming cores. Following our previous investigation into glycolaldehyde formation, we now consider a further mechanism for the formation of glycolaldehyde that involves the dimerization of the formyl radical, HCO. Quantum mechanical investigation of the HCO dimerization process upon an ice surface is predicted to be barrierless and therefore fast. In an astrophysical context, we show that this mechanism can be very efficient in star-forming cores. It is limited by the availability of the formyl radical, but models suggest that only very small amounts of CO are required to be converted to HCO to meet the observational constraints.

  18. Decomposition of formyl fluoride on the lowest triplet state surface

    NASA Astrophysics Data System (ADS)

    Sumathi, R.; Chandra, A. K.

    1992-09-01

    Decomposition of formyl fluoride on the lowest triplet potential energy hypersurface is studied using ab initio quantum chemical methods. Dissociation energies and the barrier to dissociation from the lowest triplet HFCO into H( 2S) and FCO( 2A') are reported. Results reveal that the barrier to dissociation into F( 2P) + HCO( 2A') is very high. The triatomic radical, FCO( 2A') decomposes into F( 2P) + CO( 1Σ) from the high vibrational levels of the C-F stretch.

  19. Improved Synthesis of and Nucleophilic Addition to 2-Formyl-2-Cyclohexenone.

    PubMed

    Adary, Elan M; Chang, Chih-Wei; Auria, Damian T D'; Nguyen, Phuc M; Polewacz, Klaudyna; Reinicke, Justin A; Seo, Hannah; Berger, Gideon O

    2015-01-08

    A preparation of 2-formyl-2-cyclohexenone in nearly quantitative yield and purity of approximately 95% is described. It is scalable and has been extended to the synthesis of the 5- and 7-membered ring homologs with comparable yields. Conditions have also been developed for the successful conjugate addition of dimethylmalonate to 2-formyl-2-cyclohexenone, in good and scalable yield (60%). This result has been extended to 5 other nucleophile classes, and the dimethylmalonate conjugate addition has been demonstrated with 2-formyl-2-cyclopentenone and 2-formyl-2-cycloheptenone.

  20. Bioactive Formylated Flavonoids from Eugenia rigida: Isolation, Synthesis, and X-ray Crystallography.

    PubMed

    Zaki, Mohamed A; Nanayakkara, N P Dhammika; Hetta, Mona H; Jacob, Melissa R; Khan, Shabana I; Mohammed, Rabab; Ibrahim, Mohamed A; Samoylenko, Volodymyr; Coleman, Christina; Fronczek, Frank R; Ferreira, Daneel; Muhammad, Ilias

    2016-09-23

    Two new flavonoids, rac-6-formyl-5,7-dihydroxyflavanone (1) and 2',6'-dihydroxy-4'-methoxy-3'-methylchalcone (2), together with five known derivatives, rac-8-formyl-5,7-dihydroxyflavanone (3), 4',6'-dihydroxy-2'-methoxy-3'-methyldihydrochalcone (4), rac-7-hydroxy-5-methoxy-6-methylflavanone (5), 3'-formyl-2',4',6'-trihydroxy-5'-methyldihydrochalcone (6), and 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7), were isolated from the leaves of Eugenia rigida. The individual (S)- and (R)-enantiomers of 1 and 3, together with the corresponding formylated flavones 8 (6-formyl-5,7-dihydroxyflavone) and 9 (8-formyl-5,7-dihydroxyflavone), as well as 2',4',6'-trihydroxychalcone (10), 3'-formyl-2',4',6'-trihydroxychalcone (11), and the corresponding 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7) and 2',4',6'-trihydroxydihydrochalcone (12), were synthesized. The structures of the isolated and synthetic compounds were established via NMR, HRESIMS, and electronic circular dichroism data. In addition, the structures of 3, 5, and 8 were confirmed by single-crystal X-ray diffraction crystallography. The isolated and synthetic flavonoids were evaluated for their antimicrobial and cytotoxic activities against a panel of microorganisms and solid tumor cell lines.

  1. N-formylation of amines via the aerobic oxidation of methanol over supported gold nanoparticles.

    PubMed

    Ishida, Tamao; Haruta, Masatake

    2009-01-01

    Dress code: formyl. Gold nanoparticles supported on NiO catalyze the one-pot N-formylation of amines with methanol and molecular oxygen to produce formamide at a selectivity of 90 %. This process generates methyl formate in situ, followed by reaction with amines.

  2. Role of N-terminal protein formylation in central metabolic processes in Staphylococcus aureus

    PubMed Central

    2013-01-01

    Background Bacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation. Results In order to elucidate how global metabolic processes are affected by the absence of formylated proteins the exometabolome of an S. aureus fmt mutant was compared with that of the parental strain and the transcription of corresponding enzymes was analyzed to identify possible regulatory changes. The mutant consumed glucose and other carbon sources slower than the wild type. While the turnover of several metabolites remained unaltered fmt inactivation led to increases pyruvate release and, concomitantly, reduced pyruvate dehydrogenase activity. In parallel, the release of the pyruvate-derived metabolites lactate, acetoin, and alanine was reduced. The anaerobic degradation of arginine was also reduced in the fmt mutant compared to the wild-type strain. Moreover, the lack of formylated proteins caused increased susceptibility to the antibiotics trimethoprim and sulamethoxazole suggesting that folic acid-dependant pathways were perturbed in the mutant. Conclusions These data indicate that formylated proteins are crucial for specific bacterial metabolic processes and they may help to understand why it has remained important during bacterial evolution to initiate protein biosynthesis with a formylated tRNAMet. PMID:23320528

  3. Radio detection of interstellar DCO/+/. [deuterated formyl ion

    NASA Technical Reports Server (NTRS)

    Hollis, J. M.; Snyder, L. E.; Lovas, F. J.; Buhl, D.

    1976-01-01

    The J = 1-0 transition of the deuterated formyl ion, DCO(+), has been detected in emission from NGC 2264 and DR 21(OH) and from the cool dust cloud L134. Estimates for the column-density ratios of H(C-13)O(+) to DCO(+) are given for NGC 2264 and DR 21(OH); lower limits for the ratios are given for five other clouds. The DCO(+) detection in L134 provides unique observational evidence in support of chemical fractionation. The L134 results also suggest that the dust clouds may act as repositories for primordial deuterium.

  4. The reaction of formyl radical with chlorine atom

    NASA Astrophysics Data System (ADS)

    Qu, Zhang-Wang; Dong, Feng; Zhang, Qiyuan; Kong, Fanao

    2004-03-01

    The radical-radical reaction of formyl radical with chlorine atom has been investigated by the time-resolved infrared emission spectroscopy and by the theoretical calculations at the UB3LYP/6-31++G(d,p) and single-point UCCSD(T)/6-311++G(d,p) levels. The products have been verified as the vibrationally excited CO ( v⩽4) and HCl. The reaction is initiated by radical-radical recombination forming an intermediate of formaldehyde chloride, which further dissociates into the products of HCl and CO.

  5. Concise syntheses of the cruciferous phytoalexins brassilexin, sinalexin, wasalexins, and analogues: expanding the scope of the vilsmeier formylation.

    PubMed

    Pedras, M Soledade C; Jha, Mukund

    2005-03-04

    Efficient syntheses of the phytoalexins brassilexin, sinalexin, and analogues are demonstrated through the application of the Vilsmeier formylation to indoline-2-thiones followed by a new aqueous ammonia workup procedure. Similarly, a very concise two-pot synthesis of the phytoalexins wasalexins using sequential formylation-amination of indolin-2-ones is described. Remarkably, this novel aqueous ammonia workup allows the sequential one-pot formylation-amination, expanding substantially the scope of the Vilsmeier formylation of both indoline-2-thiones and indolin-2-ones. The examination of the formylation-amination reaction and optimization of conditions, as well as the syntheses and antifungal activities of several brassilexin analogues, are reported.

  6. In vitro conversion of vinyl to formyl groups in naturally occurring chlorophylls.

    PubMed

    Loughlin, Patrick C; Willows, Robert D; Chen, Min

    2014-08-14

    The chemical structural differences distinguishing chlorophylls in oxygenic photosynthetic organisms are either formyl substitution (chlorophyll b, d, and f) or the degree of unsaturation (8-vinyl chlorophyll a and b) of a side chain of the macrocycle compared with chlorophyll a. We conducted an investigation of the conversion of vinyl to formyl groups among naturally occurring chlorophylls. We demonstrated the in vitro oxidative cleavage of vinyl side groups to yield formyl groups through the aid of a thiol-containing compound in aqueous reaction mixture at room temperature. Heme is required as a catalyst in aqueous solution but is not required in methanolic reaction mixture. The conversion of vinyl- to formyl- groups is independent of their position on the macrocycle, as we observed oxidative cleavages of both 3-vinyl and 8-vinyl side chains to yield formyl groups. Three new chlorophyll derivatives were synthesised using 8-vinyl chlorophyll a as substrate: 8-vinyl chlorophyll d, [8-formyl]-chlorophyll a, and [3,8-diformyl]-chlorophyll a. The structural and spectral properties will provide a signature that may aid in identification of the novel chlorophyll derivatives in natural systems. The ease of conversion of vinyl- to formyl- in chlorophylls demonstrated here has implications regarding the biosynthetic mechanism of chlorophyll d in vivo.

  7. Prediction of protein N-formylation using the composition of k-spaced amino acid pairs.

    PubMed

    Ju, Zhe; Cao, Jun-Zhe

    2017-10-01

    As one of important protein post-translational modifications, N-formylation has been reported to be involved in various biological processes. The accurate identification of N-formylation sites is crucial for understanding the underlying mechanisms of N-formylation. Since the traditional experimental methods are generally labor-intensive and expensive, it is important to develop computational methods to predict N-formylation sites. In this paper, a predictor named NformPred is proposed to improve the prediction of N-formylation sites by using composition of k-spaced amino acid pairs encoding scheme and support vector machine algorithm. As illustrated by 10-fold cross-validation, NformPred achieves a promising performance with a Sensitivity of 86.00%, a Specificity of 96.25%, an Accuracy of 94.48% and a Matthew's correlation coefficient of 0.8099, which are much better than those of current computational method. Feature analysis shows that some k-spaced amino acid pairs such as 'IxxL', 'LV' and 'IxxxI' play the most important roles in the prediction of N-formylation sites. These predictive and analytical results suggest that NformPred might facilitate the identification of protein N-formylation. A free online service for NformPred is accessible at http://123.206.31.171/NformPred/. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. In vitro Conversion of Vinyl to Formyl Groups in Naturally Occurring Chlorophylls

    PubMed Central

    Loughlin, Patrick C.; Willows, Robert D.; Chen, Min

    2014-01-01

    The chemical structural differences distinguishing chlorophylls in oxygenic photosynthetic organisms are either formyl substitution (chlorophyll b, d, and f) or the degree of unsaturation (8-vinyl chlorophyll a and b) of a side chain of the macrocycle compared with chlorophyll a. We conducted an investigation of the conversion of vinyl to formyl groups among naturally occurring chlorophylls. We demonstrated the in vitro oxidative cleavage of vinyl side groups to yield formyl groups through the aid of a thiol-containing compound in aqueous reaction mixture at room temperature. Heme is required as a catalyst in aqueous solution but is not required in methanolic reaction mixture. The conversion of vinyl- to formyl- groups is independent of their position on the macrocycle, as we observed oxidative cleavages of both 3-vinyl and 8-vinyl side chains to yield formyl groups. Three new chlorophyll derivatives were synthesised using 8-vinyl chlorophyll a as substrate: 8-vinyl chlorophyll d, [8-formyl]-chlorophyll a, and [3,8-diformyl]-chlorophyll a. The structural and spectral properties will provide a signature that may aid in identification of the novel chlorophyll derivatives in natural systems. The ease of conversion of vinyl- to formyl- in chlorophylls demonstrated here has implications regarding the biosynthetic mechanism of chlorophyll d in vivo. PMID:25119484

  9. Effects of molecular structures on reduction properties of formyl groups in chlorophylls and pheophytins prepared from oxygenic photosynthetic organisms.

    PubMed

    Sadaoka, Kana; Kashimura, Shigenori; Saga, Yoshitaka

    2011-07-01

    Reduction of the 7-formyl groups in chlorophyll (Chl) b and its demetalated compound pheophytin (Phe) b was kinetically analyzed by using tert-butylamine-borane complex (t-BuNH(2)·BH(3)), and was compared with that of the 3-formyl groups in Chl d and Phe d. Reduction kinetics of the 7-formyl group in Chl b was similar to that in Phe b in dichloromethane containing 5mM t-BuNH(2)·BH(3). Little difference of the reduction kinetics of the 7-formyl groups between Chl b and Phe b was in sharp contrast to the reduction kinetics of the 3-formyl groups in Chl d and Phe d: the 3-formyl group in Phe d was reduced 5.3-fold faster than that in Chl d. The 7-formyl groups in Chl b and Phe b were reduced more slowly than the 3-formyl groups in Chl d and Phe d, respectively. The difference of the reactivity between the 3- and 7-formyl groups was in line with (13)C NMR measurements of chlorophyllous pigments, in which the chemical shifts of carbon atoms in the 7-formyl groups of Chl b and Phe b were high-field shifted compared with those in the 3-formyl groups of Chl d and Phe d, respectively. These indicate that the 7-formyl groups in chlorophyllous pigments were less reactive for reduction to the corresponding hydroxymethyl groups than the 3-formyl groups due to the difference in electronic states of the formyl groups in the A- and B-rings of the chlorin macrocycle. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Formyl-methionine as a degradation signal at the N-termini of bacterial proteins

    PubMed Central

    Piatkov, Konstantin I.; Vu, Tri T. M.; Hwang, Cheol-Sang; Varshavsky, Alexander

    2015-01-01

    In bacteria, all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. The fMet residue is cotranslationally deformylated by a ribosome-associated deformylase. The formylation of N-terminal Met in bacterial proteins is not strictly essential for either translation or cell viability. Moreover, protein synthesis by the cytosolic ribosomes of eukaryotes does not involve the formylation of N-terminal Met. What, then, is the main biological function of this metabolically costly, transient, and not strictly essential modification of N terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the similarity of the formyl and acetyl groups, their identical locations in N terminally formylated (Nt formylated) and Nt-acetylated proteins, and the recently discovered proteolytic function of Nt-acetylation in eukaryotes might also signify a proteolytic role of Nt formylation in bacteria. We addressed this hypothesis about fMet based degradation signals, termed fMet/N-degrons, using specific E. coli mutants, pulse-chase degradation assays, and protein reporters whose deformylation was altered, through site-directed mutagenesis, to be either rapid or relatively slow. Our findings strongly suggest that the formylated N-terminal fMet can act as a degradation signal, largely a cotranslational one. One likely function of fMet/N-degrons is the control of protein quality. In bacteria, the rate of polypeptide chain elongation is nearly an order of magnitude higher than in eukaryotes. We suggest that the faster emergence of nascent proteins from bacterial ribosomes is one mechanistic and evolutionary reason for the pretranslational design of bacterial fMet/N degrons, in contrast to the cotranslational design of analogous Ac/N degrons in eukaryotes. PMID:26866044

  11. Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

    SciTech Connect

    Smithers, G.W.; Jahansouz, H.; Kofron, J.L.; Himes, R.H.; Reed, G.H.

    1987-06-30

    Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate, and the product was characterized by /sup 31/P, /sup 1/H, and /sup 13/C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by /sup 31/P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 /sup 0/C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by /sup 18/O incorporation from H/sub 2//sup 18/O into P/sub i/, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N/sup 10/-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k/sub cat/ values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.

  12. Experimental and theoretical study of 4-formyl pyrrole derived aroylhydrazones

    NASA Astrophysics Data System (ADS)

    Rawat, Poonam; Singh, R. N.

    2015-03-01

    Two new 4-formyl pyrrole derived aroylhydrazones (3a, b) from ethyl 4-formyl-3,5-dimetyl-1H-pyrrole-2-carboxylate and aroylhydrazides (3,5-dinitrobenzohydrazide/2-hydrazinocarbonyl-N-phenyl-acetamide) have been synthesized and characterized by various spectroscopic techniques 1H NMR, Mass, UV-Visible and FT-IR. The calculated thermodynamic parameters show that the formation of 3a as spontaneous, whereas 3b as non-spontaneous. TD-DFT has been used to calculate the absorption wavelengths, oscillator strength (f) and the nature of electronic excitations. Natural bond orbital (NBO) analysis has been carried out to explore the various conjugative and hyperconjugative interactions and their second order stabilization energy (E(2)) within monomer and its dimer. The dimer formation of 3a, 3b due to result of intermolecular hydrogen bonding N1sbnd H30⋯O84, N1sbnd H28⋯O60 is obvious in 1H NMR, NBO and FT-IR as down field chemical shifts, n(O84) → σ∗(N1sbnd H30), n(O60) → σ∗(N1sbnd H28) interactions, vibrational red shifts, respectively. To determine the strength and nature of hydrogen bonding, topological parameters at bond critical points (BCP) have been analyzed by 'Quantum theory of Atoms in molecules' (QTAIM) in detail. The global electrophilicity index (ω) has been calculated to determine the relative electrophilic strength of molecules. The local reactivity descriptors analyses such as Fukui functions (fk+, fk-), local softnesses (sk+, sk-) and electrophilicity indices (ωk+, ωk-) have been performed to determine the reactive sites within molecules. The first hyperpolarizabilities (β0) of 3a, b have been computed to evaluate the non-linear optical (NLO) response of the investigated molecules.

  13. Determination of formylated DNA and RNA by chemical labeling combined with mass spectrometry analysis.

    PubMed

    Jiang, Han-Peng; Liu, Ting; Guo, Ning; Yu, Lei; Yuan, Bi-Feng; Feng, Yu-Qi

    2017-08-15

    Nucleic acids carry diverse chemical modifications that exert critical influences in a variety of cellular processes in living organisms. In addition to methylation, the emerging DNA and RNA formylation has been reported to play functional roles in various physiological processes. However, the amounts of formylated DNA and RNA are extremely low and detection of DNA and RNA formylation is therefore a challenging task. To address this issue, we developed a strategy by chemical labeling combined with in-tube solid-phase microextraction - ultra high performance liquid chromatography - electrospray ionization - tandem mass spectrometry (in-tube SPME-UPLC-ESI-MS/MS) analysis for the sensitive determination of DNA and RNA formylation. Using the developed method, we were able to simultaneously measure six formylated nucleosides, including 5-formyl-2'-deoxycytidine (5-fodC), 5-formylcytidine (5-forC), 5-formyl-2'-deoxyuridine (5-fodU), 5-formyluridine (5-forU), 2'-O-methyl-5-formylcytidine (5-forCm) and 2'-O-methyl-5- formyluridine (5-forUm), from DNA and RNA of cultured human cells and multiple mammalian tissues. The detection limits of these formylated nucleosides improved by 307-884 folds using Girard's P (GirP) labeling coupled with in-tube SPME-UPLC-ESI-MS/MS analysis. It was worth noting that 5-forU, 5-forCm and 5-forUm which have not been detected in human sample before, were discovered in cultured human cells and tissues in the current study. In addition, we observed significant increase of 5-forC and 5-forU in RNA (p = 0.027 for 5-forC; p = 0.028 for 5-forU) and 5-fodU in DNA (p = 0.002) in human thyroid carcinoma tissues compared to normal tissues adjacent to the tumor using synthesized stable isotope GirP (d5-GirP)-assisted quantification. Our results indicated that aberrant DNA and RNA formylation may contribute to the tumor formation and development. In addition, monitoring of DNA and RNA formylation may also serve as indicator for cancer diagnostics

  14. Visible-Light-Promoted Nickel- and Organic-Dye-Cocatalyzed Formylation Reaction of Aryl Halides and Triflates and Vinyl Bromides with Diethoxyacetic Acid as a Formyl Equivalent.

    PubMed

    Huang, He; Li, Xiangmin; Yu, Chenguang; Zhang, Yueteng; Mariano, Patrick S; Wang, Wei

    2017-02-01

    A simple formylation reaction of aryl halides, aryl triflates, and vinyl bromides under synergistic nickel- and organic-dye-mediated photoredox catalysis is reported. Distinct from widely used palladium-catalyzed formylation processes, this reaction proceeds by a two-step mechanistic sequence involving initial in situ generation of the diethoxymethyl radical from diethoxyacetic acid by a 4CzIPN-mediated photoredox reaction. The formyl-radical equivalent then undergoes nickel-catalyzed substitution reactions with aryl halides and triflates and vinyl bromides to form the corresponding aldehyde products. Significantly, besides aryl bromides, less reactive aryl chlorides and triflates and vinyl halides serve as effective substrates for this process. Since the mild conditions involved in this reaction tolerate a plethora of functional groups, the process can be applied to the efficient preparation of diverse aromatic aldehydes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. New Formyl Phloroglucinol Meroterpenoids from the Leaves of Eucalyptus robusta

    PubMed Central

    Shang, Zhi-Chun; Yang, Ming-Hua; Liu, Rui-Huan; Wang, Xiao-Bing; Kong, Ling-Yi

    2016-01-01

    Seven new formyl phloroglucinol meroterpenoids (FPMs), namely eucalrobusones J-P (1–7), as well as three known ones (8–10) were isolated from the leaves of Eucalyptus robusta. Their structures were elucidated by spectroscopic data analysis, and their absolute configurations were determined by applications of the Snatzke’s helicity rule and the electron circular dichroism (ECD) calculation. These FPMs are diverse in coupling patterns between phloroglucinol and sesquiterpenoid units, forming novel polycyclic ring systems. Compound 1 possesses a new carbon skeleton that a 1-oxaspiro[5.6]dodecane core is formed through C-14 rather than C-4 of the aromadendrane moiety. Compound 2 features a novel 6/7/5 ring-fused 6-oxabicyclo[3.2.2]nonane skeleton. Compounds 3–5 are rare aristolane-based FPMs. By forming different oxo bridges, compound 3 is the first sample of FPM with benzo-dihydrofuran structure, and compound 4 possesses a novel 6/6/6/6/3-fused pentacyclic skeleton. Compounds 1, 6, and 8 exhibited significant antifungal activities against Candida glabrata with MIC50 values of 2.57, 1.95, and 2.49 μg/mL, respectively. PMID:28004790

  16. ZnO as a new catalyst for N-formylation of amines under solvent-free conditions.

    PubMed

    Hosseini-Sarvari, Mona; Sharghi, Hashem

    2006-08-18

    The treatment of amines with formic acid in the presence of ZnO under solvent-free conditions brings about highly and efficient N-formylation to give the corresponding formamides in excellent yields. The N-formylation reaction not only involves mild conditions, simple operation, and high yields but also high chemoselectivity.

  17. Human neutrophil formyl peptide receptor phosphorylation and the mucosal inflammatory response

    PubMed Central

    Leoni, Giovanna; Gripentrog, Jeannie; Lord, Connie; Riesselman, Marcia; Sumagin, Ronen; Parkos, Charles A.; Nusrat, Asma; Jesaitis, Algirdas J.

    2015-01-01

    Bacterial/mitochondrial fMLF analogs bind FPR1, driving accumulation/activation of PMN at sites of infection/injury, while promoting wound healing in epithelia. We quantified levels of UFPR1 and TFPR1 in isolated PMN by use of phosphosensitive NFPRb and phosphorylation-independent NFPRa antibodies. UFPR1 and total TFPR were assessed inflamed mucosa, observed in human IBD. In isolated PMN after fMLF stimulation, UFPR1 declined 70% (fMLFEC50 = 11 ± 1 nM; t1/2 = 15 s) and was stable for up to 4 h, whereas TFPR1 changed only slightly. Antagonists (tBoc-FLFLF, CsH) and metabolic inhibitor NaF prevented the fMLF-dependent UFPR1 decrease. Annexin A1 fragment Ac2-26 also induced decreases in UFPR1 (Ac2-26EC50 ∼ 3 µM). Proinflammatory agents (TNF-α, LPS), phosphatase inhibitor (okadaic acid), and G-protein activator (MST) modestly increased fMLFEC50, 2- to 4-fold, whereas PTX, Ca2+ chelators (EGTA/BAPTA), H2O2, GM-CSF, ENA-78, IL-1RA, and LXA4 had no effect. Aggregation-inducing PAF, however, strongly inhibited fMLF-stimulated UFPR1 decreases. fMLF-driven PMN also demonstrated decreased UFPR1 after traversing monolayers of cultured intestinal epithelial cells, as did PMN in intestinal mucosal samples, demonstrating active inflammation from UC patients. Total TFPR remained high in PMN within inflamed crypts, migrating through crypt epithelium, and in the lamina propria-adjoining crypts, but UFPR1 was only observed at some peripheral sites on crypt aggregates. Loss of UFPR1 in PMN results from C-terminal S/T phosphorylation. Our results suggest G protein–insensitive, fMLF-dependent FPR1 phosphorylation in isolated suspension PMN, which may manifest in fMLF-driven transmigration and potentially, in actively inflamed tissues, except at minor discrete surface locations of PMN-containing crypt aggregates. PMID:25395303

  18. N-Formylmethionyl Peptide Receptors on Equine Leukocytes Initiate Secretion but not Chemotaxis

    NASA Astrophysics Data System (ADS)

    Snyderman, Ralph; Pike, Marilyn C.

    1980-07-01

    The chemotaxis of leukocytes appears to be initiated by the binding of chemotactic factors to the surface of these cells. N-Formylated peptides induce chemotaxis and lysosomal enzyme secretion of leukocytes; because these peptides are available in a purified radiolabeled form, they have been useful in the characterization of receptors for chemotactic factors. Equine polymorphonuclear leukocytes secrete lysosomal enzymes but do not exhibit chemotaxis in response to the N-formylated peptides, even though they have a high-affinity cell surface receptor for these agents. The specificity of the equine receptor resembles the specificity of the receptor on chemotactically responsive leukocytes from other species. Equine polymorphonuclear leukocytes may thus be an excellent model for the study of the events that lead to a biological response following receptor occupancy.

  19. Synthesis, conformational and theoretical studies of 1,n-di(2-formyl-4-phenylazophenoxy)alkanes

    NASA Astrophysics Data System (ADS)

    Balachander, R.; Manimekalai, A.

    2016-01-01

    1,n-di(2-Formyl-4-phenylazophenoxy)alkanes 1 and 2 and 1,3-di(2-formyl-4-phenylazophenoxymethyl)benzene 3 were synthesis and characterized by FT-IR, UV-Vis, 1H, 13C NMR and mass spectral studies. The stable conformations of 1-3 were predicted theoretically and selected geometrical parameters were derived from optimized structures. The molecular parameters of HOMO-LUMO energies, polarizability, hyperpolarizability, natural bond orbital (NBO), atom in molecule (AIM) analysis and molecular electrostatic potential (MEP) surfaces were determined by the density functional theory (DFT) method and analysed.

  20. Temperature dependant electrical properties of formyl-TIPPCu(II)/p-Si heterojunction diode

    NASA Astrophysics Data System (ADS)

    Khan, Dil Nawaz; Sayyad, Muhammad Hassan; Wahab, Fazal; Tahir, Muhammad; Yaseen, Muhammad; Munawar, Munawar Ali; Ali, Mukhtar

    2014-05-01

    This paper reports the temperature dependent electrical characterization of formyl-TIPPCu(II)/p-Si heterojunction diode which was fabricated by growing thin films of formyl-TIPPCu(II) on the p-type silicon substrate by thermal sublimation technique. The variation in electrical characteristics of the fabricated devices has been systematically investigated as the function of temperature by using current-voltage (I-V) measurements in the temperature range 299-339 K. The diode parameters like ideality factor, zero bias barrier height and parasitic series resistance have been found to be strongly temperature dependant. The zero bias barrier height increases while ideality factor and series resistance decreases with increasing temperature.

  1. Formylated chloro-bridged iridium(III) dimers as OLED materials: opening up new possibilities.

    PubMed

    Wong, Michael Y; Xie, Guohua; Tourbillon, Clarisse; Sandroni, Martina; Cordes, David B; Slawin, Alexandra M Z; Samuel, Ifor D W; Zysman-Colman, Eli

    2015-05-14

    In this study, a series of four formyl-substituted chloro-bridged iridium(iii) dimers were prepared. Their absorption, photophysical and electrochemical properties were studied in dichloromethane solution. It was found that as the formyl content increased on the cyclometalating ligands, emission unexpectedly became brighter. Organic light-emitting diodes (OLEDs) were fabricated using each of these iridium dimers as the emitter. The OLED fabricated using the brightest of the series, 2b, as the dopant afforded a decent external quantum efficiency (EQE) of 2.6%. This suggests that chloro-bridged iridium dimers are potential candidates as solid-state emitters.

  2. N-Formyl-Methionyl-Leucyl-Phenylalanine Inhibits both Gamma Interferon- and Interleukin-10-Induced Expression of FcγRI on Human Monocytes

    PubMed Central

    Beigier-Bompadre, Macarena; Barrionuevo, Paula; Alves-Rosa, Fernanda; Rubel, Carolina J.; Palermo, Marina S.; Isturiz, Martín A.

    2001-01-01

    Three different classes of receptors for the Fc portion of immunoglobulin G (FcγRs), FcγRI, FcγRII, and FcγRIII, have been identified on human leukocytes. One of them, FcγRI, is a high-affinity receptor capable of induction of functions that include phagocytosis, respiratory burst, antibody-dependent cell-mediated cytotoxicity (ADCC), and secretion of cytokines. This receptor is expressed on mononuclear phagocytes, and this expression is regulated by cytokines and hormones such as gamma interferon (IFN-γ), IFN-β, interleukin-10 (IL-10), and glucocorticoids. We have recently demonstrated that the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is capable of inducing a time-dependent downregulation of both FcγRIIIB and FcγRII in human neutrophils, altering FcγR-dependent functions. Considering the biological relevance of the regulation of FcγRI, we investigated the effect of FMLP on the overexpression of FcγRI induced by both IFN-γ and IL-10 on human monocytes. We demonstrate that FMLP significantly abrogated IFN-γ- and IL-10-induced FcγRI expression, although its basal level of expression was not altered. However, other IFN-γ-mediated effects such as the overexpression of the major histocompatibility complex class II antigens and the enhancement of lipopolysaccharide-induced secretion of tumor necrosis factor alpha were not affected by FMLP treatment. The formyl peptide completely inhibited the IFN-γ- and IL-10-induced enhancement of ADCC and phagocytosis carried out by adherent cells. The inhibitory effect of FMLP on FcγRI upregulation could exert an important regulatory effect during the evolution of bacterial infections. PMID:11238229

  3. N1-acetyl-N2-formyl-5-methoxykynuramine, a biogenic amine and melatonin metabolite, functions as a potent antioxidant.

    PubMed

    Tan, D X; Manchester, L C; Burkhardt, S; Sainz, R M; Mayo, J C; Kohen, R; Shohami, E; Huo, Y S; Hardeland, R; Reiter, R J

    2001-10-01

    The biogenic amine The biogenic amine N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) was investigated for its potential antioxidative capacity. AFMK is a metabolite generated through either an enzymatic or a chemical reaction pathway from melatonin. The physiological function of AFMK remains unknown. To our knowledge, this report is the first to document the potent antioxidant action of this biogenic amine. Cyclic voltammetry (CV) shows that AFMK donates two electrons at potentials of 456 mV and 668 mV, and therefore it functions as a reductive force. This function contrasts with all other physiological antioxidants that donate a single electron only when they neutralize free radicals. AFMK reduced 8-hydroxydeoxyguanosine formation induced by the incubation of DNA with oxidants significantly. Lipid peroxidation resulting from free radical damage to rat liver homogenates was also prevented by the addition of AFMK. The inhibitory effects of AFMK on both DNA and lipid damage appear to be dose-response related. In cell culture, AFMK efficiently reduced hippocampal neuronal death induced by either hydrogen peroxide, glutamate, or amyloid b25-35 peptide. AFMK is a naturally occurring molecule with potent free radical scavenging capacity (donating two electrons/molecule) and thus may be a valuable new antioxidant for preventing and treating free radical-related disorders.

  4. Quantum State Resolved Photodissociation Dynamics of the Formyl Radical.

    NASA Astrophysics Data System (ADS)

    Neyer, David William

    The photodissociation dynamics of the formyl (HCO) radical have been investigated both experimentally and theoretically. HCO molecules, produced in a molecular beam by the laser photolysis of acetaldehyde, were excited to metastable levels with quantum state resolution. The rotational and vibrational states of the CO products from the dissociation of these levels were probed by laser-induced fluorescence using a tunable vacuum ultraviolet laser. Measurement of detailed state-to-state dissociation cross sections and theoretical modeling of these dynamics have provided valuable information about the potential energy surface of the ground electronic state (X) of the HCO system. HCO was excited to predissociative levels of the first electronic state (A) characterized by their vibrational and K-rotational quantum numbers, and the rotational and vibrational populations of the CO products were measured. While the K-state excited in the HCO has little effect on the CO products, the vibrational character of the parent causes specific changes in the product state distributions. Addition of bending or C-H stretching quanta to the HCO parent leads to increased rotational excitation in the CO(nu =O) products. Adding C-O stretch to the parent state produces increased vibrational excitation in the CO products, The dynamics of this dissociation process, which involves Renner-Teller coupling between the X and A states, was modeled using classical trajectories calculated on a global X-state potential energy surface. Stimulated emission pumping (SEP) was used to prepare HCO in metastable resonances on the X state of HCO with vibrational and rotational resolution. The energies and linewidths of these resonances were measured, and the rotational and vibrational distributions of the CO products were determined. The linewidths and product state distributions show highly non-statistical behavior which depends on the vibrational character of the HCO resonance. The rotational distributions

  5. Synthesis of fluorescence organocyclotriphosphazene derivatives having functional groups such as formyl, Schiff base and both formyl and Schiff base without using Ar or N2 atmosphere

    NASA Astrophysics Data System (ADS)

    Aslan, Fatih; Öztürk, Ali İhsan; Söylemez, Bayram

    2017-06-01

    In the present study, hexa(4-formyl-phenoxy)cyclotriphosphazene(2) and hexa(4-formyl-2-methoxy-phenoxy)cyclotriphosphazene(3), which were previously synthesized in boiling solvent under N2 atmosphere, were produced again at room conditions without using Ar or N2 atmosphere by the same method. We detected that both 2 and 3 formed with very high yields even under this mild conditions. After that, 1 equiv of the compounds (2 and 3) were reacted with 12 equiv of some anilines derivatives with different substituted groups such as hydroxyl, cyano, mercapto, heterocyclic, carboxyl, chloro for the synthesis of new organocyclotriphosphazene derivatives incorporating six non-conjugated Schiff base groups. However, such compounds did not form from all of the selected anilines derivatives. The reactions of the compounds (2 and 3) with 4-carboxy-aniline and 4-cyano-aniline led to the formation of the organocyclotriphosphazenes bearing both formyl and Schiff base units. The structures of the synthesized molecules were determined using FT-IR, 1H NMR and 31P NMR. Then, the synthesized compounds were photophysically investigated by UV absorption and fluorescence emission spectroscopy in solution state. Photophysical studies indicate that the compound 1 and all of the obtained compounds have luminescence properties and large Stoke's shifts. Some compounds showed blue-red emission peak having rather large Stoke's shifts in the range of 390-800 nm.

  6. Receptor-Dependent and -Independent Immunomodulatory Effects of Phenol-Soluble Modulin Peptides from Staphylococcus aureus on Human Neutrophils Are Abrogated through Peptide Inactivation by Reactive Oxygen Species

    PubMed Central

    Christenson, Karin; Bylund, Johan; Dahlgren, Claes

    2012-01-01

    The virulence and pathogenesis mechanisms of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains depend on a newly described group of phenol-soluble modulin (PSM) peptides (the PSMα peptides) with cytolytic activity. These toxins are α-helical peptides with a formyl group at the N terminus, and they activate neutrophils through formyl peptide receptor 2 (FPR2), a function closely correlated to the capacity of staphylococcal species to cause invasive infections. The effects of two synthetic PSMα peptides were investigated, and we show that they utilize FPR2 and promote neutrophils to produce reactive oxygen species (ROS) which in turn trigger inactivation of the peptides. Independently of FPR2, the PSMα peptides also downregulate the neutrophil response to other stimuli and exert a cytolytic effect to which apoptotic neutrophils are more sensitive than viable cells. The novel immunomodulatory functions of the PSMα peptides were sensitive to ROS generated by the neutrophil myeloperoxidase (MPO)-H2O2 system, suggesting a role for this enzyme system in counteracting bacterial virulence. PMID:22431645

  7. Ca2+ response in neutrophils after exposure to bacterial N-formyl-methionyl-leucyl-phenylalanine: delayed response in ulcerative colitis.

    PubMed

    Vainer, Ben; Lamberth, Kasper; Brimnes, Jens; Nielsen, Ole H; Claësson, Mogens H

    2003-03-01

    In acute stages of ulcerative colitis (UC), neutrophils migrate from the circulation into inflamed colonic tissue, initiated by yet unknown stimuli. The bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is a component of the surface membrane of colonic bacteria such as Escherichia coli and stimulates Ca2+ influx into neutrophils, reflecting the fact that ionized calcium is an important secondary messenger for several neutrophil functions, including locomotion, phagocytosis and free oxygen radical production. Recent studies have revealed that Ca2+ dependent ICAM-1/beta 2-integrin mediated neutrophil migration is impaired in UC patients. The aim of the present work was to study the influx of Ca2+ into peripheral blood neutrophils of UC patients after exposure to FMLP and after binding of either beta 2-integrins or intercellular adhesion molecule-1 (ICAM-1). The relative intracellular Ca2+ levels ([Ca2+]i ) were measured spectrofluorometrically in neutrophils isolated from eight UC patients and eight controls. The cells were exposed to 1 nm FMLP, 5 pm free ICAM-1, or antibodies binding ICAM-1 or the beta 2-integrins CD11a, CD11b, CD11c and CD18. A pronounced increase in [Ca2+]i was observed by exposure of cells to FMLP, and neutrophils from UC patients showed a consistent and significant delayed response as compared to cells from control subjects (P < 0.01). Antibody mediated cross-linking of CD18 triggered a small but detectable increase in [Ca2+]i, which did not differ between patients and controls. A delayed response to bacterial peptides appears to be a phenotypic trait for neutrophils of UC patients. A connection between FMLP stimulated Ca2+ influx and CD11/CD18 upregulation is discussed.

  8. Characterization of N-Boc/Fmoc/Z-N'-formyl-gem-diaminoalkyl derivatives using electrospray ionization multi-stage mass spectrometry.

    PubMed

    Verardo, Giancarlo; Gorassini, Andrea

    2013-11-01

    N-Boc/Fmoc/Z-N'-formyl-gem-diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro-inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion-trap multi-stage mass spectrometry (MS(n)). The MS(2) collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N-Fmoc/Z-amino acids, dipeptide and tripeptide acids show the [M + Na-NH2CHO](+) ion, arising from the loss of formamide, as the base peak. Differently, the MS(2) CID spectra of [M + Na](+) ion of all the N-Boc derivatives yield the abundant [M + Na-C4H8](+) and [M + Na-Boc + H](+) ions because of the loss of isobutylene and CO2 from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N-protected dipeptidyl and tripeptidyl-N'-formamides is provided by MS(2) and subsequent MS(n) experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M-H](-), [M + HCOO](-) and [M + Cl](-) ions, the presence of in-source CID fragment ions deriving from the involvement of the N-protecting group. Furthermore, MS(n) spectra of [M + Cl](-) ion of N-protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C-terminal gem-diamino residue. The present paper represents an initial attempt to study the ESI-MS behavior of these important intermediates and lays the groundwork for structural-based studies on more complex partially modified retro-inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Haemolytic activity of formyl- and acetyl-derivatives of bile acids and their gramine salts.

    PubMed

    Kozanecka-Okupnik, Weronika; Jasiewicz, Beata; Pospieszny, Tomasz; Matuszak, Monika; Mrówczyńska, Lucyna

    2017-10-01

    Bile acids (lithocholic: LCA, deoxycholic: DCA and cholic: CA) and their formyl- and acetyl-derivatives can be used as starting material in chemical synthesis of compounds with different biological activity strongly depended on their chemical structures. Our previous studies showed that biological activity of bile acids salts with gramine toward human erythrocytes was significantly different from the activity of bile acids alone. Moreover, gramine effectively modified the membrane perturbing activity of other steroids. As a continuation of our work, the haemolytic activity of formyl- and acetyl-substituet bile acids as well as their gramine salts was studied in vitro. The structures of new compounds were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. The results shown that the haemolytic activity of formyl- and acetyl-LCA and DCA was significantly higher in comparison with their native forms at the whole concentration range. At high concentration, formyl derivative of CA was as effective as LCA and DCA derivatives whereas at lower concentration its haemolytic activity was at the level of original acid. The acetyl-CA was not active as membrane perturbing agents. Furthermore, gramine significantly decreased the membrane-perturbing activity of hydrophobic bile acids derivatives. The results obtained with the cellular system are in line with physicochemical calculation. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. The C2(1)-formyl group in chlorophyll f originates from molecular oxygen.

    PubMed

    Garg, Harsh; Loughlin, Patrick C; Willows, Robert D; Chen, Min

    2017-09-28

    Chlorophylls (Chls) are the most important cofactor for capturing solar energy to drive photosynthetic reactions. Five spectral types of Chls have been identified to date with Chl f having the most red-shifted absorption maximum due to a C2(1)-formyl group substitution of Chl f However, the biochemical provenance of this formyl group is unknown. Here, we used a stable isotope labeling technique ((18)O and (2)H) to determine the origin of the C2(1)-formyl group of Chl f, and to verify whether Chl f is synthesized from Chl a in the cyanobacterial species Halomicronema hongdechloris In the presence of either H2(18)O or (18)O2, the origin of oxygen atoms in the newly synthesized chlorophylls was investigated. The pigments were isolated with HPLC, followed by MS analysis. We found that the oxygen atom of the C2(1)-formyl group originates from molecular oxygen and not from H2O. Moreover, we examined the kinetics of the labeling of Chl a and Chl f from H. hongdechloris grown in 50% D2O-seawater medium under different light conditions. When cells were shifted from white light D2O-seawater medium to far-red light H2O-seawater medium the observed deuteration in Chl f indicated that Chl(ide) a is the precursor of Chl f Taken together, our results advance our understanding of the biosynthesis pathway of the chlorophylls and the formation of the formyl group in Chl f. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  11. Unprecedented Vilsmeier formylation: expedient syntheses of the cruciferous phytoalexins sinalexin and brassilexin and discovery of a new heteroaromatic ring system.

    PubMed

    Pedras, M S; Zaharia, I L

    2001-04-19

    [reaction: see text]. A very concise first synthesis of sinalexin was achieved by regioselective formylation of 1-methoxyindoline-2-thione under Vilsmeier conditions followed by unprecedented ammonia workup. Similar formylation of indoline-2-thione yielded brassilexin and a novel pentacyclic heteroaromatic compound resulting from condensation of the Vilsmeier adduct of indoline-2-thione. Both sinalexin and brassilexin displayed strong antifungal activity against several pathogens of crucifers.

  12. A TD-DFT study on the cyanide-chemosensing mechanism of 8-formyl-7-hydroxycoumarin.

    PubMed

    Li, Guang-Yue; Zhao, Guang-Jiu; Han, Ke-Li; He, Guo-Zhong

    2011-03-01

    Proton transfer (PT) and excited-state PT process are proposed to account for the fluorescent sensing mechanism of a cyanide chemosensor, 8-formyl-7-hydroxycoumarin. The time-dependent density functional theory method has been applied to investigate the ground and the first singlet excited electronic states of this chemosensor as well as its nucleophilic addition product with cyanide, with a view to monitoring their geometries and spectrophotometrical properties. The present theoretical study indicates that phenol proton of the chemosensor transfers to the formyl group along the intramolecular hydrogen bond in the first singlet excited state. Correspondingly, the nucleophilic addition product undergoes a PT process in the ground state, and shows a similar structure in the first singlet excited state. This could explain the observed strong fluorescence upon the addition of the cyanide anion in the relevant fluorescent sensing mechanism. Copyright © 2010 Wiley Periodicals, Inc.

  13. Preparation of unsymmetrical ketones from tosylhydrazones and aromatic aldehydes via formyl C-H bond insertion.

    PubMed

    Allwood, Daniel M; Blakemore, David C; Ley, Steven V

    2014-06-06

    Preparation of ketones by insertion of diazo compounds into the formyl C-H bond of an aldehyde is an attractive procedure, but use of structurally diverse diazo compounds is hampered by preparation and safety issues. A convenient procedure for the synthesis of unsymmetrical ketones from bench-stable tosylhydrazones and aryl aldehydes is reported. The procedure can be performed in one pot from the parent carbonyl compound and needs only a base, with no additional promoters being required.

  14. Palladium-Catalyzed Nitromethylation of Aryl Halides: An Orthogonal Formylation Equivalent

    PubMed Central

    Walvoord, Ryan R.; Berritt, Simon; Kozlowski, Marisa C.

    2012-01-01

    An efficient cross-coupling reaction of aryl halides and nitromethane was developed with the use of parallel microscale experimentation. The arylnitromethane products are precursors for numerous useful synthetic products. An efficient method for their direct conversion to the corresponding oximes and aldehydes in a one-pot operation has been discovered. The process exploits inexpensive nitromethane as a carbonyl equivalent, providing a mild and convenient formylation method that is compatible with many functional groups. PMID:22839593

  15. Optimizing Size and Copy Number For PEG-fMLF (N-Formyl-methionyl-leucyl-phenylalanine) Nanocarrier Uptake by Macrophages

    PubMed Central

    Wan, Li; Zhang, Xiaoping; Pooyan, Shahriar; Palombo, Matthew S.; Leibowitz, Michael J.; Stein, Stanley; Sinko, Patrick J.

    2009-01-01

    Curing HIV-1 infection has remained elusive because of low and fluctuating drug levels arising from poor absorption, the development of viral reservoirs and sanctuary sites, toxicity, and patient nonadherence. The present study addresses the issue of insufficient drug exposure in macrophages. Viral reservoir sites such as macrophages are believed to be responsible for the viral rebound effect observed upon the discontinuation of anti-HIV drug therapy. In our proposed model, a drug can be covalently attached to a nanocarrier in order to facilitate the delivery of therapeutic agents to the site(s) of infection. As an initial step, we propose the covalent attachment of several copies of N-formyl-Met-Leu-Phe (fMLF), a known chemo-attractant for macrophages. In this article, one or more copies of fMLF were conjugated to multifunctional commercially available or novel, peptide-based PEG nanocarriers in which the structure was varied by appending PEGs with average molecular weights of 5, 20, and 40 kDa. U937 cell-specific binding and cellular uptake were analyzed. The results of uptake studies indicate that (i) uptake is energy dependent and mediated by a fMLF receptor, (ii) appending only 2 copies of the targeting ligand to the multifunctional nanocarrier appears sufficient for binding in vitro, and (iii) of the three configurations studied, the nanocarrier with a molecular weight of about 20 kDa, corresponding to a size of 20−60 nm, demonstrated the highest uptake. The results of the current studies demonstrate the feasibility of targeting macrophages and the suitability of using these synthetically versatile peptide–backbone PEG nanocarriers. The convenience, flexibility and possible limitations of this nanocarrier approach are discussed. PMID:18092743

  16. Flexible supercapacitor based on electrochemically synthesized pyrrole formyl pyrrole copolymer coated on carbon microfibers

    NASA Astrophysics Data System (ADS)

    Gholami, Mehrdad; Moozarm Nia, Pooria; Narimani, Leila; Sokhakian, Mehran; Alias, Yatimah

    2016-08-01

    The main objective of this work is to prepare a flexible supercapacitor using electrochemically synthesized pyrrole formyl pyrrole copolymer P(Py-co-FPy) coated on the carbon microfibers. Due to difficulties of working with carbon microfibers, glassy carbon was used to find out optimized conditions by varying mole ratio of pyrrole and formyl pyrrole monomers on the capacitance value. The prepared electrodes were characterized using Fourier transform infrared spectroscopy (FT-IR), field emission scanning electron microscope (FESEM), Brunauer-Emmett-Teller (BET) analysis, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Then the X-ray photoelectron spectroscopy (XPS) was used to characterize the optimized electrode. The specific capacitance is calculated using cyclic voltammetry, charge/discharge method, and impedance spectroscopy. The charge/discharge study reveals that the best specific capacitance is estimated to be 220.3 mF cm-2 for equal mole fraction of pyrrole and formyl pyrrole Py (0.1)-FP (0.1) at discharge current of 3 × 10-4 A. This optimized electrode keeps about 92% of its capacitance value in high current of discharging. The specific capacitances calculated by all the mentioned methods are in agreement with each other. Finally, the found optimized conditions were successfully applied to produce a flexible supercapacitor on the surface of carbon microfibers.

  17. Enterohepatic circulation of bacterial chemotactic peptide in rats with experimental colitis

    SciTech Connect

    Hobson, C.H.; Butt, T.J.; Ferry, D.M.; Hunter, J.; Chadwick, V.S.; Broom, M.F.

    1988-04-01

    The association of hepatobiliary disorders with colonic inflammation is well recognized. Although the pathophysiology is obscure, increased permeation of toxic bacterial products across the inflamed gut to the portal circulation might be one mechanism. Potentially toxic metabolites include N-formylated chemotactic peptides that are produced by several species of intestinal bacteria and can be detected in colonic fluid in vivo. To investigate the metabolic fate of one of these low molecular weight proinflammatory peptides, N-formyl L-methionine L-leucine /sup 125/I-L-tyrosine was introduced into colon loops of healthy rats (n = 10) and rats with experimental colitis (n = 15) induced by rectal instillation of 15% (vol/vol) acetic acid. Gut, liver, and blood radioactivity were monitored by external gamma-counting and radioactivity in bile was measured by biliary catheter drainage into a well counter. Bile was processed by high-performance liquid chromatography to determine the amount of intact, bioactive peptide excreted over 3 h. After colonic instillation of 1 nmol of peptide, the mean (+/- SEM) biliary excretion of intact peptide was 6.4 +/- 2.0 pmol in normal rats and 49.0 +/- 20 pmol in rats with colitis (p less than 0.01). An enterohepatic circulation of synthetic N-formyl L-methionine L-leucine L-tyrosine has been demonstrated in the rat. Experimental colitis was associated with an eightfold increase in biliary excretion of this proinflammatory bacterial peptide. Proinflammatory bacterial peptides synthesized by colonic bacteria could be important in the pathophysiology of colon inflammation and its frequently associated hepatobiliary complications.

  18. N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage.

    PubMed

    Jiang, Tao; Zhou, Xinfeng; Taghizadeh, Koli; Dong, Min; Dedon, Peter C

    2007-01-02

    The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N(6)-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3'-formylphosphate residues arising from 5'-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N(6)-amino groups of lysine side chains. A liquid chromatography (LC)-tandem mass spectrometry (MS) method was developed to quantify the resulting N(6)-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04-0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose-response relationship for the formation of N(6)-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N(6)-formylation of the linker histone H1. The N(6)-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N(6)-acetylation recognized as an important determinant of gene expression in mammalian cells. The N(6)-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress.

  19. Crystal and molecular structure of two geometrically restricted chemotactic tripeptides, analogues of formyl-methionine-leucine-phenylalanine.

    PubMed

    Michel, A G; Lajoie, G; Hassani, C A

    1990-12-01

    The crystal structures of HCO-Met-Leu-Phe-OC(CH3)3, (CH25H39N3O5S), fMLP-OtBu, and HCO-Met psi [CSNH]-Leu-Phe-OCH3, (C22H33N3O4S2), fMS LP-OMe, have been determined by single crystal X-ray diffraction, and their conformational properties investigated by molecular mechanics energy calculations. Crystals of fMLP-OtBu are monoclinic, space group P2(1), a = 12.027(4), b = 9.492(3), c = 12.660(4) A, beta = 101.99(3) degrees, Z = 2; those of fMS LP-OMe are orthorhombic, space group P2(1)2(1)2(1), a = 7.130(1), b = 12.097(2), c = 31.060(5) A, Z = 4. The first compounds fMLP-OtBu is the t-butyl ester of the tripeptide fMLP that represents one of the most potent compounds in inducing the lysozyme release from human neutrophils that reflects the chemotactic activity. From the crystal structure, it is shown that the orientation of the phenylalanine side chain is largely affected by the presence of the bulky group. fMSLP-OMe was shown to be inactive after thionation of the methionine residue in the original tripeptide. Nevertheless, the crystal structure does not reveal any influence of the presence of the thionated peptidic bond on the backbone conformation. The X-ray results have been used to generate parameters for empirical energy calculations. Subsequently, a strategy based on random generation of conformations followed by energy-minimization was applied to investigate the conformational space of thiopeptides, in comparison with normal peptides. From molecular free energy calculations, it is shown that the main influence of the introduction of a thioamide bond on the molecular structure is to prevent the existence of C7(eq) conformations involving the thiomethionine residue. Consequently, a larger number of conformers are found to form intramolecular hydrogen bonds involving the formyl group, reducing its availability to interact with the receptor. For the first time, the theoretical prediction of the existence of C7eq conformations for fMLP is made. The resulting

  20. Millimeter-wave spectroscopy of syn formyl azide (HC(O)N3) in seven vibrational states

    NASA Astrophysics Data System (ADS)

    Walters, Nicholas A.; Amberger, Brent K.; Esselman, Brian J.; Woods, R. Claude; McMahon, Robert J.

    2017-01-01

    Millimeter-wave spectra for formyl azide (HC(O)N3) were obtained from 240 to 360 GHz at ambient temperature. For the ground state of syn formyl azide, over 1500 independent rotational transitions were measured and least-squares fit to a complete S-reduced 8th order centrifugal distortion/rigid rotor Hamiltonian. The decomposition of formyl azide was monitored over a period of several hours, the half-life (t½ = 30 min) was determined, and its decomposition products were investigated. Transitions from five vibrational satellites of syn formyl azide (ν9, ν12, 2ν9, ν9 + ν12, and ν11) were observed, measured, and least-squares fit to complete or nearly complete octic centrifugally-distorted, single-state S-reduced models. A less complete single-state fit of 3ν9 (509.3 cm-1) was obtained from an unperturbed subset of its assignable transitions. This state is apparently coupled to the fundamental ν8 (489.4 cm-1) and the overtone 2ν12 (503.6 cm-1), but the coupling remains unanalyzed. Anharmonic CCSD(T)/ANO1 estimates of the vibrational frequencies of syn formyl azide were in close agreement with previously published experimental and computational values. Experimentally determined vibration-rotation interaction (αi) values were in excellent agreement with coupled-cluster predicted αi values for the fundamentals ν9, ν12, and ν11.

  1. Antioxidant and anticancer activity of 3'-formyl-4', 6'-dihydroxy-2'-methoxy-5'-methylchalcone and (2S)-8-formyl-5-hydroxy-7-methoxy-6-methylflavanone.

    PubMed

    Ye, Chun-Lin; Liu, Yu; Wei, Dong-Zhi

    2007-04-01

    Two new flavonoids - 3'-formyl-4',6'-dihydroxy-2'-methoxy-5'-methylchalcone (FMC) and (2S)-8-formyl-5-hydroxy-7-methoxy-6-methylflavanone (FMF) - isolated from the buds of Cleistocalyx operculatus, were investigated for their antioxidant and anticancer activity. Total antioxidant activity and reducing ability were measured. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anion radical scavenging assays were carried out to evaluate the antioxidant potential of the two compounds. The antioxidant activity of the two compounds increased in a concentration-dependent manner. FMC and FMF at a concentration of 500 microM inhibited lipid peroxidation by 64.3 +/- 2.5% and 60.3 +/- 2.3%, respectively, an antioxidant activity approximately similar to that of 500 microM alpha-tocopherol (66.3 +/- 2.5%). Similarly, the effect of FMC and FMF on reducing power increased in a concentration-dependent manner. In DPPH radical scavenging assays, the IC50 values of FMC and FMF were 50.2 +/- 2.8 microM and 75.8 +/- 2.5 microM, respectively. Moreover, FMC and FMF scavenged the superoxide generated by the phenazine methosulfate (PMS)/reduced beta-nicotinamide adenine dinucleotide (NADH) nitroblue tetrazolium (NBT) system, with IC50 values of 56.3 +/- 2.3 microM and 317.5 +/- 2.9 microM, respectively. The anticancer activity of the two compounds were determined in five human cancer cell lines, SMMC-7721 (liver cancer), 8898 (pancreatic cancer), K562 (chronic leukaemia), HeLa (tumour of cervix uteri) and 95-D (high metastic lung carcinoma). FMC and FMF showed broad-spectrum anticancer activity against all the human cancer cell lines tested. The results obtained in the current study indicate that the two flavonoids could be a potential source of natural antioxidant and anticancer agents. To our knowledge, this is the first report on bioactivity of FMC and FMF.

  2. Cavity ringdown laser absorption spectroscopy detection of formyl (HCO) radical in a low pressure flame

    NASA Astrophysics Data System (ADS)

    Scherer, J. J.; Rakestraw, D. J.

    1997-01-01

    The formyl radical has been detected via the ÖX˜ system in a low pressure CH 4/N 2/O 2 flame using cavity ringdown laser absorption spectroscopy. The direct absorption data obtained in these initial studies allows determination of the absolute HCO concentration in the flame of ca. 2.1(±0.9) × 10 13 mol cm -3, which is in excellent agreement with flame chemistry simulations. The extrapolated detection limit at 300 K of ≈ 1.4(±0.6) × 10 11 mol cm -3 is comparable to that of other methods, including intracavity laser absorption spectroscopy.

  3. Synthesis of Aldehydes by Organocatalytic Formylation Reactions of Boronic Acids with Glyoxylic Acid.

    PubMed

    Huang, He; Yu, Chenguang; Li, Xiangmin; Zhang, Yongqiang; Zhang, Yueteng; Chen, Xiaobei; Mariano, Patrick S; Xie, Hexin; Wang, Wei

    2017-07-03

    Reported herein is a conceptually novel organocatalytic strategy for the formylation of boronic acids. New reactivity is engineered into the α-amino-acid-forming Petasis reaction occurring between aryl boronic acids, amines, and glyoxylic acids to prepare aldehydes. The operational simplicity of the process and its ability to generate structurally diverse and valued aryl, heteroaryl, and α,β-unsaturated aldehydes containing a wide array of functional groups, demonstrates the practical utility of the new synthetic strategy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Theoretical study on the vibrational spectra of methoxy- and formyl-dihydroxy- trans-stilbenes and their hydrolytic equilibria

    NASA Astrophysics Data System (ADS)

    Molnár, Viktor; Billes, Ferenc; Tyihák, Ernő; Mikosch, Hans

    2008-02-01

    Compounds formed by exchanging one of the resveratrol hydroxy groups to methoxy or formyl groups are biologically important. Quantum chemical DFT calculations were applied for the simulation of some of their properties. Their optimized structures and charge distributions were computed. Based on the calculated vibrational force constants and optimized molecular structure infrared and Raman spectra were calculated. The characteristics of the vibrational modes were determined by normal coordinate analysis. Applying the calculated thermodynamic functions also for resveratrol, methanol, formaldehyde and water, thermodynamic equilibria were calculated for the equilibria between resveratrol and its methyl and formyl substituted derivatives, respectively.

  5. Thyrotropin-releasing hormone enhances the superoxide anion production of rabbit peritoneal macrophages stimulated with N-formyl-methionyl-leucyl-phenylalanine and opsonized zymosan.

    PubMed

    Koshida, H; Kotake, Y

    1993-01-01

    Effects of thyrotropin-releasing hormone (TRH) on the superoxide anion (O2-) production, which is essential for effective microbicidal and cytotoxic activity in macrophages (M phi s), were investigated. TRH by itself failed to induce the O2- production of rabbit peritoneal M phi s elicited with thioglycollate medium. However, M phi s preincubated with TRH showed the significant enhancements of O2- production following stimulation with the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), at TRH concentrations from 10(-7) to 10(-4) M with a peak enhancement at 10(-5) M. O2- generations of M phi s stimulated with opsonized zymosan (OZ) were also enhanced at TRH concentrations from 10(-10) to 10(-5) M with a peak enhancement at 10(-7) M. Maximal enhancements of O2- production were obtained with 10 min preincubation with TRH both for FMLP and OZ stimulations. TRH had no effect on O2- production when stimulated with phorbol 12-myristate 13-acetate. Thyrotropin did not augment the O2- production induced with either FMLP or OZ. These results indicate that TRH has the priming effect on FMLP- and OZ-induced O2- production of rabbit peritoneal M phi s, although the mechanism remains to be clarified.

  6. Morphology and electrical properties of electrochemically synthesized pyrrole-formyl pyrrole copolymer

    NASA Astrophysics Data System (ADS)

    Gholami, Mehrdad; Nia, Pooria Moozarm; Alias, Yatimah

    2015-12-01

    A direct electrochemical copolymerization of pyrrole-formyl pyrrole (Py-co-FPy) was carried out by oxidative copolymerization of formyl pyrrole and pyrrole in LiClO4 aqueous solution through galvanostatic method. The (Py-co-FPy) copolymer was characterized using Fourier-transform infrared spectroscopy (FT-IR), field emission scanning electron microscope (FESEM), energy-filtering transmission electron microscope (EFTEM), thermal gravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The FESEM images showed that the synthesized copolymer had a hollow whelk-like helixes structure, which justifies the enhancement of charge transportation through the copolymer film. Cyclic voltammetry studies revealed that the electrocatalytic activity of synthesized copolymer has improved and the surface coverage in copolymer enhanced 1.6 times compared to polypyrrole alone. Besides, (Py-co-FPy) copolymer showed 2.5 times lower electrochemical charge transfer resistance (Rct) value in impedance spectroscopy. Therefore, this copolymer has a strong potential to be used in several applications such as sensor applications.

  7. Preparation and characterization of 2-formyl-1,4,5,6-tetrahydropyridine, a compound with a cracker-like odor

    USDA-ARS?s Scientific Manuscript database

    2-Acetyltetrahydropyridine is an important flavor constituent that imparts roasty, popcorn-like, and cracker-like odors to foods such as bread, rice, popcorn, taco shells and tortilla chips. We postulated that the homolog with a formyl group in the C-2 position, 2-formyltetrahydropyridine, might hav...

  8. Potassium iodide catalyzed simultaneous C3-formylation and N-aminomethylation of indoles with 4-substituted-N,N-dimethylanilines.

    PubMed

    Li, Lan-Tao; Li, Hong-Ying; Xing, Li-Juan; Wen, Li-Juan; Wang, Peng; Wang, Bin

    2012-12-28

    A one-pot dual functionalization of indoles has been developed. The simultaneous C3-formylation and N-aminomethylation of indoles can be achieved using readily available potassium iodide as a catalyst and tert-butyl peroxybenzoate as a co-oxidant.

  9. Organocatalytic Asymmetric Domino Michael/Henry Reaction of Indolin-3-ones with o-Formyl-β-nitrostyrenes.

    PubMed

    Mahajan, Suruchi; Chauhan, Pankaj; Loh, Charles C J; Uzungelis, Server; Raabe, Gerhard; Enders, Dieter

    2015-04-01

    A highly diastereo- and enantioselective domino Michael/Henry reaction of 1-acetylindolin-3-ones with o-formyl-(E)-β-nitrostyrenes catalyzed by low loading of a quinine-derived amine-squaramide provides the corresponding indolin-3-one derivatives bearing four adjacent stereogenic centers in good to high yields and with excellent stereoselectivities.

  10. Effects of substituents on synthetic analogs of chlorophylls. Part 4: How formyl group location dictates the spectral properties of chlorophylls b, d and f.

    PubMed

    Yuen, Jonathan M; Harris, Michelle A; Liu, Mengran; Diers, James R; Kirmaier, Christine; Bocian, David F; Lindsey, Jonathan S; Holten, Dewey

    2015-01-01

    Photosynthetic organisms are adapted to light characteristics in their habitat in part via the spectral characteristics of the associated chlorophyll pigments, which differ in the position of a formyl group around the chlorin macrocycle (chlorophylls b, d, f) or no formyl group (chlorophyll a). To probe the origin of this spectral tuning, the photophysical and electronic structural properties of a new set of synthetic chlorins are reported. The zinc and free base chlorins have a formyl group at either the 2- or 3-position. The four compounds have fluorescence yields in the range 0.19-0.28 and singlet excited-state lifetimes of ca 4 ns for zinc chelates and ca 8 ns for the free base forms. The photophysical properties of the 2- and 3-formyl zinc chlorins are similar to those observed previously for 13-formyl or 3,13-diformyl chlorins, but differ markedly from those for 7-formyl analogs. Molecular-orbital characteristics obtained from density functional theory (DFT) calculations were used as input to spectral simulations employing the four-orbital model. The analysis has uncovered the key changes in electronic structure engendered by the presence/location of a formyl group at various macrocycle positions, which is relevant to understanding the distinct spectral properties of the natural chlorophylls a, b, d and f. © 2014 The American Society of Photobiology.

  11. Bioactive Peptides

    PubMed Central

    Daliri, Eric Banan-Mwine; Oh, Deog H.; Lee, Byong H.

    2017-01-01

    The increased consumer awareness of the health promoting effects of functional foods and nutraceuticals is the driving force of the functional food and nutraceutical market. Bioactive peptides are known for their high tissue affinity, specificity and efficiency in promoting health. For this reason, the search for food-derived bioactive peptides has increased exponentially. Over the years, many potential bioactive peptides from food have been documented; yet, obstacles such as the need to establish optimal conditions for industrial scale production and the absence of well-designed clinical trials to provide robust evidence for proving health claims continue to exist. Other important factors such as the possibility of allergenicity, cytotoxicity and the stability of the peptides during gastrointestinal digestion would need to be addressed. This review discusses our current knowledge on the health effects of food-derived bioactive peptides, their processing methods and challenges in their development. PMID:28445415

  12. Bioactive Peptides.

    PubMed

    Daliri, Eric Banan-Mwine; Oh, Deog H; Lee, Byong H

    2017-04-26

    The increased consumer awareness of the health promoting effects of functional foods and nutraceuticals is the driving force of the functional food and nutraceutical market. Bioactive peptides are known for their high tissue affinity, specificity and efficiency in promoting health. For this reason, the search for food-derived bioactive peptides has increased exponentially. Over the years, many potential bioactive peptides from food have been documented; yet, obstacles such as the need to establish optimal conditions for industrial scale production and the absence of well-designed clinical trials to provide robust evidence for proving health claims continue to exist. Other important factors such as the possibility of allergenicity, cytotoxicity and the stability of the peptides during gastrointestinal digestion would need to be addressed. This review discusses our current knowledge on the health effects of food-derived bioactive peptides, their processing methods and challenges in their development.

  13. Differential regulation by phorbol ester of formyl-methionyl peptide and leukotriene B sub 4 receptors on human neutrophils

    SciTech Connect

    Goldman, D.W.; Chung, S.; Richards, S. )

    1991-03-15

    Activation of protein kinase C (PKC) with suboptimal does of phorbol myristyl acetate (PMA) will increase fMP receptor expression with parallel potentiation of superoxide generation. PMA-induced changes in leukotriene B{sub 4} (LTB{sub 4}) receptor expression were assessed in parallel with fMP receptor expression to determine if these two independent receptor classes are regulated in a similar manner by PKC. The relative density of fMP receptors was assessed by flow cytometry. The relative density of receptors for LTB{sub 4} was quantitated by incubating 2 {times} 10{sup 6} Ns with 10nM({sup 3}H)-LTB{sub 4} and determining the amount of radioactivity bound after filtration on glass fiber filters. Incubation of N with 10ng/mL PMA induced a 3.2-fold increase in fMP receptor expression by 5 min which was sustained for up to 15 min. In contrast, LTB{sub 4} receptor density decreased by 36% within 5 min. in response to 10 ng/mL PMA. Staurosporine, a potent antagonist of PKC, had no effect of fMP receptor expression but markedly enhanced LTB{sub 4} receptor expression by 1.7-fold at 200nM. PKC acts to decrease the surface expression of LTB{sub 4} receptors in contrast to the enhancement of fMP receptor expression, suggesting in contrast to the enhancement of fMP receptor expression, suggesting that potentiation of N function by PMA may be stimulus-specific.

  14. Hot Band Spectroscopy of the Formyl Cation, H12C16O+

    NASA Astrophysics Data System (ADS)

    Neese, Christopher F.; Kreynin, Peter S.; Oka, Takeshi

    2013-10-01

    Midinfrared spectra of the formyl cation, HCO+, were recorded by direct absorption in a glow discharge (composition CO, 50 mTorr; H2, 50 mTorr; He, 7 Torr) using a color center laser spectrometer and velocity modulation with heterodyne detection. The bands measured were 1000-0000, 1110-0110, 1200-0200, 1220-0220, 1001-0001, 1111-0111, 2000-1000, and 1002-0002. These bands were analyzed and fitted together with all other available rotationally resolved spectra to arrive at a new set of spectroscopic constants for HCO+. The relative intensities were fit to obtain a rotational temperature, Trot = 434 ± 7 K, and vibrational temperatures for each mode, T1 = 3300 ± 240 K, T2 = 841 ± 15 K, and T3 = 3157 ± 68 K.

  15. Experimental determination of the absolute infrared absorption intensities of formyl radical HCO.

    PubMed

    Ryazantsev, Sergey V; Tyurin, Daniil A; Feldman, Vladimir I

    2017-12-05

    Formyl radical HCO is an important reactive intermediate in combustion, atmospheric and extraterrestrial chemistry. Like in the case of other transients, the lack of knowledge of the absolute IR intensities limits the quantitative spectroscopic studies on this species. We report the first experimental determination of the absorption intensities for the fundamental vibrational bands of HCO. The measurements have been performed using matrix-isolation FTIR spectroscopy. Determination of the values was based on the repeated photodissociation and thermal recovery of the HCO radical using the known value of the absorption coefficient of CO. The experimentally determined values (93.2±6.0, 67.2±4.5, and 109.2±6.6kmmol(-1) for the ν1, ν2, and ν3 modes, respectively) have been compared to the calculated IR intensities obtained by DFT and UCCSD(T) computations. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Millimeter-Wave Spectroscopy of Formyl Azide (HC(O)N_3)

    NASA Astrophysics Data System (ADS)

    Walters, Nicholas A.; Amberger, Brent K.; Esselman, Brian J.; Woods, R. Claude; McMahon, Robert J.

    2015-06-01

    Formyl azide (HC(O)N_3) is a highly unstable molecule (t1/2˜2 hours at room temperature as a gas) that has only recently been studied spectroscopically by UV, IR, Raman and NMR methods. We have synthesized formyl azide and obtained its absorption spectrum at room temperature over the range 250-360 GHz. As in the case of carbonyl diazide, two conformers are expected for HC(O)N_3, with the syn-isomer 2.8 kcal/mol lower in energy than the anti-isomer (CCSD(T)/ANO1). Calculations at the same level of theory and the same basis set predict the dipole moments for the syn-isomer (μ = 1.56 D) and anti-isomer (μ = 2.56 D). These calculations also indicate that b-type transitions should dominate the syn-isomer spectrum, while a-type transitions become more significant in the case of the anti-isomer. Despite the anti-isomer having a larger dipole moment, the syn-isomer still gives rise to all the dominant features of the spectrum. Thus far, five vibrational states (νb{9}, νb{12}, 2νb{9}, νb{9} + νb{12}, νb{11}) have been studied for the syn-isomer, with the highest energy state νb{11} = 582.6 wn. Searches for the spectra of the anti-isomer are ongoing. Banert, K. et al. Angew. Chem. Int. Ed. 2012, 51, 4718-4721 Zeng, X. et al. Angew. Chem. Int. Ed. 2013, 52, 3503-3506 Amberger, B.K. et al. J. Mol. Spectrosc. 259, (2014) 15-20

  17. Demetalation kinetics of natural chlorophylls purified from oxygenic photosynthetic organisms: effect of the formyl groups conjugated directly to the chlorin pi-macrocycle.

    PubMed

    Hirai, Yuki; Tamiaki, Hitoshi; Kashimura, Shigenori; Saga, Yoshitaka

    2009-12-01

    Demetalation kinetics of natural chlorophyll (Chl) d purified from Acaryochloris marina was first studied and compared with those of Chls a and b. The demetalation rate constant of Chl d, which possessed a formyl group at the 3-position, was five-fold smaller than that of Chl a possessing a vinyl group at the same position in aqueous acetone at the proton concentration of 1.2 x 10(-3) M at 25 degrees C. In contrast, the demetalation rate constant of Chl b possessing a formyl group at the 7-position was 26 times smaller than that of Chl a. The activation energy of demetalation reaction of Chl d was larger than that of Chl a, but smaller than that of Chl b. These indicate that the substitution effect of 3-formyl group on the acidic removal of central magnesium in Chls was smaller than that of 7-formyl group.

  18. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  19. Dimethyl sulfoxide participant iron-mediated cascade oxidation/α-formylation reaction of substituted 2,3-dihydropyrroles under air and protonic acid free condition.

    PubMed

    Zhang, Zhiguo; Tian, Qing; Qian, Jingjing; Liu, Qingfeng; Liu, Tongxin; Shi, Lei; Zhang, Guisheng

    2014-09-05

    An efficient and Brønsted acid free one-pot protocol to directly generate structurally sophisticated α-formylpyrrole derivatives in moderate to good yields has been demonstrated, involving an iron-mediated domino oxidation/formylation reaction of readily available 2,3-dihydro-1H-pyrroles in dimethyl sulfoxide and air atmosphere, in which dimethyl sulfoxide acts as the formyl donor. A possible mechanism is presented.

  20. Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay.

    PubMed

    Nguyen, Kiet T; Hu, Xubo; Pei, Dehua

    2004-06-01

    A new spectrophotometric/fluorimetric assay for peptide deformylase (PDF) has been developed by coupling the PDF reaction with that of dipeptidyl peptidase I (DPPI) and using N-formyl-Met-Lys-AMC as substrate. Removal of the N-terminal formyl group by PDF renders the dipeptide an efficient substrate of DPPI, which subsequently removes the dipeptidyl units to release 7-amino-4-methylcoumarin as the chromophore/fluorophore. The PDF reaction is conveniently monitored on a UV-Vis spectrophotometer or a fluorimeter in a continuous fashion. The utility of the assay was demonstrated by determining the catalytic activity of PDF and the inhibition constants of PDF inhibitors. These studies revealed the slow-binding behavior of a previously reported macrocyclic PDF inhibitor. This method offers several advantages over the existing PDF assays and should be particularly useful for screening PDF inhibitors in the continuous fashion.

  1. The Sensing of Humidity by Surface-Type Ag/FORMYL-TIPPCu(II)/Ag Sensor for Environmental Monitoring

    NASA Astrophysics Data System (ADS)

    Khan, Dil Nawaz; Sayyad, Muhammad Hassan; Tahir, Muhammad; Wahab, Fazal; Yaseen, Muhammad; Ali, Mukhtar; Munawar, Munawar Ali

    2014-06-01

    In this paper, we have studied the effects of changing relative humidity on the electrical parameters and their multi frequency response of the surface type Ag/formyl-TIPPCu(II)/Ag humidity sensors. The silver electrodes of thickness 100 nm were primarily deposited on cleaned glass substrates by thermal evaporator. A gap of 40 μm was created between the electrodes of each device by using mask during the evaporation process. Thin films of formyl-TIPPCu(II) of 140 nm thickness were grown on silver electrodes by thermal sublimation technique. The values of capacitance and resistance of the sensors were found at different humidity levels at frequency of 1, 10 and 100 kHz of AC input signal. A remarkable increase in capacitance and decrease in resistance were observed during the rise of relative humidity from 45% to 95% RH. The hysteresis response of these humidity sensors was also studied at 1 kHz AC signal.

  2. Vibronic structure in triatomic molecules: The hydrocarbon flame bands of the formyl radical (HCO). A theoretical study

    NASA Astrophysics Data System (ADS)

    Serrano-Andrés, Luis; Forsberg, Niclas; Malmqvist, Per-A.˚Ke

    1998-05-01

    A theoretical study of the vibrational structure of the X˜ 2A' ground and B˜ 2A' excited states of the formyl radical, HCO, and its deuterated form, DCO, has been performed. The potential energy surfaces have been computed by means of a multiconfigurational perturbative method, CASPT2. The computed geometries and the harmonic and anharmonic frequencies are successfully compared to the available experimental information. The vibrational intensities of the transition B˜ 2A'↔X˜2A' have been computed both for absorption and emission. The results lead to accurate determinations of several structural parameters and some reassignments of the vibrational transitions of the so-called hydrocarbon flame bands of the formyl radical.

  3. Synthesis and biological evaluation of novel formyl-pyrazoles bearing coumarin moiety as potent antimicrobial and antioxidant agents.

    PubMed

    Nagamallu, Renuka; Kariyappa, Ajay Kumar

    2013-12-01

    A series of coumarin appended formyl-pyrazoles 14-18 were synthesized by a simple and accessible approach. The reaction of 8-acetyl-4-methyl-7-hydroxy coumarin 3 and phenyl hydrazine hydrochlorides 4-8 produces the intermediate compounds 8-acetyl-4-methyl-7-hydroxy coumarin hydrazones 9-13. The reaction of compounds 9-13 and DMF in the presence of POCl3 yielded formyl-pyrazoles bearing coumarin moiety 14-18 in good yield. The synthesized new compounds 14-18 and the intermediates 8-acetyl-4-methyl-7-hydroxy coumarin hydrazones 9-13 prepared were screened in vitro for their antibacterial, antifungal antioxidant activities. The compounds 12 and 17 having chloro substitution exhibited promising antifungal and antibacterial activity against the different organisms tested. The compound 17 showed remarkable DPPH radical scavenging ability. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Efficient sonochemical synthesis of alkyl 4-aryl-6-chloro-5-formyl-2-methyl-1,4-dihydropyridine-3-carboxylate derivatives.

    PubMed

    Ruiz, Enrique; Rodríguez, Hortensia; Coro, Julieta; Niebla, Vladimir; Rodríguez, Alfredo; Martínez-Alvarez, Roberto; de Armas, Hector Novoa; Suárez, Margarita; Martín, Nazario

    2012-03-01

    A facile, efficient and environment-friendly protocol for the synthesis of 6-chloro-5-formyl-1,4-dihydropyridine derivatives has been developed by the convenient ultrasound-mediated reaction of 2(1H)pyridone derivatives with the Vilsmeier-Haack reagent. This method provides several advantages over current reaction methodologies including a simpler work-up procedure, shorter reaction times and higher yields. Copyright © 2011. Published by Elsevier B.V.

  5. The immune-stimulating peptide WKYMVm has therapeutic effects against ulcerative colitis

    PubMed Central

    Kim, Sang Doo; Kwon, Soonil; Lee, Sung Kyun; Kook, Minsoo; Lee, Ha Young; Song, Ki-Duk; Lee, Hak-Kyo; Baek, Suk-Hwan; Park, Chan Bae; Bae, Yoe-Sik

    2013-01-01

    In this study, we examined the therapeutic effects of an immune-stimulating peptide, WKYMVm, in ulcerative colitis. The administration of WKYMVm to dextran sodium sulfate (DSS)-treated mice reversed decreases in body weight, bleeding score and stool score in addition to reversing DSS-induced mucosa destruction and shortened colon. The WKYMVm-induced therapeutic effect against ulcerative colitis was strongly inhibited by a formyl peptide receptor (FPR) 2 antagonist, WRWWWW, indicating the crucial role of FPR2 in this effect. Mechanistically, WKYMVm effectively decreases intestinal permeability by stimulating colon epithelial cell proliferation. WKYMVm also strongly decreases interleukin-23 and transforming growth factor-β production in the colon of DSS-treated mice. We suggest that the potent immune-modulating peptide WKYMVm and its receptor FPR2 may be useful in the development of efficient therapeutic agents against chronic intestinal inflammatory diseases. PMID:24030327

  6. Synthesis of adrenal peptide E and some of its biological activities.

    PubMed

    Heimer, E P; Lambros, T J; Felix, A M; Fleminger, G; Li, C H; Westphal, M; Meienhofer, J

    1983-09-01

    A synthesis of peptide E, a highly potent, 25-amino acid adrenal opioid peptide containing both a [Met]enkephalin at the NH2-terminus and [Leu]enkephalin sequence at the COOH-terminus, originally isolated from bovine adrenal medulla [D. L. Kilpatrick, T. Taniguchi, B. N. Jones, A. S. Stern, J. E. Shively, J. Hullihan, S. Kimura, S. Stein, and S. Udenfriend (1981) Proc. Natl. Acad. Sci. USA 78, 3265-3268], is reported. The synthesis was accomplished by the solid-phase method employing the 4-(aminoacyloxymethyl)phenylacetamidomethyl(Pam)-copoly(styrene-1% divinylbenzene) resin. Two synthetic strategies (N-indole formyl protected vs unprotected tryptophan) were followed and results compared and evaluated. It was determined that peptide E prepared with protection of tryptophan (residues 13 and 14) was preferred and gave final product that was readily purified by HPLC. The biological activity of the synthetic material was found to be equivalent to the reported activity of the natural compound.

  7. Bioactive peptides derived from natural proteins with respect to diversity of their receptors and physiological effects.

    PubMed

    Yoshikawa, Masaaki

    2015-10-01

    We have found various bioactive peptides derived from animal and plant proteins, which interact with receptors for endogenous bioactive peptides such as opioids, neurotensin, complements C3a and C5a, oxytocin, and formyl peptides etc. Among them, rubiscolin, a δ opioid peptide derived from plant RuBisCO, showed memory-consolidating, anxiolytic-like, and food intake-modulating effects. Soymorphin, a μ opioid peptide derived from β-conglycinin showed anxiolytic-like, anorexigenic, hypoglycemic, and hypotriglyceridemic effects. β-Lactotensin derived from β-lactoglobulin, the first natural ligand for the NTS2 receptor, showed memory-consolidating, anxiolytic-like, and hypocholesterolemic effects. Weak agonist peptides for the complements C3a and C5a receptors were released from many proteins and exerted various central effects. Peptides showing anxiolytic-like antihypertensive and anti-alopecia effects via different types of receptors such as OT, FPR and AT2 were also obtained. Based on these study, new functions and post-receptor mechanisms of receptor commom to endogenous and exogenous bioactive peptides have been clarified. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Artificially produced [7-formyl]-chlorophyll d functions as an antenna pigment in the photosystem II isolated from the chlorophyllide a oxygenase-expressing Acaryochloris marina.

    PubMed

    Tsuchiya, Tohru; Akimoto, Seiji; Mizoguchi, Tadashi; Watabe, Kazuyuki; Kindo, Hayato; Tomo, Tatsuya; Tamiaki, Hitoshi; Mimuro, Mamoru

    2012-08-01

    Acaryochloris marina, a chlorophyll (Chl) d-dominated cyanobacterium, is a model organism for studying photosynthesis driven by far-red light using Chl d. Furthermore, studies on A. marina may provide insights into understanding how the oxygenic photosynthetic organisms adapt after the acquisition of new Chl. To solve the reaction mechanism of its unique photosynthesis, photosystem (PS) II complexes were isolated from A. marina and analyzed. However, the lack of a molecular genetic method for A. marina prevented us from conducting further studies. We recently developed a transformation system for A. marina and we introduced a chlorophyllide a oxygenase gene into A. marina. The resultant transformant accumulated [7-formyl]-Chl d, which has never been found in nature. In the current study, we isolated PS II complexes that contained [7-formyl]-Chl d. The pigment composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96±0.04 Chl a, 53.21±1.00 Chl d, and 5.48±0.33 [7-formyl]-Chl d per two pheophytin a molecules. In contrast, the composition of the control PS II complexes was 2.01±0.06 Chl a and 62.96±2.49 Chl d. The steady-state fluorescence and excitation spectra of the PS II complexes revealed that energy transfer occurred from [7-formyl]-Chl d to the major Chl d species; however, the electron transfer was not affected by the presence of [7-formyl]-Chl d. These findings demonstrate that artificially produced [7-formyl]-Chl d molecules that are incorporated into PS II replace part of the Chl d molecules and function as the antenna. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Combined spectroscopic and DFT studies on 6-bromo-4-chloro-3-formyl coumarin.

    PubMed

    Raj, R K; Gunasekaran, S; Gnanasambandan, T; Seshadri, S

    2015-03-15

    The FTIR and FT-Raman spectra of 6-bromo-4-chloro-3-formyl coumarin (6B4C3FC) have been recorded in the region 4000-400 and 4000-100 cm(-1), respectively. The optimized geometry, frequency and intensity of the vibrational bands were obtained by the density functional theory (DFT) using 6-31G(d,p) basis set. The harmonic vibrational frequencies were scaled and compared with experimental values. The observed and the calculated frequencies were found to be in good agreement. The UV-Visible spectrum was also recorded and compared with the theoretical values. The calculated HOMO and LUMO energies show that charge transfer occurs within molecule. The first order hyperpolarizability (β0) of 6B4C3FC is 21 times greater than that of urea. Stability of the molecule arising from hyperconjugative interactions, charge delocalization have been analyzed using natural bond orbital (NBO) analysis. Information about the charge density distribution of the molecule and its chemical reactivity has been obtained by mapping molecular electrostatic potential surface. In addition, the non-linear optical properties were discussed from the dipole moment values and the excitation wavelength in the UV-Visible region. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Combined spectroscopic and DFT studies on 6-bromo-4-chloro-3-formyl coumarin

    NASA Astrophysics Data System (ADS)

    Raj, R. K.; Gunasekaran, S.; Gnanasambandan, T.; Seshadri, S.

    2015-03-01

    The FTIR and FT-Raman spectra of 6-bromo-4-chloro-3-formyl coumarin (6B4C3FC) have been recorded in the region 4000-400 and 4000-100 cm-1, respectively. The optimized geometry, frequency and intensity of the vibrational bands were obtained by the density functional theory (DFT) using 6-31G(d,p) basis set. The harmonic vibrational frequencies were scaled and compared with experimental values. The observed and the calculated frequencies were found to be in good agreement. The UV-Visible spectrum was also recorded and compared with the theoretical values. The calculated HOMO and LUMO energies show that charge transfer occurs within molecule. The first order hyperpolarizability (β0) of 6B4C3FC is 21 times greater than that of urea. Stability of the molecule arising from hyperconjugative interactions, charge delocalization have been analyzed using natural bond orbital (NBO) analysis. Information about the charge density distribution of the molecule and its chemical reactivity has been obtained by mapping molecular electrostatic potential surface. In addition, the non-linear optical properties were discussed from the dipole moment values and the excitation wavelength in the UV-Visible region.

  11. Different equation-of-motion coupled cluster methods with different reference functions: The formyl radical

    NASA Astrophysics Data System (ADS)

    Kuś, Tomasz; Bartlett, Rodney J.

    2008-09-01

    The doublet and quartet excited states of the formyl radical have been studied by the equation-of-motion (EOM) coupled cluster (CC) method. The Sz spin-conserving singles and doubles (EOM-EE-CCSD) and singles, doubles, and triples (EOM-EE-CCSDT) approaches, as well as the spin-flipped singles and doubles (EOM-SF-CCSD) method have been applied, subject to unrestricted Hartree-Fock (HF), restricted open-shell HF, and quasirestricted HF references. The structural parameters, vertical and adiabatic excitation energies, and harmonic vibrational frequencies have been calculated. The issue of the reference function choice for the spin-flipped (SF) method and its impact on the results has been discussed using the experimental data and theoretical results available. The results show that if the appropriate reference function is chosen so that target states differ from the reference by only single excitations, then EOM-EE-CCSD and EOM-SF-CCSD methods give a very good description of the excited states. For the states that have a non-negligible contribution of the doubly excited configurations one is able to use the SF method with such a reference function, that in most cases the performance of the EOM-SF-CCSD method is better than that of the EOM-EE-CCSD approach.

  12. Optimized synthesis of the melatonin metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK).

    PubMed

    Ximenes, Valdecir F

    2008-10-01

    N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) is the product of oxidative pyrrole ring cleavage of melatonin. AFMK and its deformylated derivative N(1)-acetyl-5-methoxykynuramine (AMK) are compounds for which there are increasing demands because of their antioxidant, immunomodulatory and anti-inflammatory properties. Here, we sought to determine the best reaction conditions for preparation of AFMK using chlorpromazine (CPZ) as a co-catalyst in the peroxidase-mediated oxidation of melatonin. The parameters studied were pH, identity and concentration of buffers, hydrogen peroxide (H(2)O(2)) and CPZ concentrations and the presence or absence of dissolved molecular oxygen in the reaction medium. The rate and efficiency of AFMK production were compared with a noncatalyzed method which uses a high concentration of H(2)O(2). We found that by using CPZ and bubbling molecular oxygen during the course of the reaction, the yield of AFMK was significantly increased (about 60%) and the reaction time decreased (about 30 min), as compared with the noncatalyzed reaction (yield 32% and reaction time 4 hr). Based on these data, we suggest that this could be a new, easily performed and efficient route for AFMK preparation. Additionally, we provide evidence that a radical chain reaction could be responsible for the formation of AFMK.

  13. Different equation-of-motion coupled cluster methods with different reference functions: the formyl radical.

    PubMed

    Kuś, Tomasz; Bartlett, Rodney J

    2008-09-14

    The doublet and quartet excited states of the formyl radical have been studied by the equation-of-motion (EOM) coupled cluster (CC) method. The S(z) spin-conserving singles and doubles (EOM-EE-CCSD) and singles, doubles, and triples (EOM-EE-CCSDT) approaches, as well as the spin-flipped singles and doubles (EOM-SF-CCSD) method have been applied, subject to unrestricted Hartree-Fock (HF), restricted open-shell HF, and quasirestricted HF references. The structural parameters, vertical and adiabatic excitation energies, and harmonic vibrational frequencies have been calculated. The issue of the reference function choice for the spin-flipped (SF) method and its impact on the results has been discussed using the experimental data and theoretical results available. The results show that if the appropriate reference function is chosen so that target states differ from the reference by only single excitations, then EOM-EE-CCSD and EOM-SF-CCSD methods give a very good description of the excited states. For the states that have a non-negligible contribution of the doubly excited configurations one is able to use the SF method with such a reference function, that in most cases the performance of the EOM-SF-CCSD method is better than that of the EOM-EE-CCSD approach.

  14. Ultrafast intramolecular charge transfer of formyl perylene observed using femtosecond transient absorption spectroscopy.

    PubMed

    Mohammed, Omar F

    2010-11-04

    The excited-state photophysics of formylperylene (FPe) have been investigated in a series of nonpolar, polar aprotic, and polar protic solvents. A variety of experimental and theoretical methods were employed including femtosecond transient absorption (fs-TA) spectroscopy with 130 fs temporal resolution. We report that the ultrafast intramolecular charge transfer from the perylene unit to the formyl (CHO) group can be facilitated drastically by hydrogen-bonding interactions between the carbonyl group oxygen of FPe and hydrogen-donating solvents in the electronically excited state. The excited-state absorption of FPe in methanol (MeOH) is close to the reported perylene radical cation produced by bimolecular quenching by an electron acceptor. This is a strong indication for a substantial charge transfer in the S(1) state in protic solvents. The larger increase of the dipole moment change in the protic solvents than that in aprotic ones strongly supports this observation. Relaxation mechanisms including vibrational cooling and solvation coupled to the charge-transfer state are also discussed.

  15. N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils

    PubMed Central

    Mora-Cartín, Ricardo; Chacón-Díaz, Carlos; Gutiérrez-Jiménez, Cristina; Gurdián-Murillo, Stephany; Lomonte, Bruno; Chaves-Olarte, Esteban

    2016-01-01

    Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus. In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus. Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution. PMID:27001541

  16. Synthesis of RNA containing 5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine.

    PubMed

    Michaelides, Iacovos N; Tago, Nobuhiro; Viverge, Bastien; Carell, Thomas

    2017-09-14

    5-Hydroxymethyl-, 5-formyl-, and 5-carboxycytidine are new epigenetic bases (hmdC, fdC, cadC) that were recently discovered in the DNA of higher eukaryotes. The same bases (hmC, fC and caC) have now also been detected in mammalian RNA with a high abundance in mRNA. While for the DNA bases phosphoramidites (PAs) are available that allow the synthesis of xdC-containing oligonucleotides for deeper biological studies, the corresponding RNA PAs for fC and caC have not yet been disclosed. Here we report novel RNA PAs for hmC, fC and caC that can be used in routine RNA synthesis. The new building blocks are compatible with the canonical PAs and also with themselves, which enables even the synthesis of RNA strands containing all three of these bases. The study will pave the way for detailed physical, biochemical and biological studies to unravel the function of these non-canonical modifications in RNA. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Photodissociation dynamics of formyl fluoride (HFCO) at 193 nm: Branching ratios and distributions of kinetic energy

    SciTech Connect

    Lee, H.; Wu, C.-Y.; Yang, S.K.; Lee, Y.-P.

    2005-08-15

    Following photodissociation of formyl fluoride (HFCO) at 193 nm, we detected products with fragmentation translational spectroscopy utilizing a tunable vacuum ultraviolet beam from a synchrotron for ionization. Among three primary dissociation channels observed in this work, the F-elimination channel HFCO{yields}HCO+F dominates, with a branching ratio {approx}0.66 and an average release of kinetic energy {approx}55 kJ mol{sup -1}; about 17% of HCO further decomposes to H+CO. The H-elimination channel HFCO{yields}FCO+H has a branching ratio {approx}0.28 and an average release of kinetic energy {approx}99 kJ mol{sup -1}; about 21% of FCO further decomposes to F+CO. The F-elimination channel likely proceeds via the S{sub 1} surface whereas the H-elimination channel proceeds via the T{sub 1} surface; both channels exhibit moderate barriers for dissociation. The molecular HF-elimination channel HFCO{yields}HF+CO, correlating with the ground electronic surface, has a branching ratio of only {approx}0.06; the average translational release of 93 kJ mol{sup -1}, {approx}15% of available energy, implies that the fragments are highly internally excited. Detailed mechanisms of photodissociation are discussed.

  18. Synthesis, characterization, DNA-binding studies and acetylcholinesterase inhibition activity of new 3-formyl chromone derivatives.

    PubMed

    Parveen, Mehtab; Malla, Ali Mohammed; Yaseen, Zahid; Ali, Akhtar; Alam, Mahboob

    2014-01-05

    A series of new substituted 3-formyl chromone derivatives (4-6) were synthesized by one step reaction methodology by knoevenagel condensation, structurally similar to known bisintercalators. The new compounds were characterized by IR, (1)H NMR, (13)C NMR, MS and analytical data. The in vitro DNA binding profile of compounds (4-6) was carried out by absorption, fluorescence and viscosity measurements. It was found that synthesized compounds, especially compound 6 (evident from binding constant value) bind strongly with calf thymus DNA, presumably via an intercalation mode. Additionally, molecular docking studies of compounds (4-6) were carried out with B-DNA (PDBID: 1BNA) which revealed that partial intercalative mode of mechanism is operational in synthesized compounds (4-6) with CT-DNA. The binding constants evaluated from fluorescence spectroscopy of compounds with CT-DNA follows the order compound 6>compound 5>compound 4. All the compounds (4-6) were screened for acetylcholinesterase inhibition assay. It can be inferred from data, that compound (6) showed potent AChE inhibition having IC50=0.27μM, almost in vicinity to reference drug Tacrine (IC50=0.19μM). Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Field test results of the physical solvent N-Formyl morpholine for gas treating applications

    SciTech Connect

    Palla, N.; Lee, A.L.

    1997-12-31

    The Institute of Gas Technology (IGT) is developing gas processing technology that will reduce gas processing costs for current production and allow subquality gas to be economically produced that would have been otherwise, not produced. The experimental program has primarily focused on the evaluation of N-Formyl Morpholine (NFM) as a physical solvent for the cost-effective upgrading of subquality natural gas to pipeline quality. The selection of NFM for this program was based on previous work conducted by IGT in the selective removal of hydrogen sulfide, and carbon dioxide from coal gasifier effluents. That work showed that the use of NFM resulted in a significant cost advantage over 107 other solvents for that application. The project approach for the development of NFM process has been divided into following main categories: obtain vapor-liquid equilibrium, physical properties and additional published literature data; obtain mass-transfer coefficients using 2 inch absorber/stripper apparatus and calculate equation of state parameters and binary interaction parameters using VLE data; develop a gas processing model using Aspen Plus simulation program and evaluate economic advantages of the NFM process compared to commercial physical solvent; and design a pilot plant skid mounted field test unit and conduct field test experiments.

  20. New peptide deformylase inhibitors design, synthesis and pharmacokinetic assessment.

    PubMed

    Lv, Fengping; Chen, Chen; Tang, Yang; Wei, Jianhai; Zhu, Tong; Hu, Wenhao

    2016-08-01

    The docking approach for the screening of designed small molecule ligands, led to the identification of a critical arginine residue in peptide deformylase for spiro cyclopropyl PDF inhibitor's extra hydrophobic binding, providing us a useful tool for searching more efficient PDF inhibitors to fight for horrifying antibiotics resistance. Further synthetic modification was undertaken to optimize the potency of amide compounds. To lower metabolic susceptibility and in turn reduce unwanted metabolic toxicity that was observed clinically, while retaining desired antibacterial activity, the use of azoles as amide bioisosteres had also been investigated. After the completion of chemical synthesis, all the compounds were evaluated through in vitro antibacterial activity assay, some of which were further subject to in vivo rat pharmacokinetic assessment. Those findings in this letter showed that spiro cyclopropyl proline N-formyl hydroxylamines, and especially the bioisosteric azoles, can represent a promising class of PDF inhibitors.

  1. Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

    PubMed

    Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S

    2017-07-25

    Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

  2. Photoinduced reactions of both 2-formyl-2H-azirine and isoxazole: A theoretical study based on electronic structure calculations and nonadiabatic dynamics simulations.

    PubMed

    Cao, Jun

    2015-06-28

    In the present work, the combined electronic structure calculations and dynamics simulations have been performed to explore photocleavages of 2-formyl-2H-azirine and isoxazole in the gas phase and the subsequent rearrangement reactions. The carbonyl n → π(*) transition induces a cleavage of the C-N single bond of 2-formyl-2H-azirine to yield β-formylvinylnitrene in open-shell singlet state. However, the n → π(*) excitation of the imine chromophore results in a cleavage of the C-C single bond, producing a nitrile ylide intermediate through an internal conversion to the ground state. β-formylvinylnitrene and nitrile ylide with the carbonyl group are easily transformed into 2-formyl-2H-azirine and oxazole, respectively. The N-O bond cleavages on both S1((1)ππ(*)) and S2((1)nNπ(*)) of isoxazole are ultrafast processes, and they give products of 2-formyl-2H-azirine, 3-formylketenimine, HCN + CHCHO, and HCO + CHCHN. Both 2H-azirines and ketenimines were suggested to be formed from the triplet vinylnitrenes by intersystem crossing in the previous studies. However, our calculations show that the singlet β-formylvinylnitrene is responsible for the formation of 2-formyl-2H-azirine and 3-formylketenimine, and the singlet vinylnitrenes can play a key role in the photoinduced reactions of both 2H-azirines and isoxazoles.

  3. Photoinduced reactions of both 2-formyl-2H-azirine and isoxazole: A theoretical study based on electronic structure calculations and nonadiabatic dynamics simulations

    NASA Astrophysics Data System (ADS)

    Cao, Jun

    2015-06-01

    In the present work, the combined electronic structure calculations and dynamics simulations have been performed to explore photocleavages of 2-formyl-2H-azirine and isoxazole in the gas phase and the subsequent rearrangement reactions. The carbonyl n → π* transition induces a cleavage of the C—N single bond of 2-formyl-2H-azirine to yield β-formylvinylnitrene in open-shell singlet state. However, the n → π* excitation of the imine chromophore results in a cleavage of the C—C single bond, producing a nitrile ylide intermediate through an internal conversion to the ground state. β-formylvinylnitrene and nitrile ylide with the carbonyl group are easily transformed into 2-formyl-2H-azirine and oxazole, respectively. The N—O bond cleavages on both S1(1ππ*) and S2(1nNπ*) of isoxazole are ultrafast processes, and they give products of 2-formyl-2H-azirine, 3-formylketenimine, HCN + CHCHO, and HCO + CHCHN. Both 2H-azirines and ketenimines were suggested to be formed from the triplet vinylnitrenes by intersystem crossing in the previous studies. However, our calculations show that the singlet β-formylvinylnitrene is responsible for the formation of 2-formyl-2H-azirine and 3-formylketenimine, and the singlet vinylnitrenes can play a key role in the photoinduced reactions of both 2H-azirines and isoxazoles.

  4. Photoinduced reactions of both 2-formyl-2H-azirine and isoxazole: A theoretical study based on electronic structure calculations and nonadiabatic dynamics simulations

    SciTech Connect

    Cao, Jun

    2015-06-28

    In the present work, the combined electronic structure calculations and dynamics simulations have been performed to explore photocleavages of 2-formyl-2H-azirine and isoxazole in the gas phase and the subsequent rearrangement reactions. The carbonyl n → π{sup *} transition induces a cleavage of the C—N single bond of 2-formyl-2H-azirine to yield β-formylvinylnitrene in open-shell singlet state. However, the n → π{sup *} excitation of the imine chromophore results in a cleavage of the C—C single bond, producing a nitrile ylide intermediate through an internal conversion to the ground state. β-formylvinylnitrene and nitrile ylide with the carbonyl group are easily transformed into 2-formyl-2H-azirine and oxazole, respectively. The N—O bond cleavages on both S{sub 1}({sup 1}ππ{sup *}) and S{sub 2}({sup 1}n{sub N}π{sup *}) of isoxazole are ultrafast processes, and they give products of 2-formyl-2H-azirine, 3-formylketenimine, HCN + CHCHO, and HCO + CHCHN. Both 2H-azirines and ketenimines were suggested to be formed from the triplet vinylnitrenes by intersystem crossing in the previous studies. However, our calculations show that the singlet β-formylvinylnitrene is responsible for the formation of 2-formyl-2H-azirine and 3-formylketenimine, and the singlet vinylnitrenes can play a key role in the photoinduced reactions of both 2H-azirines and isoxazoles.

  5. Antimicrobial Peptides

    PubMed Central

    Bahar, Ali Adem; Ren, Dacheng

    2013-01-01

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics). PMID:24287494

  6. N-formylation and N-methylation of amines using metal-free N-heterocyclic carbene catalysts and CO2 as carbon source.

    PubMed

    Bobbink, Felix D; Das, Shoubhik; Dyson, Paul J

    2017-02-01

    N-formylation and N-methylation of amines are important reactions that are used to produce a wide range of key intermediates and compounds. This protocol describes the environmentally benign N-formylation and N-methylation of primary and secondary amines using carbon dioxide (CO2) as the carbon source, hydrosilanes as reductants and N-heterocyclic carbenes (NHCs) as catalysts. Using CO2 as a reagent has the advantage of low cost and negligible toxicity. However, the catalyst is air-sensitive and must be generated fresh before use; consequently, the techniques used to prepare and manipulate the catalyst are described. The synthetic approach described in this protocol does not use any toxic reagents; using the appropriate catalyst, N-formylated or N-methylated products can be obtained with high selectivity. The overall time for catalyst preparation and for conducting several catalytic reactions in parallel is 15-48 h, depending on the nature of the substrates.

  7. 3'-Formyl phosphate-ended DNA: high-energy intermediate in antibiotic-induced DNA sugar damage.

    PubMed Central

    Chin, D H; Kappen, L S; Goldberg, I H

    1987-01-01

    Under anaerobic conditions where the nitroaromatic radiation-sensitizer misonidazole substitutes for dioxygen, DNA strand breakage (gaps with phosphate residues at each end) by the nonprotein chromophore of the antitumor antibiotic neocarzinostatin (NCS-Chrom) is associated with the generation of a reactive form of formate from the C-5' of deoxyribose of thymidylate residues. Such lesions account for a minority (10-15%) of the strand breakage found in the aerobic reaction without misonidazole. Amino-containing nucleophiles such as tris(hydroxymethyl)aminomethane (Tris) and hydroxylamine act as acceptors for the activated formate. The amount of [3H]formyl hydroxamate produced from DNA labeled with [5'-3H]thymidine is comparable to the spontaneously released thymine. During the course of the reaction, misonidazole undergoes a DNA-dependent reduction and subsequent conjugation with glutathione used to activate NCS-Chrom. From these and earlier results, we propose a possible mechanism in which the carbon-centered radical formed at C-5' by hydrogen atom abstraction by thiol-activated NCS-Chrom reacts anaerobically with misonidazole to form a nitroxyl-radical-adduct intermediate, which fragments to produce an oxy radical at C-5'. beta-Fragmentation results in cleavage between C-5' and C-4' with the generation of 3'-formyl phosphate-ended DNA, a high-energy form of formate, which spontaneously hydrolyzes, releasing formate and creating a 3'-phosphate end, or transfers the formyl moiety to available nucleophiles. A similar mechanism, involving dioxygen addition, is probably responsible for the 10-15% DNA gap formation in the aerobic reaction. PMID:2959956

  8. Identification of 1-chloro-2-formyl indenes and tetralenes as novel antistaphylococcal agents exhibiting sortase A inhibition.

    PubMed

    Kahlon, Amandeep Kaur; Negi, Arvind S; Kumari, Ruma; Srivastava, Kishore K; Kumar, Shiv; Darokar, Mahendra P; Sharma, Ashok

    2014-03-01

    Tetralene and indene compounds have shown inhibitory activity against human pathogen, Mycobacterium tuberculosis. Their potential use as antistaphylococcal agent against drug-resistant Staphylococcus aureus has not been explored so far. We determined in vitro antistaphylococcal activity and mechanism of action of these compounds as sortase A inhibitors through in silico analysis followed by biological assays. Tetralene and indene series were tested against S. aureus strains MTCC96, MRSA, and VA30. Three compounds showed significant reduction in MIC in both wild-type and drug-resistant S. aureus strains. In silico absorption, distribution, metabolism, excretion, and toxicity analysis of identified leads and cytotoxicity testing with colorimetric method using Vero and WRL-68 cell lines showed no significant cytotoxic effects. Molecular docking of these molecules with sortase A (PDB: 2KID) showed H-bond interaction with functional site residue Arg197 of sortase A. Sortase A inhibition assay was developed by expressing SrtA∆N from S. aureus strain MTCC96. Tetralene and indene compounds were found to have sortase A inhibitory potential. S. aureus strain MTCC96 treated with these compounds showed surface-sorting inhibition of fibronectin-binding protein and reduction in adherence to host extracellular matrix protein, fibronectin. 1-Chloro, 2-formyl, 6-methoxy, 1-tetralene (Tet-5), 1,5-dichloro, 2-formyl, 1-indene (Tet-20) and 1-chloro, 2-formyl, 5,6-methylenedioxy, and 1-indene (Tet-21) exhibited antistaphylococcal activity along with sortase A inhibition. The results also indicate the possible role of these leads in other reactions essential for cell viability.

  9. NLO properties of formyl-methoxy derivatives of [2.2]paracyclophane containing the donor group in different positions

    NASA Astrophysics Data System (ADS)

    Puntus, Lada N.; Suponitsky, Kyrill Y.; Antonov, Dmitrii; Pekareva, Irina S.; Lyssenko, Konstantin A.; Kajzar, François

    2014-10-01

    Formyl-methoxy derivatives of [2.2]paracyclophane (pCp) have been synthesized and studied by optical spectroscopy as well as the X-ray diffraction method. These molecules crystallize in acentric space groups. The efficiency of SHG ability of these compounds was measured by the powder technique at 1064 nm fundamental wavelength and average NLO susceptibility for some of them was found comparable with such well-known NLO crystal as NPP. The calculations of molecular and crystalline nonlinearities within density functional theory using M052X/6-31+G* level of approximation were also conducted for the considered series of compounds.

  10. 1-Formyl-3-phenyl-5-(4-isopropylphenyl)-2-pyrazoline: Synthesis, characterization, antimicrobial activity and DFT studies

    NASA Astrophysics Data System (ADS)

    Sid, Assia; Messai, Amel; Parlak, Cemal; Kazancı, Nadide; Luneau, Dominique; Keşan, Gürkan; Rhyman, Lydia; Alswaidan, Ibrahim A.; Ramasami, Ponnadurai

    2016-10-01

    The structure of 1-formyl-3-phenyl-5-(4-isopropylphenyl)-2-pyrazoline synthesized as single crystal was investigated by FTIR, NMR, XRD. Experimental data were complemented by quantum mechanical calculations. XRD data show that the compound crystallizes in the triclinic system (P-1) via trans isomer (a = 6.4267(4) Å, b = 10.9259(12) Å, c = 12.4628(9) Å and α = 102.894(8)°, β = 102.535(6)°, γ = 101.633(7)°). Anti-microbial screening results indicate that the compound shows promising activity. The theoretically predicted and experimentally obtained parameters reveal further insight into pyrazoline systems.

  11. [The characteristic infrared spectra of N-(2-phenyl-(1,2,3-triazole-formyl)-N'-arylthioueas].

    PubMed

    An, Y; Li, Y; Hu, J; Meng, L; Wang, G

    2001-04-01

    The characteristic infrared spectra of N-(2-phenyl-(1,2,3-triazole-formyl)-N'-aryl)-thioueas have been studied and the changing rules with the change of chemical structure have been discussed. Specially, the effect of the characteristic infrared of long-range radical by different substitute is discussed in detail. Results show that characteristic bands of radical which as far as five radicals away form substitute group are still affected. It is very suggestive meaning for long-range spectra research.

  12. Bis(μ-4-formyl­benzoato-κ2 O:O′)bis­[(4-formyl­benzoato-κ2 O,O′)bis­(iso­nicotin­amide-κN 1)copper(II)

    PubMed Central

    Sertçelik, Mustafa; Çaylak Delibaş, Nagihan; Necefoğlu, Hacali; Hökelek, Tuncer

    2013-01-01

    The asymmetric unit of the centrosymmetric dinuclear title compound, [Cu2(C8H5O3)4(C6H6N2O)4], contains one half of the complex mol­ecule. The CuII atoms are bridged by the carboxyl­ate groups of two 4-formyl­benzoate (FOB) anions. Besides the two bridging FOB anions, one additional chelating FOB anion and two isonicotinamide (INA) ligands complete the distorted CuN2O4 octa­hedral coordination of each Cu2+ cation. Within the asymmetric unit, the benzene and pyridine rings are oriented at dihedral angles of 25.1 (3) and 12.6 (3)°, respectively. In the crystal, N—H⋯O and C—H⋯O hydrogen bonds link the mol­ecules into a three-dimensional network. π–π contacts between the pyridine rings [shortest centroid–centroid distance = 3.821 (3) Å] may further stabilize the crystal structure. One of the formyl groups of the two FOB anions is disordered over two sets of sites with an occupancy ratio of 0.65:0.35. PMID:23723788

  13. Bis(μ-4-formyl-benzoato-κ(2) O:O')bis-[(4-formyl-benzoato-κ(2) O,O')bis-(iso-nicotin-amide-κN (1))copper(II)].

    PubMed

    Sertçelik, Mustafa; Caylak Delibaş, Nagihan; Necefoğlu, Hacali; Hökelek, Tuncer

    2013-05-01

    The asymmetric unit of the centrosymmetric dinuclear title compound, [Cu2(C8H5O3)4(C6H6N2O)4], contains one half of the complex mol-ecule. The Cu(II) atoms are bridged by the carboxyl-ate groups of two 4-formyl-benzoate (FOB) anions. Besides the two bridging FOB anions, one additional chelating FOB anion and two isonicotinamide (INA) ligands complete the distorted CuN2O4 octa-hedral coordination of each Cu(2+) cation. Within the asymmetric unit, the benzene and pyridine rings are oriented at dihedral angles of 25.1 (3) and 12.6 (3)°, respectively. In the crystal, N-H⋯O and C-H⋯O hydrogen bonds link the mol-ecules into a three-dimensional network. π-π contacts between the pyridine rings [shortest centroid-centroid distance = 3.821 (3) Å] may further stabilize the crystal structure. One of the formyl groups of the two FOB anions is disordered over two sets of sites with an occupancy ratio of 0.65:0.35.

  14. C-Peptide Test

    MedlinePlus

    ... vital for the body to use its main energy source, glucose . Since C-peptide and insulin are produced ... these cases, C-peptide measurement is a useful alternative to testing for insulin. C-peptide measurements can ...

  15. Excited state proton transfer in di-(2-hydroxy-3-formyl-5-tert butyl phenyl) methane and solvent effect

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, M.; Banerjee, D.; Mukherjee, S.

    2006-05-01

    The proton transfer reaction and the spectroscopic properties of di-(2-hydroxy-3-formyl-5-tert butyl phenyl) methane (HFPM) have been examined in different nonpolar and polar solvents at room temperature and 77 K, by means of absorption, emission and time resolved fluorescence spectroscopy. In the ground state, the primary closed form has been identified in all the nonpolar and polar solvents and the anion is detected only in presence of base in some of the polar solvents. After photoexcitation, the excited state intramolecular proton transfer (ESIPT) is indicated by a large Stokes shifted emission (˜10,600 cm -1) in all the nonpolar and polar solvents used, except in water and ethylene glycol (EG). The ESIPT band is likely to be originated from the enol tautomer of the HFPM. Two types of anion and H-bonded complex have been detected in the excited state. In water and EG, only anion and H-bonded complex have been detected in the excited state. At 77 K, HFPM shows phosphorescence in pure ethanol, and in n-hexane in presence of triethylamine. It has been suggested that the appearance of phosphorescence is due to the rotation of the formyl group. The measured nonradiative decay rates have always been found to dominate in the decay processes of the excited state of HFPM. Some semiempirical calculations have been undertaken to rationalize the experimental findings.

  16. Equivalent absorption and in vivo kinetics of tritiated folic acid and 5-formyl-tetrahydrofolic acid in rats

    SciTech Connect

    Bhandari, S.D.; Gregory, J.F. )

    1990-02-26

    The intestinal absorption and in vivo turnover kinetics of ({sup 3}H)folic acid (FA) and (6S)-5-formyl-({sup 3}H)tetrahydrofolate (5-CHO-THF) were examined to determine whether differences exist in the inherent bioavailability of these forms of the vitamin. Following oral administration of 2 {mu}Ci/100 g body weight (in 50 mM sodium ascorbate, pH 7), a biphasic pattern of urinary tritium excretion was observed for each labeled folate. The following kinetic results were obtained (n=9). Little tritium was found in the GI tract after 8 hours, which indicated nearly complete absorption of each folate. HPLC analysis of urine revealed similar excretory patterns over 0-8 days post-dose for each folate administered, and the patterns of hepatic ({sup 3}H)folates were equivalent when examined after 8 hours and 4 days post-dose. These findings indicate that the bioavailability FA and 5-formyl-THF is equivalent.

  17. Synthesis, structures, and solid state self-assemblies of formyl and acetyl substituted triptycenes and their derivatives.

    PubMed

    Li, Peng-Fei; Chen, Chuan-Feng

    2012-10-19

    Starting from triptycene, 2-, (2,6- or 2,7-)di-, and (2,6,14- or 2,7,14-)triformyl or acetyl substituted triptycenes were selectively synthesized. The derivatization of the formyl or acetyl substituted triptycenes was then investigated. Consequently, it was found that the formyl-substituted triptycenes could be transformed into cyano substituted triptycene derivatives by the aldoxime formation and dehydration. Acetoxyl- and acetamino-substituted triptycenes were synthesized by Baeyer-Villiger oxidation of acetyl-substituted triptycenes and Beckmann rearrangement of acetyl-oxime triptycenes, respectively. Deacetylation of triacetaminotriptycene provided an alternative way to the synthesis of triaminotriptycene. In addition, 2-ethynyltriptycene could be conveniently synthesized by Corey-Fuchs reaction of 2-formyltriptycene, and 1,3,5-tritriptycenebenzene was obtained in high yield by the dehydration cyclotrimerization of 2-acetyltriptycene. The different functionalized triptycene derivatives and their regioisomers were well characterized by the FT-IR, (1)H NMR, (13)C NMR, MS spectra, and single crystal X-ray analyses. Moreover, it was also found that 2,6,14-triacetaminotriptycene with the three amide groups paralleled to their connected aromatic rings could self-assemble into a 2D layer with porous structure, and further 3D microporous architecture by the hydrogen-bond network in the solid state.

  18. Synthesis of all four stereoisomers of 5-formyl-4-hydroxymethyl-1,3-oxazolidin-2-ones from D-glucosamine.

    PubMed

    Murakami, Teiichi

    2013-06-28

    All four stereoisomers of 5-formyl-4-hydroxymethyl-1,3-oxazolidin-2-ones (FHOs) were conveniently prepared from D-glucosamine by base-catalyzed epimerizations. 2-N,3-O-Carbonyl-D-glucosamine (7) was successively treated with NaBH4 and NaIO4 to give (4S,5R)-FHO 18, which was epimerized with DBU in DMF to give (4S,5S)-FHO 20. The glucosamine derivative 7 was epimerized to 2-N,3-O-carbonyl-D-mannosamine 23, from which (4R,5R)- and (4R,5S)-FHO derivatives (27 and 31) were prepared. The NMR measurements revealed that the 4,5-cis-4(or 5)-formyl-5(or 4)-hydroxymethyl-oxazolidinone derivatives form five-membered lactol ring, whereas the 4,5-trans-disubstituted derivatives form the hydrate or methanol adduct of the open-chain aldehyde, or the symmetrical dimer. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Excited state proton transfer in di-(2-hydroxy-3-formyl-5-tert butyl phenyl) methane and solvent effect.

    PubMed

    Mukhopadhyay, M; Banerjee, D; Mukherjee, S

    2006-05-15

    The proton transfer reaction and the spectroscopic properties of di-(2-hydroxy-3-formyl-5-tert butyl phenyl) methane (HFPM) have been examined in different nonpolar and polar solvents at room temperature and 77 K, by means of absorption, emission and time resolved fluorescence spectroscopy. In the ground state, the primary closed form has been identified in all the nonpolar and polar solvents and the anion is detected only in presence of base in some of the polar solvents. After photoexcitation, the excited state intramolecular proton transfer (ESIPT) is indicated by a large Stokes shifted emission (approximately 10,600 cm-1) in all the nonpolar and polar solvents used, except in water and ethylene glycol (EG). The ESIPT band is likely to be originated from the enol tautomer of the HFPM. Two types of anion and H-bonded complex have been detected in the excited state. In water and EG, only anion and H-bonded complex have been detected in the excited state. At 77 K, HFPM shows phosphorescence in pure ethanol, and in n-hexane in presence of triethylamine. It has been suggested that the appearance of phosphorescence is due to the rotation of the formyl group. The measured nonradiative decay rates have always been found to dominate in the decay processes of the excited state of HFPM. Some semiempirical calculations have been undertaken to rationalize the experimental findings.

  20. 7-formyl-10-methylisoellipticine, a novel ellipticine derivative, induces mitochondrial reactive oxygen species (ROS) and shows anti-leukaemic activity in mice.

    PubMed

    Russell, Eileen G; Guo, Jianfeng; O'Sullivan, Elaine C; O'Driscoll, Caitriona M; McCarthy, Florence O; Cotter, Thomas G

    2016-02-01

    Acute myeloid leukaemia (AML) is the most common type of leukaemia in adults and is associated with high relapse rates. Current treatment options have made significant progress but the 5 year survival for AML remains low and therefore, there is an urgent need to develop novel therapeutics. Ellipticines, a class of cancer chemotherapeutic agents, have had limited success clinically due to low solubility and toxic side effects. Isoellipticines, novel isomers of ellipticine, have been designed to overcome these limitations. One particular isoellipticine, 7-formyl-10-methylisoellipticine, has previously showed strong ability to inhibit the growth of leukaemia cell lines. In this study the anti-leukaemia effect of this compound was investigated in detail on an AML cell line, MV4-11. Over a period of 24 h 7-formyl-10-methyl isoellipticine at a concentration of 5 μM can kill up to 40 % of MV4-11 cells. Our research suggests that the cytotoxicity of 7-formyl-10-methylisoellipticine is partially mediated by an induction of mitochondrial reactive oxygen species (ROS). Furthermore, 7-formyl-10-methylisoellipticine demonstrated promising anti-tumour activity in an AML xenograft mouse model without causing toxicity, implying the potential of isoellipticines as novel chemotherapeutic agents in the treatment of leukaemia.

  1. [Optimization of alkaline hydrolysis based on the side chain of diethyl ester 4-amino-N(5)-formyl-N(8), N(10)-dideazatetrahydrofolic acid].

    PubMed

    Yuan, M M; Wang, M; Liu, J Y; Zhang, Z L

    2017-08-18

    To optimize and establish the best hydrolysis method of diethyl ester 4-amino-N(5)-formyl-N(8), N(10)-dideazatetrahydrofolate through the optimization of simple compound of diethyl N-(4-aminobenzoyl)-L-glutamate. To increase the low yield of hydrolysis reaction of diethyl ester 4-amino-N(5)-formyl-N(8), N(10)-dideazatetrahydrofolate due to the by-products and difficult purification, we studied the effect of NaOH and KOH, two kinds of alkalis, three concentrations between 0.175-1 mol/L and five types of reaction time involved in 20, 30, 60, 120 and 180 min on the common side chain diethyl N-(4-aminobenzoyl)-L-glutamate. A high performance liquid chromatography was established for measuring the target product and the by-products in reaction liquid in different reaction conditions. Finally, on the basis of the best hydrolysis method of diethyl ester 4-amino-N(5)-formyl-N(8), N(10)-dideazatetrahydrofolate, we completed the optimization of the hydrolysis reaction conditions of diethyl ester 4-amino-N(5)-formyl-N(8), N(10)-dideazatetrahydrofolate. We developed the best reaction condition for the hydrolysis of diethyl ester 4-amino-N(5)-formyl-N(8), N(10)-dideazatetrahydrofolate, which could be carried out easily and efficiently. The results indicated that treated with the optimized condition of 0.3 mol/L KOH in 60 min at the room temperature, diethyl ester 4-amino-N(5)-formyl-N(8), N(10)-dideazatetrahydrofolate was converted into its diacid derivative in 95.6 % yield, which turned to be a better reaction condition compared with the previous reaction condition. The structures of those compounds were identified to be correct by 1H nuclear magnetic resonance(1H NMR), 13C nuclear magnetic resonance(13C NMR) and electrospray ionization time of flight mass spectrometry (ESI-MS). The purity of the diacid derivative of the compound was determined to be 96% by high performance liquid chromatography(HPLC).The new hydrolysis reaction condition could not only avoid the formation of

  2. Intramolecular interactions between chalcogen atoms: organoseleniums derived from 1-bromo-4-tert-butyl-2,6-di(formyl)benzene.

    PubMed

    Zade, Sanjio S; Panda, Snigdha; Singh, Harkesh B; Sunoj, Raghavan B; Butcher, Ray J

    2005-04-29

    [structure: see text] The synthesis and characterization of a series of low-valent organoselenium compounds derived from 1-bromo-4-tert-butyl-2,6-di(formyl)benzene (22) is described. The synthesis of diselenide 25 was achieved by the lithiation route whereas bis(4-tert-butyl-2,6-di(formyl)phenyl) diselenide (26) was synthesized by treating 22 with disodium diselenide. A series of monoselenides (27, 28, and 29) was obtained by facile nucleophilic substitution of bromine in 22, using the corresponding selenolates as nucleophiles. The halogenation reactions of bis(4-tert-butyl-2,6-di(formyl)phenyl) diselenide (26) did not afford the corresponding selenenyl halides but resulted in the isolation of an unexpected cyclic selenenate ester 34 as a product. The selenide 32 was synthesized by the treatment of dimethoxymethyl diselenide with trilithiated 2-bromo-5-tert-butyl-N,N'-di(phenyl)isophthalamide. The existence of potential Se...O intramolecular nonbonding interactions was examined by IR, (1)H, and (77)Se NMR spectroscopy, X-ray crystallography, and computational studies. The X-ray crystal structures of 26 and 27, having two ortho formyl groups, reveal the absence of any Se...O interactions. However, the Se...O interactions were observed in the selenenate ester 34 where one of the formyl groups has been utilized for the selenenate ring formation. The crystal structures of 26 and 27 exhibited intermolecular short-range C-H...Se interactions (hydrogen bonding). Although there are four heteroatoms in carbamoyl moieties ortho to selenium capable of forming a five-membered ring on intramolecular coordination, no such intramolecular Se...X (X = N, O) interaction was observed in the crystal structure of 32. The density functional theory calculations at the B3LYP/6-31G* level predicted that for all the diformyl systems (47a-c, 48a-c), the anti,anti conformer (when both formyl oxygen atoms point away from the selenium) is more stable. This preference was found to be reversed in

  3. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested.

  4. Synthesis and antibacterial activity of Schiff bases and amines derived from alkyl 2-(2-formyl-4-nitrophenoxy)alkanoates.

    PubMed

    Goszczyńska, Agata; Kwiecień, Halina; Fijałkowski, Karol

    A series of novel Schiff bases and secondary amines were obtained in good yields, as a result of the reductive amination of alkyl 2-(2-formyl-4-nitrophenoxy)alkanoates with both aniline and 4-methoxyaniline under established mild reaction conditions. Sodium triacetoxyborohydride as well as hydrogen in the presence of palladium on carbon were used as efficient reducing agents of the Schiff bases, in both direct and stepwise reductive amination processes. The Schiff bases, amines, and amine hydrochlorides were designed as potential antibacterial agents, and structure-activity relationship could be established following in vitro assays against Gram-positive and Gram-negative bacteria. The minimal inhibitory concentration and zone of inhibition were also determined. In these tests, some of Schiff bases and secondary amine hydrochlorides showed moderate-to-good activity against Gram-positive bacteria, including S. aureus, M. luteus, and S. mutans.

  5. The effect of coadsorbed water on the stability, configuration and interconversion of formyl (HCO) and hydroxymethylidyne (COH) on platinum (111)

    SciTech Connect

    Árnadóttir, Líney; Stuve, Eric M.; Jónsson, Hannes

    2012-07-01

    Two forms of the methanol electro-oxidation intermediate with stoichiometry C:H:O, COH (hydroxymethylidyne) and HCO (formyl), on Pt (11 1) with and without coadsorbed water were studied using density functional theory calculations. The structure, adsorption energy and stability with respect to dissociation were calculated. Both HC=O and C–OH were stable on clean Pt (11 1) and with a single coadsorbed water molecule, while only the HCO configuration was stable in the presence of a whole water layer. The vibrational modes of HC=O on a bridge site showed no mode around 1700 cm-1 characteristic of C=O stretch making it hard to distinguish it from C–OH.

  6. Characterization of peptide deformylase homologues from Staphylococcus epidermidis.

    PubMed

    Lin, Penghui; Hu, Tiancen; Hu, Jian; Yu, Wenqi; Han, Cong; Zhang, Jian; Qin, Guangrong; Yu, Kunqian; Götz, Friedrich; Shen, Xu; Jiang, Hualiang; Qu, Di

    2010-10-01

    The emergence of multi-drug-resistant strains of Staphylococcus epidermidis emphasizes the need to develop new antibiotics. The unique and essential role of the peptide deformylase (PDF) in catalysing the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria makes it an attractive antibacterial drug target. In the present study, both deformylase homologues from S. epidermidis (SePDF-1 and SePDF-2) were cloned and expressed, and their enzymic activities were characterized. Co(2+)-substituted SePDF-1 exhibited much higher enzymic activity (k(cat)/K(m) 6.3 × 10(4) M(-1) s(-1)) than those of Ni(2+)- and Zn(2+)-substituted SePDF-1, and SePDF-1 showed much weaker binding ability towards Ni(2+) than towards Co(2+) and Zn(2+), which is different from PDF in Staphylococcus aureus (SaPDF), although they share 80 % amino-acid sequence identity. The determined crystal structure of SePDF-1 was similar to that of (SaPDF), except for differences in the metal-binding sites. The other deformylase homologue, SePDF-2, was shown to have no peptide deformylase activity; the function of SePDF-2 needs to be further investigated.

  7. Regulatory Peptides in Plants.

    PubMed

    Vanyushin, B F; Ashapkin, V V; Aleksandrushkina, N I

    2017-02-01

    Many different peptides regulating cell differentiation, growth, and development are found in plants. Peptides participate in regulation of plant ontogenesis starting from pollination, pollen tube growth, and the very early stages of embryogenesis, including formation of embryo and endosperm. They direct differentiation of meristematic stem cells, formation of tissues and individual organs, take part in regulation of aging, fruit maturation, and abscission of plant parts associated with apoptosis. Biological activity of peptides is observed at very low concentrations, and it has mainly signal nature and hormonal character. "Mature" peptides appear mainly due to processing of protein precursors with (or without) additional enzymatic modifications. Plant peptides differ in origin, structure, and functional properties. Their specific action is due to binding with respective receptors and interactions with various proteins and other factors. Peptides can also regulate physiological functions by direct peptide-protein interactions. Peptide action is coordinated with the action of known phytohormones (auxins, cytokinins, and others); thus, peptides control phytohormonal signal pathways.

  8. Crystal Structures of Peptide Deformylase from Rice Pathogen Xanthomonas oryzae pv. oryzae in Complex with Substrate Peptides, Actinonin, and Fragment Chemical Compounds.

    PubMed

    Ngo, Ho-Phuong-Thuy; Ho, Thien-Hoang; Lee, Inho; Tran, Huyen-Thi; Sur, Bookyo; Kim, Seunghwan; Kim, Jeong-Gu; Ahn, Yeh-Jin; Cha, Sun-Shin; Kang, Lin-Woo

    2016-10-05

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight on rice; this species is one of the most destructive pathogenic bacteria in rice cultivation worldwide. Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides in bacterial cells and is an important target to develop antibacterial agents. We determined crystal structures of Xoo PDF (XoPDF) at up to 1.9 Å resolution, which include apo, two substrate-bound (methionine-alanine or methionine-alanine-serine), an inhibitor-bound (actinonin), and six fragment chemical-bound structures. Six fragment chemical compounds were bound in the substrate-binding pocket. The fragment chemical-bound structures were compared to the natural PDF inhibitor actinonin-bound structure. The fragment chemical molecules will be useful to design an inhibitor specific to XoPDF and a potential pesticide against Xoo.

  9. Tulipalin A induced phytotoxicity.

    PubMed

    McCluskey, James; Bourgeois, Marie; Harbison, Raymond

    2014-04-01

    Tulipalin A induced phytotoxicity is a persistent allergic contact dermatitides documented in floral workers exposed to Alstroemeria and its cultivars.[1] The causative allergen is tulipalin A, a toxic glycoside named for the tulip bulbs from which it was first isolated.[2] The condition is characterized by fissured acropulpitis, often accompanied by hyperpigmentation, onychorrhexis, and paronychia. More of the volar surface may be affected in sensitized florists. Dermatitis and paronychia are extremely common conditions and diagnostic errors may occur. A thorough patient history, in conjunction with confirmatory patch testing with a bulb sliver and tuliposide A exposure, can prevent misdiagnosis. We report a case of Tulipalin A induced phytotoxicity misdiagnosed as an unresolved tinea manuum infection in a patient evaluated for occupational exposure.

  10. Tulipalin A induced phytotoxicity

    PubMed Central

    McCluskey, James; Bourgeois, Marie; Harbison, Raymond

    2014-01-01

    Tulipalin A induced phytotoxicity is a persistent allergic contact dermatitides documented in floral workers exposed to Alstroemeria and its cultivars.[1] The causative allergen is tulipalin A, a toxic glycoside named for the tulip bulbs from which it was first isolated.[2] The condition is characterized by fissured acropulpitis, often accompanied by hyperpigmentation, onychorrhexis, and paronychia. More of the volar surface may be affected in sensitized florists. Dermatitis and paronychia are extremely common conditions and diagnostic errors may occur. A thorough patient history, in conjunction with confirmatory patch testing with a bulb sliver and tuliposide A exposure, can prevent misdiagnosis. We report a case of Tulipalin A induced phytotoxicity misdiagnosed as an unresolved tinea manuum infection in a patient evaluated for occupational exposure. PMID:25024947

  11. 10-Formyl-2,4,6,8,12-penta­nitro-2,4,6,8,10,12-hexa­azatetra­cyclo­[5.5.0.03,11.05,9]dodeca­ne

    PubMed Central

    Jin, Shaohua; Chen, Shusen; Chen, Huaxiong; Li, Lijie; Shi, Yanshan

    2009-01-01

    The title compound, C7H7N11O11 (PNMFIW), is a caged heterocycle substituted with five nitro and one formyl groups. It is related to the hexa­azaisowurtzitane family of high-density high-energy polycyclic cage compounds. Four nitro groups are appended to the four N atoms of the two five-membered rings, while a nitro group and a formyl are attached to the two N atoms of the six-membered ring. PMID:21578838

  12. Antinociception by neutrophil-derived opioid peptides in noninflamed tissue--role of hypertonicity and the perineurium.

    PubMed

    Rittner, H L; Hackel, D; Yamdeu, R-S; Mousa, S A; Stein, C; Schäfer, M; Brack, A

    2009-05-01

    Inflammatory pain can be controlled by intraplantar opioid injection or by secretion of endogenous opioid peptides from leukocytes in inflamed rat paws. Antinociception requires binding of opioid peptides to opioid receptors on peripheral sensory nerve terminals. In the absence of inflammation, hydrophilic opioid peptides do not penetrate the perineurial barrier and, thus, do not elicit antinociception. This study was designed to examine the conditions under which endogenous, neutrophil-derived hydrophilic opioid peptides (i.e. Met-Enkephalin and beta-endorphin) can raise nociceptive thresholds in noninflamed tissue in rats. Intraplantar injection of the chemokine CXCL2/3 (macrophage inflammatory protein-2) induced selective neutrophil recruitment without overt signs of inflammation or changes in mechanical nociceptive thresholds (paw pressure threshold). Following intraplantar injection of hypertonic saline, the perineurial barrier was permeable for hours and intraplantar injection of opioid peptides increased mechanical nociceptive thresholds. While formyl-Met-Leu-Phe (fMLP) triggered opioid peptide release from neutrophils in vitro, nociceptive thresholds were unchanged in vivo. In vitro, hypertonicity interfered with fMLP-induced p38 mitogen activated kinase (MAPK) phosphorylation and opioid peptide release from neutrophils. These inhibitory effects were fully reversible by washout. In vivo, return to normotonicity occurred within 30min while the perineurium remained permeable for hours. Under these conditions, fMLP triggered MAPK phosphorylation and induced opioid peptide-mediated increases in nociceptive thresholds in the noninflamed paw. Taken together, antinociception mediated by endogenous opioids in noninflamed tissue has two important requirements: (i) opening of the perineurial barrier for opioid peptide access and (ii) opioid peptide release from neutrophils involving p38 MAPK.

  13. TRH-like peptides.

    PubMed

    Bílek, R; Bičíková, M; Šafařík, L

    2011-01-01

    TRH-like peptides are characterized by substitution of basic amino acid histidine (related to authentic TRH) with neutral or acidic amino acid, like glutamic acid, phenylalanine, glutamine, tyrosine, leucin, valin, aspartic acid and asparagine. The presence of extrahypothalamic TRH-like peptides was reported in peripheral tissues including gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. Work deals with the biological function of TRH-like peptides in different parts of organisms where various mechanisms may serve for realisation of biological function of TRH-like peptides as negative feedback to the pituitary exerted by the TRH-like peptides, the role of pEEPam such as fertilization-promoting peptide, the mechanism influencing the proliferative ability of prostatic tissues, the neuroprotective and antidepressant function of TRH-like peptides in brain and the regulation of thyroid status by TRH-like peptides.

  14. Peptide Antimicrobial Agents

    PubMed Central

    Jenssen, Håvard; Hamill, Pamela; Hancock, Robert E. W.

    2006-01-01

    Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents. PMID:16847082

  15. Prevention of cardiac damage induced by formyl-leurosine, a potent cytostatic agent, by radio-detoxified endotoxin (Tolerin) in dogs

    SciTech Connect

    Bertok, L.; Juhasz-Nagy, A.; Sotonyi, P.

    1984-08-01

    Radio-detoxified endotoxin (Tolerin), produced by /sup 60/Co-gamma irradiation of Escherichia coli 089 endotoxin, can protect dogs against the acute cardiotoxic side-effects of formyl-leurosine, a semi-synthetic Vinca derivative with promising antineoplastic potency. Formyl-leurosine induces a rapid decrease in arterial blood pressure and diminishes the contractile force of the myocardium in the anaesthetized dog. These responses indicate a direct pharmacologic relaxant effect of the drug on the heart and vasculature smooth muscle. The early cardiovascular depression is of short duration and is unaffected by Tolerin. Tolerin can prevent, however, the secondary, more dangerous phase of circulatory depression that is associated with the severe cardiotoxic manifestations of the drug, as demonstrated by hemodynamic and morphologic (light and electronmicroscopic) patterns.

  16. Alternative synthesis of 3-acetyl, 3-epoxy, and 3-formyl chlorins from a 3-vinyl chlorin, methyl pyropheophorbide-a, via iodination.

    PubMed

    Oba, Toru; Masuya, Takuto; Yasuda, Satoru; Ito, Satoshi

    2015-08-01

    We developed novel methods to convert the C3-vinyl group of a chlorophyll derivative, methyl pyropheophorbide-a, into an acetyl group, an epoxy group, and a formyl group via iodination with I2 and phenyliodine(III) bis(trifluoroacetate). Reaction of the iodinated intermediate with ethylene glycol and subsequent treatment with base led to formation of the C3-acetyl chlorin. Reaction of the iodinated intermediate with ethylenediamine afforded the C3-oxiranyl chlorin. The C3-formyl chlorin was readily derived from the epoxide without hazardous reagents such as OsO4. These reactions were facile and useful alternatives to the previous methods. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Structure and Activity of Human Mitochondrial Peptide Deformylase, a Novel Cancer Target

    SciTech Connect

    Escobar-Alvarez, Sindy; Goldgur, Yehuda; Yang, Guangli; Ouerfelli, Ouathek; Li, Yueming; Scheinberg, David A.

    2009-07-21

    Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 A), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 A) identified the substrate-binding site. A defined S1' pocket, but no S2' or S3' substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2' and S3' binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2'and P3' positions of a formylated peptide substrate to turnover.

  18. Identification of distinct physiochemical properties of toxic prefibrillar species formed by A{beta} peptide variants

    SciTech Connect

    Goeransson, Anna-Lena; Nilsson, K. Peter R.; Kagedal, Katarina; Brorsson, Ann-Christin

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer Identification of toxic prefibrillar A{beta} species. Black-Right-Pointing-Pointer Fluorescence measurements using a combined set of fluorophores. Black-Right-Pointing-Pointer Morphology studies using transmission electron microscopy. -- Abstract: The formation of amyloid-{beta} peptide (A{beta}) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of A{beta}. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of A{beta}-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various A{beta} aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those A{beta} peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the A{beta} peptide to form nontoxic versus toxic species.

  19. Remarkable Features of the McMurry Reaction Conditions in Dimerization of Formyl- and 2-Formylvinylpurpurinimides. Electrochemistry of Monomeric Ni(II) Purpurinimide and the Corresponding Dyads

    PubMed Central

    Goswami, Lalit N.; Ethirajan, Manivannan; Dobhal, Mahabeer P.; Zhang, Min; Missert, Joseph R.; Shibata, Masayuki; Kadish, Karl M.; Pandey, Ravindra K.

    2009-01-01

    To investigate the electrochemical properties of purpurinimide dyads and electron transfer sites for their reduction and oxidation, a series of dimers with variable C-C linkages were synthesized. For the preparation of these novel structures, the formyl and 2-formylvinyl substituents were regioselectively introduced at positions-3 and 20 of Ni(II) purpurinimides by the Vilsmeier reaction. The Ni(II) complexes were then subjected to the McMurry reaction under two different conditions with unexpected results being obtained. For example, the reaction of formyl purpurinimides with TiCl3(DME)1.5 failed to produce the desired C-C dimers and the starting compounds were recovered almost quantitatively. Under similar reaction conditions, the 20-(2-formylvinyl)purpurinimide also did not dimerize but gave instead unexpected benzoisobacteriochlorins via an intramolecular cyclization. However, treatment of the 3-formyl- and 20-formyl-purpurinimides with TiCl4/Zn gave the corresponding dimers linked with one double bond (trans-) in modest yields. Under similar conditions, Ni(II) purpurinimides containing a 2-formylvinyl substituent either at position-3 or position-20 afforded the respective C-C dimers, where the purpurinimide moieties were joined with a trans-trans-trans- hexatriene linker. Molecular modeling data suggest that the nature of the conformational energy difference found in all trans- vs. trans-cis-trans- conformers of the dimers connected by a hexatriene linker at the meso- or β-position of the macrocycle is not due to the intrinsic conformational energy difference of the linker region, which is identical for both dimers. PMID:19072690

  20. PH dependent adhesive peptides

    DOEpatents

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  1. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  2. Adsorption of formaldehyde and formyl intermediates on Pt, PtRu-, and PtRuMo-alloy surfaces: A density functional study

    NASA Astrophysics Data System (ADS)

    Cahyanto, Wahyu Tri; Shukri, Ganes; Agusta, Mohammad Kemal; Kasai, Hideaki

    2013-02-01

    Stable binding configuration for formaldehyde (H2CO) and formyl (HCO) adsorption on Pt, PtRu, and PtRuMo are studied within the frame of density functional theory (DFT). We address this study to investigate the role of Ru and Mo on the binding characteristic of formaldehyde and formyl adsorption with respect to interaction strength and charge analysis. Several binding conformation on all possible surface adsorption sites are considered in determining the most stable adsorption geometry on three surfaces. Our results show that the presence of Ru in PtRu and Mo in PtRuMo stabilize the formaldehyde and formyl, which are indicated by stronger bond strength. Further electronic structure analysis shows that the addition of Ru in PtRu and Mo in PtRuMo modifies the electronic structure of Pt's surface significantly. The presence of both impurities shifted the derived anti-bonding state - which is originally located below the fermi level in pure Pt surface - to be above the fermi level in PtRu and PtRuMo systems. This fact explains the stronger adsorption found on PtRu & PtRuMo as compared to pure Pt surface.

  3. Equilibrium thermodynamics to form a rhodium formyl complex from reactions of CO and H2: metal σ donor activation of CO.

    PubMed

    Imler, Gregory H; Zdilla, Michael J; Wayland, Bradford B

    2014-04-23

    A rhodium(II) dibenzotetramethylaza[14]annulene dimer ([(tmtaa)Rh]2) (1) reacts with CO and H2 in toluene and pyridine to form equilibrium distributions with hydride and formyl complexes ((tmtaa)Rh-H (2); (tmtaa)Rh-C(O)H (3)). The rhodium formyl complex ((tmtaa)Rh-C(O)H) was isolated under a CO/H2 atmosphere, and the molecular structure was determined by X-ray diffraction. Equilibrium constants were evaluated for reactions of (tmtaa)Rh-H with CO to produce formyl complexes in toluene (K2(298 K)(tol) = 10.8 (1.0) × 10(3)) and pyridine (K2(298 K)(py) = 2.2 (0.2) × 10(3)). Reactions of 1 and 2 in toluene and pyridine are discussed in the context of alternative radical and ionic pathways. The five-coordinate 18-electron Rh(I) complex ([(py)(tmtaa)Rh(I)](-)) is proposed to function as a nucleophile toward CO to give a two-electron activated bent Rh-CO unit. Results from DFT calculations on the (tmtaa)Rh system correlate well with experimental observations. Reactions of 1 with CO and H2 suggest metal catalyst design features to reduce the activation barriers for homogeneous CO hydrogenation.

  4. Drug forecast – the peptide deformylase inhibitors as antibacterial agents

    PubMed Central

    Guay, David R P

    2007-01-01

    The relatively rapid development of microbial resistance after the entry of every new antimicrobial into the marketplace necessitates a constant supply of new agents to maintain effective pharmacotherapy. Despite extensive efforts to identify novel lead compounds from molecular targets, only the peptide deformylase inhibitors (PDIs) have shown any real promise, with some advancing to phase I human trials. Bacterial peptide deformylase, which catalyzes the removal of the N-formyl group from N-terminal methionine following translation, is essential for bacterial protein synthesis, growth, and survival. The majority of PDIs are pseudopeptide hydroxamic acids and two of these (IV BB-83698 and oral NVP LBM-415) entered phase I human trials. However, agents to the present have suffered from major potential liabilities. Their in vitro activity has been limited to gram-positive aerobes and some anaerobes and has been quite modest against the majority of such species (MIC90 values ranging from 1–8 mg/L). They have exerted bacteriostatic, not bacteriocidal, activity, thus reducing their potential usefulness in the management of serious infections in the immunocompromised. The relative ease with which microorganisms have been able to develop resistance and the multiple available mechanisms of resistance (mutations in fmt, defB, folD genes; AcrAB/TolC efflux pump; overexpression of peptide deformylase) are worrisome. These could portend a short timespan of efficacy after marketing. Despite these current liabilities, further pursuit of more potent and broader spectrum PDIs which are less susceptible to bacterial mechanisms of resistance is still warranted. PMID:18472972

  5. Ion/Ion Reactions of MALDI-Derived Peptide Ions: Increased Sequence Coverage via Covalent and Electrostatic Modification upon Charge Inversion

    PubMed Central

    Stutzman, John R.; McLuckey, Scott A.

    2012-01-01

    Atmospheric pressure matrix assisted laser desorption/ionization (AP-MALDI)-derived tryptic peptide ions have been subjected to ion/ion reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas phase. The ion/ion reaction produces a negatively charged electrostatic complex composed of the peptide cation and reagent dianion, whereupon dehydration of the complex via collision-induced dissociation (CID) produces a Schiff base product anion. Collisional activation of modified lysine-terminated tryptic peptide anions is consistent with a covalent modification of unprotonated primary amines (i.e. N-terminus and ε-NH2 of lysine). Modified arginine-terminated tryptic peptides have shown evidence of a covalent modification at the N-terminus and a non-covalent interaction with the arginine residue. The modified anions yield at least as much sequence information upon CID as the unmodified cations for the small tryptic peptides examined here and more sequence information for the large tryptic peptides. This study represents the first demonstration of gas phase ion/ion reactions involving MALDI-derived ions. In this case, covalent modification upon charge inversion is shown to enhance MALDI tandem mass spectrometry of tryptic peptides. PMID:23078018

  6. Ion/ion reactions of MALDI-derived peptide ions: increased sequence coverage via covalent and electrostatic modification upon charge inversion.

    PubMed

    Stutzman, John R; McLuckey, Scott A

    2012-12-18

    Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI)-derived tryptic peptide ions have been subjected to ion/ion reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas-phase. The ion/ion reaction produces a negatively charged electrostatic complex composed of the peptide cation and reagent dianion, whereupon dehydration of the complex via collision-induced dissociation (CID) produces a Schiff base product anion. Collisional activation of modified lysine-terminated tryptic peptide anions is consistent with a covalent modification of unprotonated primary amines (i.e., N-terminus and ε-NH(2) of lysine). Modified arginine-terminated tryptic peptides have shown evidence of a covalent modification at the N-terminus and a noncovalent interaction with the arginine residue. The modified anions yield at least as much sequence information upon CID as the unmodified cations for the small tryptic peptides examined here and more sequence information for the large tryptic peptides. This study represents the first demonstration of gas-phase ion/ion reactions involving MALDI-derived ions. In this case, covalent and electrostatic modification charge inversion is shown to enhance MALDI tandem mass spectrometry of tryptic peptides.

  7. Peptide signaling in Hydra.

    PubMed

    Fujisawa, Toshitaka; Hayakawa, Eisuke

    2012-01-01

    Peptides play a number of crucial roles as signaling molecules in metazoans. In order to elaborate a more complete picture of the roles played by peptides in a single organism, we launched the "Hydra Peptide Project". For this project, we used Hydra magnipapillata, a species belonging to Cnidaria, one of the most basal metazoan phyla, and using a peptidomic approach, we systematically identified a number of peptide signaling molecules, their encoding genes and their functions. In this article, we report the peptides isolated from Hydra and other cnidarians, as well as their synthesis, processing and release from the cells to the target. Possible peptide signaling pathways are overviewed and finally we discuss the evolution of the peptide signaling system.

  8. A switchable stapled peptide.

    PubMed

    Kalistratova, Aleksandra; Legrand, Baptiste; Verdié, Pascal; Naydenova, Emilia; Amblard, Muriel; Martinez, Jean; Subra, Gilles

    2016-03-01

    The O-N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence-containing peptides, cyclic peptides, epimerization-free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α-amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O-N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide.

  9. The HIV-1 gp41 ectodomain is cleaved by matriptase to produce a chemotactic peptide that acts through FPR2.

    PubMed

    Wood, Matthew P; Cole, Amy L; Eade, Colleen R; Chen, Li-Mei; Chai, Karl X; Cole, Alexander M

    2014-07-01

    Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. Whereas this chemotactic activity has been reported, it is not known how these peptides could be produced under biological conditions. The heptad repeat 1 (HR1) region of gp41 is exposed to the extracellular environment and could therefore be susceptible to proteolytic processing into smaller peptides. Matriptase is a serine protease expressed at the surface of most epithelia, including the prostate and mucosal surfaces. Here, we present evidence that matriptase efficiently cleaves the HR1 portion of gp41 into a 22-residue chemotactic peptide MAT-1, the sequence of which is highly conserved across HIV-1 clades. We found that MAT-1 induced migration of primary neutrophils and monocytes, the latter of which act as a cellular reservoir of HIV during early stage infection. We then used formyl peptide receptor 1 (FPR1) and FPR2 inhibitors, along with HEK 293 cells, to demonstrate that MAT-1 can induce chemotaxis specifically using FPR2, a receptor found on the surface of monocytes, macrophages and neutrophils. These findings are the first to identify a proteolytic cleavage product of gp41 with chemotactic activity and highlight a potential role for matriptase in HIV-1 transmission and infection at epithelial surfaces and within tissue reservoirs of HIV-1. © 2014 John Wiley & Sons Ltd.

  10. Cell Penetrating Peptides and Cationic Antibacterial Peptides

    PubMed Central

    Rodriguez Plaza, Jonathan G.; Morales-Nava, Rosmarbel; Diener, Christian; Schreiber, Gabriele; Gonzalez, Zyanya D.; Lara Ortiz, Maria Teresa; Ortega Blake, Ivan; Pantoja, Omar; Volkmer, Rudolf; Klipp, Edda; Herrmann, Andreas; Del Rio, Gabriel

    2014-01-01

    Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs. PMID:24706763

  11. Identification of quantitative trait loci influencing foliar concentrations of terpenes and formylated phloroglucinol compounds in Eucalyptus nitens.

    PubMed

    Henery, Martin L; Moran, Gavin F; Wallis, Ian R; Foley, William J

    2007-01-01

    Leaves of eucalypt species contain a variety of plant secondary metabolites, including terpenoids and formylated phloroglucinol compounds (FPCs). Both terpene and FPC concentrations are quantitative traits that can show large variation within a population and have been shown to be heritable. The molecular genetic basis of this variation is currently unknown. Progeny from a field trial of a three-generation mapping pedigree of Eucalyptus nitens were assayed for terpenes and FPCs. Quantitative trait loci (QTL) analyses were conducted using a map constructed from 296 markers to locate regions of the genome influencing foliar concentrations of these plant secondary compounds. A large number of significant QTL for 14 traits were located across nine linkage groups, with significant clustering of QTL on linkage groups 7, 8 and 9. As expected, QTL for biosynthetically related compounds commonly colocated, but QTL for unrelated monterpenes and FPCs also mapped closely together. Colocation of these QTL with mapped candidate genes from the various biosynthetic pathways, and subsequent use of these genes in association mapping, will assist in determining the causes of variation in plant secondary metabolites in eucalypts.

  12. Quantum chemical studies and vibrational analysis of 4-acetyl benzonitrile, 4-formyl benzonitrile and 4-hydroxy benzonitrile - A comparative study

    NASA Astrophysics Data System (ADS)

    Arjunan, V.; Carthigayan, K.; Periandy, S.; Balamurugan, K.; Mohan, S.

    2012-12-01

    The FTIR and FT-Raman vibrational spectra of 4-actetyl benzonitrile, 4-formyl benzonitrile and 4-hydroxy benzonitrile molecules have been recorded in the range 4000-400 and 4000-100 cm-1, respectively. The complete vibrational assignment and analysis of the fundamental modes of the most stable geometry of the compounds were carried out using the experimental FTIR and FT-Raman data on the basis of peak positions, relative intensities and quantum chemical studies. The observed vibrational frequencies were compared with the theoretical wavenumbers of the optimised geometry of the compounds obtained from the DFT-B3LYP gradient calculations employing the standard 6-31G**, high level 6-311++G** and cc-pVDZ basis sets. The structural parameters and vibrational wavenumbers obtained from the DFT methods are in good agreement with the experimental data. With hope of providing more and effective information on the fundamental vibrations, total energy distributions of the fundamental modes have been performed by assuming Cs point group symmetry. The effect of substituents -COCH3, -CHO and -OH in the benzonitrile moiety have been analysed and compared. The kinetic and thermodynamic stability and chemical hardness of the molecule have been determined.

  13. Weakly Bound Free Radicals in Combustion: "Prompt" Dissociation of Formyl Radicals and Its Effect on Laminar Flame Speeds.

    PubMed

    Labbe, Nicole J; Sivaramakrishnan, Raghu; Goldsmith, C Franklin; Georgievskii, Yuri; Miller, James A; Klippenstein, Stephen J

    2016-01-07

    Weakly bound free radicals have low-dissociation thresholds such that at high temperatures, time scales for dissociation and collisional relaxation become comparable, leading to significant dissociation during the vibrational-rotational relaxation process. Here we characterize this "prompt" dissociation of formyl (HCO), an important combustion radical, using direct dynamics calculations for OH + CH2O and H + CH2O (key HCO-forming reactions). For all other HCO-forming reactions, presumption of a thermal incipient HCO distribution was used to derive prompt dissociation fractions. Inclusion of these theoretically derived HCO prompt dissociation fractions into combustion kinetics models provides an additional source for H-atoms that feeds chain-branching reactions. Simulations using these updated combustion models are therefore shown to enhance flame propagation in 1,3,5-trioxane and acetylene. The present results suggest that HCO prompt dissociation should be included when simulating flames of hydrocarbons and oxygenated molecules and that prompt dissociations of other weakly bound radicals may also impact combustion simulations.

  14. Ruthenium-Catalyzed Asymmetric Hydrohydroxyalkylation of Butadiene: The Role of the Formyl Hydrogen Bond in Stereochemical Control

    PubMed Central

    Grayson, Matthew N.; Krische, Michael J.; Houk, K. N.

    2016-01-01

    The catalyst generated in situ from RuH2(CO)(PPh3)3, (S)-SEGPHOS, and a chiral phosphoric acid promotes asymmetric hydrohydroxyalkylation of butadiene and affords enantioenriched α-methyl homoallylic alcohols. The observed diastereo- and enantioselectivities are determined by both the chiral phosphine and chiral phosphate ligands. Density functional theory calculations (M06/SDD-6-311G(d,p)-IEFPCM(acetone)//B3LYP/SDD-6-31G(d)) predict that the product distribution is controlled by the kinetics of carbon-carbon bond formation, and this process occurs via a closed-chair Zimmerman-Traxler-type transition structure (TS). Chiral phosphate-dependent stereoselectivity arising from this TS is enabled through a hydrogen bond between the phosphoryl oxygen and the aldehyde formyl proton present in TADDOL-derived catalysts. This interaction is absent in the corresponding BINOL-derived systems and the opposite sense of attack on the aldehyde occurs. Additional factors influencing stereochemical control are determined. PMID:26107070

  15. High-performance liquid chromatographic resolution of 1-(1,4-benzodioxane-2-formyl)-piperazine enantiomers after chiral derivatization.

    PubMed

    Chen, Zhiqiong; Yu, Yu; Li, Longjiang

    2005-02-01

    Chiral separation of racemic mixtures is of the greatest importance to the pharmaceutical industry, as the isomers of a given racemate may exhibit substantially different pharmacological effects, not to mention possibly differing toxicity behaviour. A novel chiral separation method is developed for the determination of 1-(1,4-benzodioxane-2-formyl)piperazine (BFP) enantiomers. The indirect resolution is performed by applying precolumn derivatization with the chiral reagent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). The resulting diastereoisomers are separated on a reversed-phase ODS column with methanol-potassium dihydrogen phosphate (0.02mol/L, 50:50) as mobile phase. UV detection is at 250 nm. The effect of mobile phase composition upon resolution and analysis time is investigated. Two diastereoisomers show nearly base-line separation under optimal chromatographic conditions. The presented study provides a simple and accurate method for the enantiomeric quality control and the optical purity assay of BFP.

  16. Novel detection of formylated phloroglucinol compounds (FPCs) in the wound wood of Eucalyptus globulus and E. nitens.

    PubMed

    Eyles, Alieta; Davies, Noel W; Mohammed, Caroline

    2003-04-01

    This study characterized the chemical responses of Eucalyptus globulus and Eucalyptus nitens to artificial inoculation with a basidiomycete decay fungus. Nine-year-old trees responded to mechanical wounding or inoculation with the decay fungus by producing new wound wood characterized by the presence of dark extractives 17 months after wounding. Analysis of crude wound wood extracts by HPLC coupled to negative ion electrospray mass spectrometry revealed the presence of a complex mixture of many unidentified formylated phlorglucinol compounds (FPCs), in addition to a diverse range of other polyphenolic compounds (hydrolyzable tannins, proanthocyanidins, flavanone glycoside, stilbene glycosides). Prior to this study, FPCs have only been reported from leaves and buds of Eucalyptus spp. Unequivocal evidence for the presence of macrocarpal A and B, and sideroxylonal A and B in the crude extracts was obtained, as well as evidence for a wide range of as yet unreported FPCs. Subsequent preliminary in vitro fungal and bacterial bioassays did not support an antimicrobial role for FPCs in host-pathogen interactions in eucalypts.

  17. Weakly Bound Free Radicals in Combustion: "Prompt" Dissociation of Formyl Radicals and Its Effect on Laminar Flame Speeds

    SciTech Connect

    Labbe, Nicole J.; Sivaramakrishnan, Raghu; Goldsmith, C. Franklin; Georgievskii, Yuri; Miller, James A.; Klippenstein, Stephen J.

    2016-01-07

    Weakly bound free radicals have low-dissociation thresholds such that at high temperatures, timescales for dissociation and collisional relaxation become comparable, leading to significant dissociation during the vibrational-rotational relaxation process. Here we characterize this “prompt” dissociation of formyl (HCO), an important combustion radical, using direct dynamics calculations for OH + CH2O and H + CH2O (key HCO-forming reactions). For all other HCO-forming reactions, presumption of a thermal incipient HCO distribution was used to derive prompt dissociation fractions. Inclusion of these theoretically derived HCO prompt dissociation fractions into combustion kinetics models provides an additional source for H-atoms that feeds chain branching reactions. Simulations using these updated combustion models are therefore shown to enhance flame propagation in 1,3,5-trioxane and acetylene. The present results suggest that HCO prompt dissociation should be included when simulating flames of hydrocarbons and oxygenated molecules and that prompt dissociations of other weakly bound radicals may also impact combustion simulations

  18. Effects of methoxy and formyl substituents on the energetics and reactivity of α-naphthalenes: a calorimetric and computational study.

    PubMed

    Silva, Ana L R; Freitas, Vera L S; Ribeiro da Silva, Maria D M C

    2014-07-01

    A combined experimental and computational study was developed to evaluate and understand the energetics and reactivity of formyl and methoxy α-naphthalene derivatives. Static bomb combustion calorimetry and the Calvet microcalorimetry were the experimental techniques used to determine the standard (p(o)=0.1 MPa) molar enthalpies of formation, in the liquid phase, ΔfHm(o)(l), and of vaporization, Δl(g)Hm(o), at T=298.15K, respectively, of the two liquid naphthalene derivatives. Those experimental values were used to derive the values of the experimental standard molar enthalpies of formation, in the gaseous phase, ΔfHm(o)(g), of 1-methoxynaphthalene, (-3.0 ± 3.1)kJmol(-1), and of 1-formylnaphthalene, (36.3 ± 4.1)kJ mol(-1). High-level quantum chemical calculations at the composite G3(MP2)//B3LYP level were performed to estimate the values of the ΔfHm(o)(g) of the two compounds studied resulting in values in very good agreement with experimental ones. Natural bond orbital (NBO) calculations were also performed to determine more about the structure and reactivity of this class of compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

    SciTech Connect

    Feltner, D.E.; Marasco, W.A.

    1989-06-01

    The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of (3H)FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM (3H)FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. (3H)FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of (3H)FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM (3H)FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.

  20. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions.

  1. Crystallization and preliminary X-ray crystallographic analysis of peptide deformylase (PDF) from Bacillus cereus in ligand-free and actinonin-bound forms

    SciTech Connect

    Park, Joon Kyu; Moon, Jin Ho; Kim, Jae-Hong; Kim, Eunice EunKyeong

    2005-01-01

    Peptide deformylase (PDF) from B. cereus has been overexpressed, purified and crystallized in ligand-free and actinonin-bound forms. Diffraction data have been collected from these crystals to 1.7 and 2.0 Å resolution, respectively. In bacteria, protein expression initiates with an N-formyl group and this needs to be removed in order to ensure proper bacterial growth. These formylation and deformylation processes are unique to eubacteria; therefore, inhibition of these would provide a novel antibacterial therapy. Deformylation is carried out by peptide deformylase (PDF). PDF from Bacillus cereus, one of the major pathogenic bacteria, was cloned into expression plasmid pET-28a (Novagen), overexpressed in Escherichia coli BL21 (DE3) and purified to high quality. Crystals have been obtained of both ligand-free PDF and PDF to which actinonin, a highly potent naturally occurring inhibitor, is bound. Both crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.72, b = 44.04, c = 85.19 Å and a = 41.31, b = 44.56, c = 84.47 Å, respectively. Diffraction data were collected to 1.7 Å resolution for the inhibitor-free crystals and to 2.0 Å resolution for the actinonin-bound crystals.

  2. Antimicrobial Peptides in Reptiles

    PubMed Central

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  3. Polycyclic peptide therapeutics.

    PubMed

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Topical peptides as cosmeceuticals.

    PubMed

    Pai, Varadraj Vasant; Bhandari, Prasana; Shukla, Pankaj

    2017-01-01

    Peptides are known to have diverse biological roles, most prominently as signaling/regulatory molecules in a broad variety of physiological processes including defense, immunity, stress, growth, homeostasis and reproduction. These aspects have been used in the field of dermatology and cosmetology to produce short, stable and synthetic peptides for extracellular matrix synthesis, pigmentation, innate immunity and inflammation. The evolution of peptides over the century, which started with the discovery of penicillin, has now extended to their usage as cosmeceuticals in recent years. Cosmeceutical peptides may act as signal modulators of the extracellular matrix component, as structural peptides, carrier peptides and neurotransmitter function modulators. Transdermal delivery of peptides can be made more effective by penetration enhancers, chemical modification or encapsulation of peptides. The advantages of using peptides as cosmeceuticals include their involvement in many physiological functions of the skin, their selectivity, their lack of immunogenicity and absence of premarket regulatory requirements for their use. However, there are disadvantages: clinical evidence for efficacy is often weak, absorption may be poor due to low lipophilicity, high molecular weight and binding to other ingredients, and prices can be quite high.

  5. The natriuretic peptides.

    PubMed

    Baxter, Gary F

    2004-03-01

    The natriuretic peptides are a family of widely distributed, but evolutionarily conserved, polypeptide mediators that exert a range of actions throughout the body. In cardiovascular homeostasis, the endocrine roles of the cardiac-derived atrial and B-type natriuretic peptide (ANP and BNP) in regulating central fluid volume and blood pressure have been recognised for two decades. However, there is a growing realisation that natriuretic peptide actions go far beyond their volume regulating effects. These pleiotropic actions include local (autocrine/paracrine) regulatory actions of ANP and BNP within the heart, and of another natriuretic peptide, CNP, within the vessel wall. Effects on function and growth of the local tissue environment are likely to be of great importance, especially in disease states where tissue and circulating levels of ANP and BNP rise markedly. At present, the relevance of other natriuretic peptides (notably uroguanylin and DNP) to human physiology and pathology remain uncertain. Other articles in this issue of Basic Research in Cardiology review the molecular physiology of natriuretic peptide signalling, with a particular emphasis on the lessons from genetically targetted mice; the vascular activity of natriuretic peptides; the regulation and roles of natriuretic peptides in ischaemic myocardium; and the diagnostic, prognostic and therapeutic roles of natriuretic peptides in heart failure.

  6. Transcriptional regulation of the human mitochondrial peptide deformylase (PDF).

    PubMed

    Pereira-Castro, Isabel; Costa, Luís Teixeira da; Amorim, António; Azevedo, Luisa

    2012-05-18

    The last years of research have been particularly dynamic in establishing the importance of peptide deformylase (PDF), a protein of the N-terminal methionine excision (NME) pathway that removes formyl-methionine from mitochondrial-encoded proteins. The genomic sequence of the human PDF gene is shared with the COG8 gene, which encodes a component of the oligomeric golgi complex, a very unusual case in Eukaryotic genomes. Since PDF is crucial in maintaining mitochondrial function and given the atypical short distance between the end of COG8 coding sequence and the PDF initiation codon, we investigated whether the regulation of the human PDF is affected by the COG8 overlapping partner. Our data reveals that PDF has several transcription start sites, the most important of which only 18 bp from the initiation codon. Furthermore, luciferase-activation assays using differently-sized fragments defined a 97 bp minimal promoter region for human PDF, which is capable of very strong transcriptional activity. This fragment contains a potential Sp1 binding site highly conserved in mammalian species. We show that this binding site, whose mutation significantly reduces transcription activation, is a target for the Sp1 transcription factor, and possibly of other members of the Sp family. Importantly, the entire minimal promoter region is located after the end of COG8's coding region, strongly suggesting that the human PDF preserves an independent regulation from its overlapping partner. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Assaying peptide translocation by the peptide transporter TAP.

    PubMed

    Jongsma, Marlieke L M; Neefjes, Jacques

    2013-01-01

    MHC class I molecules display peptides at the cell surface that are mostly derived from cytosolic or nuclear proteins. Since peptide loading of MHC class I molecules occurs in the ER lumen, cytosolic peptides have to pass the ER membrane. The peptide transporter TAP translocates peptides over this ER membrane which is critical for successful MHC class I antigen presentation. How peptide translocation by TAP can be assayed and inhibitors of chemical or viral origin can be identified, will be described here.

  8. Identification of structurally diverse methanofuran coenzymes in methanococcales that are both N-formylated and N-acetylated.

    PubMed

    Allen, Kylie D; White, Robert H

    2014-10-07

    Methanofuran (MF) is a coenzyme necessary for the first step of methanogenesis from CO2. The well-characterized MF core structure is 4-[N-(γ-l-glutamyl-γ-l-glutamyl)-p-(β-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan (APMF-γ-Glu2). Three different MF structures that differ on the basis of the composition of their side chains have been determined previously. Here, we use liquid chromatography coupled with high-resolution mass spectrometry and a variety of biochemical methods to deduce the unique structures of MFs present in four different methanogens in the order Methanococcales. This is the first detailed characterization of the MF occurring in methanogens of this order. MF in each of these organisms contains the expected APMF-γ-Glu2; however, the composition of the side chain is different from that of the previously described MF structures. In Methanocaldococcus jannaschii, additional γ-linked glutamates that range from 7 to 12 residues are present. The MF coenzymes in Methanococcus maripaludis, Methanococcus vannielii, and Methanothermococcus okinawensis also have additional glutamate residues but interestingly also contain a completely different chemical moiety in the middle of the side chain that we have identified as N-(3-carboxy-2- or 3-hydroxy-1-oxopropyl)-l-aspartic acid. This addition results in the terminal γ-linked glutamates being incorporated in the opposite orientation. In addition to these nonacylated MF coenzymes, we also identified the corresponding N-formyl-MF and, surprisingly, N-acetyl-MF derivatives. N-Acetyl-MF has never been observed or implied to be functioning in nature and may represent a new route for acetate formation in methanogens.

  9. Stereoretentive formylation of (S)-proline: new application of the self-regeneration of stereo-centres (SRS) principle via chelation to cobalt(III).

    PubMed

    Temizsoy, Mehmet; Sethi, Waqas; Reinholdt, Anders; Schau-Magnussen, Magnus; Bendix, Jesper; Hammershøi, Anders

    2015-11-14

    In a Vilsmeier-Haack-type formylation reaction the α-(dihydroxymethyl)-(S)-prolinato complex (+)578-p-[Co(tren){(RC,SN)-Pro[CH(OH)2]O}]Cl2·2H2O (22) was produced stereoselectively (85% ee) from the (S)-prolinato complex, (+)578-p-[Co(tren){(SC,SN)-ProO}]2(H3O)2(HOEt2)(O3SCF3)7 (18). Similar reaction of the (S)-alaninato complex, (-)578-p-[Co(tren)(S-AlaO)](H3O)(O3SCF3)3 (13), produced the racemate, rac-p-[Co(tren)(Ala{CH(OH)2}O)]SO4·2H2O (17). The contrasting stereochemical outcomes of the formylation reaction with 18versus13 were ascribed to the stereogenic character of the coordinated sec. amine of the (S)-prolinate chelate in 18, which serves to uphold a chiral environment during reaction, whereas reaction intermediates derived from 13 lack this stereochemical feature. The stereoselective formylation of (S)-proline, relying on coordination to an inert metal centre, as conducted here, constitutes a novel application of the concept of Self-Regeneration of Stereocentres (SRS). The α-(hydroxymethyl)-(S)-prolinato complex, (+)578-p-[Co(tren){(RC,SN)-Pro(CH2OH)O}]Cl2·2H2O (23) resulted from borohydride reduction of 22. The molecular structures of (+)578-p-[Co(tren){(RC,SN)-Pro[CH(OH)2]O}]Cl2·2H2O (22), rac-p-[Co(tren)-(Ala{CH(OH)2}O)]I2·H2O (17) and (+)578-p-[Co(tren){(RC,SN)-Pro(CH2OH)O}]Cl2·2H2O (23) were established by X-ray crystallography.

  10. catena-Poly[[di-aqua-bis-(4-formyl-benzo-ato-κO (1))nickel(II)]-μ-pyrazine-κ(2) N:N'].

    PubMed

    Celik, Fatih; Dilek, Nefise; Caylak Delibaş, Nagihan; Necefoğlu, Hacali; Hökelek, Tuncer

    2014-02-01

    In the title polymeric compound, [Ni(C8H5O3)2(C4H4N2)(H2O)2] n , the Ni(II) atom is located on a twofold rotation axis and has a slightly distorted octa-hedral coordination sphere. In the equatorial plane, it is coordinated by two carboxyl-ate O atoms of two symmetry-related monodentate formyl-benzoate anions and by two N atoms of the bridging pyrazine ligand, which is bis-ected by the twofold rotation axis. The axial positions are occupied by two O atoms of the coordinating water mol-ecules. In the formyl-benzoate anion, the carboxyl-ate group is twisted away from the attached benzene ring by 7.0 (6)°, while the benzene and pyrazine rings are oriented at a dihedral angle of 66.2 (3)°. The pyrazine ligands bridge the Ni(II) cations, forming polymeric chains running along the b-axis direction. Intra-molecular O-H⋯O hydrogen bonds link the water ligands to the carboxyl-ate O atoms. In the crystal, water-water O-H⋯O hydrogen bonds link adjacent chains into layers parallel to the bc plane. Pyrazine-formyl C-H⋯O hydrogen bonds link the layers, forming a three-dimensional network. There are also weak C-H⋯π inter-actions present. The title compound is isotypic with the copper(II) complex [Çelik et al. (2014a). Acta Cryst. E70, m4-m5].

  11. Bacteriocin Inducer Peptides

    USDA-ARS?s Scientific Manuscript database

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  12. Peptide bioregulators inhibit apoptosis.

    PubMed

    Khavinson, V K; Kvetnoii, I M

    2000-12-01

    The effects of peptide bioregulators epithalon and vilon on the dynamics of irradiation-induced apoptotic death of spleen lymphocytes in rats indicate that these agents inhibit physiologically programmed cell death. The antiapoptotic effect of vilon was more pronounced, which corroborates the concept on tissue-specific effect of peptide bioregulators.

  13. Palladium-catalyzed stereoconvergent formylation of (E/Z)-β-bromo-β-fluorostyrenes: straightforward access to (Z)-α-fluorocinnamic aldehydes and (Z)-β-fluorocinnamic alcohols.

    PubMed

    Zemmouri, Rajae; Kajjout, Mohammed; Castanet, Yves; Eddarir, Said; Rolando, Christian

    2011-10-07

    We report here the stereoconvergent formylation of (E/Z)-β-bromo-β-fluorostyrene mixtures with carbon monoxide and sodium formate catalyzed by palladium. Optimization of reaction conditions leads to the corresponding pure (Z)-α-fluorocinnamaldehydes in good yields. The reaction was extended to styrenes bearing electro-attracting or electro-donating groups. The obtained α-fluoroaldehydes were smoothly reduced to the corresponding (Z)-β-fluorocinnamic alcohol by NaBH(4). The reaction could be performed on functionalized substrates as demonstrated by the access to the glucoside of β-fluoroconiferyl alcohol, (Z)-β-fluoroconiferin, a strong inhibitor of lignin polymerization.

  14. Synthesis, structural characterization and antitumor activity of a Ca(II) coordination polymer based on 4-formyl-1,3-benzenedisulfonate-2-furoic acid hydrazide ligands

    NASA Astrophysics Data System (ADS)

    Tai, Xi-Shi; Wang, Xin

    2017-03-01

    A new Ca(II) coordination polymer, {[CaL(H2O)4] · (H2O)4} n (L = 4-formyl-1,3-benzenedisulfonate-2-furoic acid hydrazide) has been prepared by one-pot synthesis method. And it was characterized by elemental analysis, IR and thermal analysis. The result of X-ray single-crystal diffraction analysis shows that the Ca(II) complex molecules form one-dimensional chain structure by the bridging oxygen atoms. The anti-tumor activity of L ligand and the Ca(II) coordination polymer has also been studied.

  15. Comparative study of reductive amination reaction on 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid and its monomethoxy analog using the Multipin approach.

    PubMed

    Bui, C T; Bray, A M; Pham, Y; Campbell, R; Ercole, F; Rasoul, F A; Maeji, N J

    1998-01-01

    The 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid (Barany) linker and its monomethoxy analog were applied to the Multipin method of solid phase synthesis. A comparative assessment of reductive amination and cleavage of these linkers under conditions of multiple synthesis indicated that both were applicable to a broad range of primary amines including aniline and 4-nitroaniline. Apart from the greater lability of the dimethoxy version under TFA cleavage, there was no observable advantage of one linker over the other within the described experiment.

  16. Antimicrobial Peptides from Fish

    PubMed Central

    Masso-Silva, Jorge A.; Diamond, Gill

    2014-01-01

    Antimicrobial peptides (AMPs) are found widely distributed through Nature, and participate in the innate host defense of each species. Fish are a great source of these peptides, as they express all of the major classes of AMPs, including defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins. As with other species, the fish peptides exhibit broad-spectrum antimicrobial activity, killing both fish and human pathogens. They are also immunomodulatory, and their genes are highly responsive to microbes and innate immuno-stimulatory molecules. Recent research has demonstrated that some of the unique properties of fish peptides, including their ability to act even in very high salt concentrations, make them good potential targets for development as therapeutic antimicrobials. Further, the stimulation of their gene expression by exogenous factors could be useful in preventing pathogenic microbes in aquaculture. PMID:24594555

  17. Annexin A1 mimetic peptide controls the inflammatory and fibrotic effects of silica particles in mice

    PubMed Central

    Trentin, P G; Ferreira, T P T; Arantes, A C S; Ciambarella, B T; Cordeiro, R S B; Flower, R J; Perretti, M; Martins, M A; Silva, P M R

    2015-01-01

    Background and Purpose Endogenous glucocorticoids are pro-resolving mediators, an example of which is the endogenous glucocorticoid-regulated protein annexin A1 (ANXA1). Because silicosis is an occupational lung disease characterized by unabated inflammation and fibrosis, in this study we tested the therapeutic properties of the N-terminal ANXA1-derived peptide annexin 1-(2-26) (Ac2-26) on experimental silicosis. Experimental Approach Swiss-Webster mice were administered silica particles intranasally and were subsequently treated with intranasal peptide Ac2-26 (200 μg per mouse) or dexamethasone (25 μg per mouse) for 7 days, starting 6 h post-challenge. Ac2-26 abolished the leukocyte infiltration, collagen deposition, granuloma formation and generation of pro-inflammatory cytokines evoked by silica; these variables were only partially inhibited by dexamethasone. Key Results A clear exacerbation of the silica-induced pathological changes was observed in ANXA1 knockout mice as compared with their wild-type (WT) littermate controls. Incubation of lung fibroblasts from WT mice with Ac2-26 in vitro reduced IL-13 or TGF-β-induced production of CCL2 (MCP-1) and collagen, but this peptide did not affect the production of CCL2 (MCP-1) by stimulated fibroblasts from formyl peptide receptor type 1 (FPR1) knockout mice. Ac2-26 also inhibited the production of CCL2 (MCP-1) from fibroblasts of FPR2 knockout mice. Conclusions and Implications Collectively, our findings reveal novel protective properties of the ANXA1 derived peptide Ac2-26 on the inflammatory and fibrotic responses induced by silica, and suggest that ANXA1 mimetic agents might be a promising strategy as innovative anti-fibrotic approaches for the treatment of silicosis. PMID:25659822

  18. Consumption of peptide-included and free tryptophan induced by peroxyl radicals: A kinetic study.

    PubMed

    Fuentes, E; López-Alarcón, C

    2014-10-01

    It is well-known that tryptophan residues are efficiently oxidized by peroxyl radicals, generating kynurenine, and N-formyl kynurenine as well as hydroperoxide derivatives as products. In the present work we studied the kinetic of such reaction employing free and peptide-included tryptophan. Two azocompounds were used to produce peroxyl radicals: AAPH (2,2'-Azobis(2-methylpropionamidine) dihydrochloride) and ABCVA (4,4'-Azobis(4-cyanovaleric acid)), which generate cationic and anionic peroxyl radicals, respectively. Tryptophan consumption was assessed by fluorescence spectroscopy and the reactions were carried out in phosphate buffer (75mM, pH 7.4) at 45°C. Only a slight effect of the peroxyl radical charge was evidenced on the consumption of free tryptophan and the dipeptide Gly-Trp. Employing AAPH as peroxyl radical source, at low free tryptophan concentrations (1-10µM) near 0.3 mol of tryptophan were consumed per each mol of peroxyl radicals introduced into the system. However, at high free tryptophan concentrations (100µM-1mM) such stoichiometry increased in a tryptophan concentration-way. At 1mM three moles of tryptophan were consumed per mol of AAPH-derived peroxyl radicals, evidencing the presence of chain reactions. A similar behavior was observed when di and tri-peptides (Gly-Trp, Trp-Gly, Gly-Trp-Gly, Trp-Ala, Ala-Trp-Ala) were studied. Nonetheless, at low initial concentration (5µM), the initial consumption rate of tryptophan included in the peptides was two times higher than free tryptophan. In contrast, at high concentration (1mM) free and peptide-included tryptophan showed similar initial consumption rates. These results could be explained considering a disproportionation process of tryptophanyl radicals at low free tryptophan concentrations, a process that would be inhibited when tryptophan is included in peptides.

  19. Cyclic Opioid Peptides.

    PubMed

    Remesic, Michael; Lee, Yeon Sun; Hruby, Victor J

    2016-01-01

    For decades the opioid receptors have been an attractive therapeutic target for the treatment of pain. Since the first discovery of enkephalin, approximately a dozen endogenous opioid peptides have been known to produce opioid activity and analgesia, but their therapeutics have been limited mainly due to low blood brain barrier penetration and poor resistance to proteolytic degradation. One versatile approach to overcome these drawbacks is the cyclization of linear peptides to cyclic peptides with constrained topographical structure. Compared to their linear parents, cyclic analogs exhibit better metabolic stability, lower offtarget toxicity, and improved bioavailability. Extensive structure-activity relationship studies have uncovered promising compounds for the treatment of pain as well as further elucidate structural elements required for selective opioid receptor activity. The benefits that come with employing cyclization can be further enhanced through the generation of polycyclic derivatives. Opioid ligands generally have a short peptide chain and thus the realm of polycyclic peptides has yet to be explored. In this review, a brief history of designing ligands for the opioid receptors, including classic linear and cyclic ligands, is discussed along with recent approaches and successes of cyclic peptide ligands for the receptors. Various scaffolds and approaches to improve bioavailability are elaborated and concluded with a discourse towards polycyclic peptides.

  20. Expression, crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Xanthomonas oryzae pv. oryzae.

    PubMed

    Ngo, Phuong-Thuy Ho; Kim, Jin-Kwang; Kim, Hyesoon; Jung, Junho; Ahn, Yeh-Jin; Kim, Jeong-Gu; Lee, Byoung-Moo; Kang, Lin-Woo

    2008-11-01

    Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides; this process is crucial for cell survival. As it is an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), PDF from Xoo was cloned, expressed, purified and crystallized. Native PDF crystals diffracted to 2.7 A resolution and belonged to the hexagonal space group P6(1)22, with unit-cell parameters a = b = 59.0, c = 266.3 A. One monomer is present in the asymmetric unit, with a corresponding crystal volume per protein weight of 3.50 A(3) Da(-1) and a solvent content of 64.9%.

  1. Inhibition of Chlamydial Infection in the Genital Tract of Female Mice by Topical Application of a Peptide Deformylase Inhibitor

    PubMed Central

    Balakrishnan, Amit; Wang, Lingling; Li, Xiaojin; Ohman-Strickland, Pamela; Malatesta, Paul; Fan, Huizhou

    2009-01-01

    Summary Chlamydia trachomatis is an obligate intracellular bacterium responsible for a number of health problems, including sexually transmitted infection in humans. We recently discovered that C. trachomatis infection in cell culture is highly susceptible to inhibitors of peptide deformylase, an enzyme that removes the N-formyl group from newly synthesized polypeptides. In this study, one of the deformylase inhibitors, GM6001, was tested for potential antichlamydial activity using a murine genital C. muridarum infection model. Topical application of GM6001 significantly reduced C. muridarum loading in BALB/c mice that were vaginally infected with the pathogen. In striking contrast, growth of the probiotic Lactobacillus plantarum is strongly resistant to the PDF inhibitor. GM6001 demonstrated no detectable toxicity against host cells. On the basis of these data and our previous observations, we conclude that further evaluation of PDF inhibitors for prevention and treatment of sexually transmitted chlamydial infection is warranted. PMID:17936604

  2. Targeted Peptide Specificity

    DTIC Science & Technology

    1989-01-31

    the conformation of small natural peptide ligands. In the last year two peptides were investigaýed by 2D-NMR,(j2.- conotoxin SI, which targets to the...targets. Although these venoms are very complex, we have focused on three groups of peptide toxins, the a, j and w- conotoxins which target to nicotinic...grant period are summarized below.) In addition, the 2D-NMR work is continuing and in addition to examining one of the conotoxins specific for the

  3. Synthesis and biological evaluation of esters of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives as inhibitors of 5α-reductase type 2.

    PubMed

    Sánchez-Márquez, Araceli; Arellano, Yazmín; Bratoeff, Eugene; Heuze, Yvonne; Córdova, Karen; Nieves, Gladys; Soriano, Juan; Cabeza, Marisa

    2016-12-01

    In this study, we investigated the in vitro effect of 16-formyl-17-methoxy dehydroepiandrosterone derivatives on the activity of 5α-reductase type 2 (5α-R2) obtained from human prostate. The activity of different concentrations of these derivatives was determined for the conversion of labelled testosterone to dihydrotestosterone. The results indicated that an aliphatic ester moiety at the C-3 position of these derivatives increases their in vitro potency as inhibitors of 5α-R2 activity compared to finasteride®, which is considered to be a potent inhibitor of 5α-R2. In this case, the augmentation of the lipophilicity of these dehydroepiandrosterone derivatives increased their potency as inhibitors of 5α-R2. However, the presence of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings as the cycloaliphatic ester moiety at C-3 of the formyl methoxy dehydroepiandrosterone scaffold did not inhibit the activity of this enzyme. This may be due to the presence of steric factors between the enzyme and the spatial structure of these derivatives.

  4. Crystal structure of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase, an NAD⁺-dependent dismutase from Mesorhizobium loti.

    PubMed

    Mugo, Andrew Njagi; Kobayashi, Jun; Mikami, Bunzo; Yoshikane, Yu; Yagi, Toshiharu; Ohnishi, Kouhei

    2015-01-02

    5-Formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase (FHMPCDH) from Mesorhizobium loti is the fifth enzyme in degradation pathway I for pyridoxine. The enzyme catalyzes a dismutation reaction: the oxidation of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) to 3-hydroxy-2-methylpyridine 4,5-dicarboxylic acid with NAD(+) and reduction of FHMPC to 4-pyridoxic acid with NADH. FHMPCDH belongs to the l-3-hydroxyacyl-CoA dehydrogenase (HAD) family. The crystal structure was determined by molecular replacement and refined to a resolution of 1.55Å (R-factor of 16.4%, Rfree=19.4%). There were two monomers in the asymmetric unit. The overall structure of the monomer consisted of N- and C-terminal domains connected by a short linker loop. The monomer was similar to members of the HAD family (RMSD=1.9Å). The active site was located between the domains and highly conserved to that of human heart l-3-hydroxyacyl-CoA dehydrogenase (HhHAD). His-Glu catalytic dyad, a serine and two asparagine residues of HhHAD were conserved. Ser116, His137 and Glu149 in FHMPCDH are connected by a hydrogen bonding network forming a catalytic triad. The functions of the active site residues in the reaction mechanism are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Crystal structure of catena-poly[[di-aqua-bis-(4-formyl-benzoato-κO (1))cobalt(II)]-μ-pyrazine-κ(2) N:N'].

    PubMed

    Aşkın, Gülçin Şefiye; Çelik, Fatih; Dilek, Nefise; Necefoğlu, Hacali; Hökelek, Tuncer

    2015-04-01

    In the title polymeric compound, [Co(C8H5O3)2(C4H4N2)(H2O)2] n , the Co(II) atom is located on a twofold rotation axis and has a slightly distorted octa-hedral coordination sphere. In the equatorial plane, it is coordinated by two carboxyl-ate O atoms of two symmetry-related monodentate formyl-benzoate anions and by two N atoms of two bridging pyrazine ligands. The latter are bis-ected by the twofold rotation axis. The axial positions are occupied by two O atoms of the coordinating water mol-ecules. In the formyl-benzoate anion, the carboxyl-ate group is twisted away from the attached benzene ring by 7.50 (8)°, while the benzene and pyrazine rings are oriented at a dihedral angle of 64.90 (4)°. The pyrazine ligands bridge the Co(II) cations, forming linear chains running along the b-axis direction. Strong intra-molecular O-H⋯O hydrogen bonds link the water mol-ecules to the carboxyl-ate O atoms. In the crystal, weak O-Hwater⋯Owater hydrogen bonds link adjacent chains into layers parallel to the bc plane. The layers are linked via C-Hpyrazine⋯Oform-yl hydrogen bonds, forming a three-dimensional network. There are also weak C-H⋯π inter-actions present.

  6. Tetra-μ3-methano­lato-tetra­kis­[(2-formyl-6-meth­oxy­phenolato)methano­lnickel(II)

    PubMed Central

    Ayikoe, Kouassi; Butcher, Ray J.; Gultneh, Yilma

    2010-01-01

    The molecule of the title compound, [Ni4(CH3O)4(C8H7O3)4(CH3OH)4], has S 4 symmetry. Each of the four NiII atoms occupies every other corner of a cube, with the alternate corners occupied by μ3-methano­late bridging groups linking to three NiII atoms. Each NiII atom is in an O6 octa­hedral coordination environment formed by three O atoms from three μ3-methano­late groups, one from methanol, and two others from a bidentate 2-formyl-6-meth­oxy­phenolate ligand. The Ni—O bond distances range from 2.0020 (14) to 2.0938 (14) Å, the cis bond angles range from 81.74 (6) to 97.63°, and the trans bond angles range from 168.76 (5) to 175.22 (6)°. There are bifurcated hydrogen-bonding inter­actions between the coordinated methanol OH groups and both the phenolic and meth­oxy O atoms of an adjoining 2-formyl-6-meth­oxy­phenolate moiety. In addition, there are weak inter­molecular C—H⋯O inter­actions involving the meth­oxy O atoms. PMID:21588902

  7. Antibiotic Activity and Characterization of BB-3497, a Novel Peptide Deformylase Inhibitor

    PubMed Central

    Clements, John M.; Beckett, R. Paul; Brown, Anthony; Catlin, Graham; Lobell, Mario; Palan, Shilpa; Thomas, Wayne; Whittaker, Mark; Wood, Stephen; Salama, Sameeh; Baker, Patrick J.; Rodgers, H. Fiona; Barynin, Vladimir; Rice, David W.; Hunter, Michael G.

    2001-01-01

    Peptide deformylase (PDF) is an essential bacterial metalloenzyme which deformylates the N-formylmethionine of newly synthesized polypeptides and as such represents a novel target for antibacterial chemotherapy. To identify novel PDF inhibitors, we screened a metalloenzyme inhibitor library and identified an N-formyl-hydroxylamine derivative, BB-3497, and a related natural hydroxamic acid antibiotic, actinonin, as potent and selective inhibitors of PDF. To elucidate the interactions that contribute to the binding affinity of these inhibitors, we determined the crystal structures of BB-3497 and actinonin bound to Escherichia coli PDF at resolutions of 2.1 and 1.75 Å, respectively. In both complexes, the active-site metal atom was pentacoordinated by the side chains of Cys 90, His 132, and His 136 and the two oxygen atoms of N-formyl-hydroxylamine or hydroxamate. BB-3497 had activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis, and activity against some gram-negative bacteria. Time-kill analysis showed that the mode of action of BB-3497 was primarily bacteriostatic. The mechanism of resistance was via mutations within the formyltransferase gene, as previously described for actinonin. While actinonin and its derivatives have not been used clinically because of their poor pharmacokinetic properties, BB-3497 was shown to be orally bioavailable. A single oral dose of BB-3497 given 1 h after intraperitoneal injection of S. aureus Smith or methicillin-resistant S. aureus protected mice from infection with median effective doses of 8 and 14 mg/kg of body weight, respectively. These data validate PDF as a novel target for the design of a new generation of antibacterial agents. PMID:11158755

  8. A peptide deformylase-ribosome complex reveals mechanism of nascent chain processing.

    PubMed

    Bingel-Erlenmeyer, Rouven; Kohler, Rebecca; Kramer, Günter; Sandikci, Arzu; Antolić, Snjezana; Maier, Timm; Schaffitzel, Christiane; Wiedmann, Brigitte; Bukau, Bernd; Ban, Nenad

    2008-03-06

    Messenger-RNA-directed protein synthesis is accomplished by the ribosome. In eubacteria, this complex process is initiated by a specialized transfer RNA charged with formylmethionine (tRNA(fMet)). The amino-terminal formylated methionine of all bacterial nascent polypeptides blocks the reactive amino group to prevent unfavourable side-reactions and to enhance the efficiency of translation initiation. The first enzymatic factor that processes nascent chains is peptide deformylase (PDF); it removes this formyl group as polypeptides emerge from the ribosomal tunnel and before the newly synthesized proteins can adopt their native fold, which may bury the N terminus. Next, the N-terminal methionine is excised by methionine aminopeptidase. Bacterial PDFs are metalloproteases sharing a conserved N-terminal catalytic domain. All Gram-negative bacteria, including Escherichia coli, possess class-1 PDFs characterized by a carboxy-terminal alpha-helical extension. Studies focusing on PDF as a target for antibacterial drugs have not revealed the mechanism of its co-translational mode of action despite indications in early work that it co-purifies with ribosomes. Here we provide biochemical evidence that E. coli PDF interacts directly with the ribosome via its C-terminal extension. Crystallographic analysis of the complex between the ribosome-interacting helix of PDF and the ribosome at 3.7 A resolution reveals that the enzyme orients its active site towards the ribosomal tunnel exit for efficient co-translational processing of emerging nascent chains. Furthermore, we have found that the interaction of PDF with the ribosome enhances cell viability. These results provide the structural basis for understanding the coupling between protein synthesis and enzymatic processing of nascent chains, and offer insights into the interplay of PDF with the ribosome-associated chaperone trigger factor.

  9. Antibiotic activity and characterization of BB-3497, a novel peptide deformylase inhibitor.

    PubMed

    Clements, J M; Beckett, R P; Brown, A; Catlin, G; Lobell, M; Palan, S; Thomas, W; Whittaker, M; Wood, S; Salama, S; Baker, P J; Rodgers, H F; Barynin, V; Rice, D W; Hunter, M G

    2001-02-01

    Peptide deformylase (PDF) is an essential bacterial metalloenzyme which deformylates the N-formylmethionine of newly synthesized polypeptides and as such represents a novel target for antibacterial chemotherapy. To identify novel PDF inhibitors, we screened a metalloenzyme inhibitor library and identified an N-formyl-hydroxylamine derivative, BB-3497, and a related natural hydroxamic acid antibiotic, actinonin, as potent and selective inhibitors of PDF. To elucidate the interactions that contribute to the binding affinity of these inhibitors, we determined the crystal structures of BB-3497 and actinonin bound to Escherichia coli PDF at resolutions of 2.1 and 1.75 A, respectively. In both complexes, the active-site metal atom was pentacoordinated by the side chains of Cys 90, His 132, and His 136 and the two oxygen atoms of N-formyl-hydroxylamine or hydroxamate. BB-3497 had activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis, and activity against some gram-negative bacteria. Time-kill analysis showed that the mode of action of BB-3497 was primarily bacteriostatic. The mechanism of resistance was via mutations within the formyltransferase gene, as previously described for actinonin. While actinonin and its derivatives have not been used clinically because of their poor pharmacokinetic properties, BB-3497 was shown to be orally bioavailable. A single oral dose of BB-3497 given 1 h after intraperitoneal injection of S. aureus Smith or methicillin-resistant S. aureus protected mice from infection with median effective doses of 8 and 14 mg/kg of body weight, respectively. These data validate PDF as a novel target for the design of a new generation of antibacterial agents.

  10. Genetic and Functional Analysis of the Biosynthesis of a Non-Ribosomal Peptide Siderophore in Burkholderia xenovorans LB400.

    PubMed

    Vargas-Straube, María José; Cámara, Beatriz; Tello, Mario; Montero-Silva, Francisco; Cárdenas, Franco; Seeger, Michael

    2016-01-01

    B. xenovorans LB400 is a model bacterium for the study of the metabolism of aromatic compounds. The aim of this study was the genomic and functional characterization of a non-ribosomal peptide synthetase containing gene cluster that encodes a siderophore in B. xenovorans LB400. The mba gene cluster from strain LB400 encodes proteins involved in the biosynthesis and transport of a hydroxamate-type siderophore. Strain LB400 has a unique mba gene organization, although mba gene clusters have been observed in diverse Burkholderiales. Bioinformatic analysis revealed the presence of promoters in the mba gene cluster that strongly suggest regulation by the ferric uptake regulator protein (Fur) and by the alternative RNA polymerase extracytoplasmic function sigma factor MbaF. Reverse transcriptase PCR analyses showed the expression of iron-regulated transcriptional units mbaFGHIJKL, mbaN, mbaABCE, mbaO, mbaP and mbaD genes under iron limitation. Chrome azurol S (CAS) assay strongly suggests that strain LB400 synthesized a siderophore under iron limitation. Mass spectrometry ESI-MS and MALDI-TOF-MS analyses revealed that the siderophore is a non-ribosomal peptide, and forms an iron complex with a molecular mass of 676 Da. Based on bioinformatic prediction, CAS assay and MS analyses, we propose that the siderophore is L-Nδ-hydroxy-Nδ-formylOrn-D-β-hydroxyAsp-L-Ser-L-Nδ-hydroxy-Nδ-formylOrn-1,4-diaminobutane that is closely related to malleobactin-type siderophores reported in B. thailandensis.

  11. Genetic and Functional Analysis of the Biosynthesis of a Non-Ribosomal Peptide Siderophore in Burkholderia xenovorans LB400

    PubMed Central

    Vargas-Straube, María José; Cámara, Beatriz; Tello, Mario; Montero-Silva, Francisco; Cárdenas, Franco; Seeger, Michael

    2016-01-01

    B. xenovorans LB400 is a model bacterium for the study of the metabolism of aromatic compounds. The aim of this study was the genomic and functional characterization of a non-ribosomal peptide synthetase containing gene cluster that encodes a siderophore in B. xenovorans LB400. The mba gene cluster from strain LB400 encodes proteins involved in the biosynthesis and transport of a hydroxamate-type siderophore. Strain LB400 has a unique mba gene organization, although mba gene clusters have been observed in diverse Burkholderiales. Bioinformatic analysis revealed the presence of promoters in the mba gene cluster that strongly suggest regulation by the ferric uptake regulator protein (Fur) and by the alternative RNA polymerase extracytoplasmic function sigma factor MbaF. Reverse transcriptase PCR analyses showed the expression of iron-regulated transcriptional units mbaFGHIJKL, mbaN, mbaABCE, mbaO, mbaP and mbaD genes under iron limitation. Chrome azurol S (CAS) assay strongly suggests that strain LB400 synthesized a siderophore under iron limitation. Mass spectrometry ESI-MS and MALDI-TOF-MS analyses revealed that the siderophore is a non-ribosomal peptide, and forms an iron complex with a molecular mass of 676 Da. Based on bioinformatic prediction, CAS assay and MS analyses, we propose that the siderophore is L-Nδ-hydroxy-Nδ-formylOrn-D-β-hydroxyAsp-L-Ser-L-Nδ-hydroxy-Nδ-formylOrn-1,4-diaminobutane that is closely related to malleobactin-type siderophores reported in B. thailandensis. PMID:26963250

  12. Anti-antimicrobial Peptides

    PubMed Central

    Ryan, Lloyd; Lamarre, Baptiste; Diu, Ting; Ravi, Jascindra; Judge, Peter J.; Temple, Adam; Carr, Matthew; Cerasoli, Eleonora; Su, Bo; Jenkinson, Howard F.; Martyna, Glenn; Crain, Jason; Watts, Anthony; Ryadnov, Maxim G.

    2013-01-01

    Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance. PMID:23737519

  13. Insulin C-peptide

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003701.htm Insulin C-peptide test To use the sharing features ... a product that is created when the hormone insulin is produced and released into the body. The ...

  14. Peptide Optical waveguides.

    PubMed

    Handelman, Amir; Apter, Boris; Shostak, Tamar; Rosenman, Gil

    2017-02-01

    Small-scale optical devices, designed and fabricated onto one dielectric substrate, create integrated optical chip like their microelectronic analogues. These photonic circuits, based on diverse physical phenomena such as light-matter interaction, propagation of electromagnetic waves in a thin dielectric material, nonlinear and electro-optical effects, allow transmission, distribution, modulation, and processing of optical signals in optical communication systems, chemical and biological sensors, and more. The key component of these optical circuits providing both optical processing and photonic interconnections is light waveguides. Optical confinement and transmitting of the optical waves inside the waveguide material are possible due to the higher refractive index of the waveguides in comparison with their surroundings. In this work, we propose a novel field of bionanophotonics based on a new concept of optical waveguiding in synthetic elongated peptide nanostructures composed of ordered peptide dipole biomolecules. New technology of controllable deposition of peptide optical waveguiding structures by nanofountain pen technique is developed. Experimental studies of refractive index, optical transparency, and linear and nonlinear waveguiding in out-of-plane and in-plane diphenylalanine peptide nanotubes have been conducted. Optical waveguiding phenomena in peptide structures are simulated by the finite difference time domain method. The advantages of this new class of bio-optical waveguides are high refractive index contrast, wide spectral range of optical transparency, large optical nonlinearity, and electro-optical effect, making them promising for new applications in integrated multifunctional photonic circuits. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  15. Melanins from opioid peptides.

    PubMed

    Rosei, M A

    1996-12-01

    Opioid peptides and other Tyr-NH2-terminal peptides are substrates in vitro for mushroom and sepia tyrosine, giving rise to synthetic melanins retaining the peptide moiety (opiomelanins). The melanopeptides are characterized by a total solubility in hydrophylic solvents at neutral and basic pH. Opioid peptides (enkephalins, endorphins, and esorphins), if oxidized by tyrosinase in the presence of Dopa, are easily incorporated into Dopa-melanin, producing mixed-type pigments that can also be solubilized in hydrophylic solvents. Melanins derived from opioid peptides exhibit paramagnetism, as evidenced by an EPR spectrum identical to that of Dopa-melanin. However, the presence of the linked peptide chain is able to influence dramatically the electron transfer properties and the oxidizing behaviour of the melanopeptides, so that whereas Tyr-Gly-melanin appears to behave as Dopa-melanin, Enk-melanin does not exhibit any oxidizing activity. Opiomelanins are characterized by a peculiar UV-VIS spectrum; that is, by the presence of a distinct peak (330 nm) that disappears upon chemical treatment by acid hydrolysis. Opiomelanins are stable pigments at neutral and basic pH in the dark, whereas the addition of H2O2 leads to a 15% degradation. Under stimulated solar illumination, opiomelanins are more easily destroyed with respect to Dopa-melanin, with increasing degradation when exposed to increased hydrogen peroxide concentrations and more alkaline pH. Some speculations on the possible existence and role of opiomelanins have been outlined.

  16. Reagent Cluster Anions for Multiple Gas-Phase Covalent Modifications of Peptide and Protein Cations

    NASA Astrophysics Data System (ADS)

    Prentice, Boone M.; Stutzman, John R.; McLuckey, Scott A.

    2013-07-01

    Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the `cost' of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K10 + 3H]3+ and the reagent cluster [5R5Na - Na]-). This type of gas-phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anion which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included as illustrative of the results obtained for the clusters comprised of two to six reagent molecules.

  17. Reagent Cluster Anions for Multiple Gas-phase Covalent Modifications of Peptide and Protein Cations

    PubMed Central

    Prentice, Boone M.; Stutzman, John R.; McLuckey, Scott A.

    2013-01-01

    Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated here using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used here to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the ‘cost’ of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K10+3H]3+ and the reagent cluster [5R5Na-Na]−). This type of gas phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anions which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included here as illustrative of the results obtained for the clusters comprised of 2–6 reagent molecules. PMID:23702708

  18. Magainin 2a - Induced Permeabilization of Phospholipid Vesicles

    DTIC Science & Technology

    1991-01-02

    unilamellar vesicles (SUVs) of phosphatidylserine ( PS ). Addition of peptide to the SUVs causes an initial rapid release of dye, lasting about 100 sec...Measurements 39 VII. Results 40 A. Effect of MGN2a on the Permeability of PS Vesicles 40 B. Effect of Extravesicular Osmotic Pressure on 6CF Release from...Depiction of all-or-none versus graded release mechanisms 34 4. Standard quenching curve for 6CF entrapped within PS SUVs 38 5. MGN2a-induced release

  19. Linear Peptides in Intracellular Applications.

    PubMed

    Zuconelli, Cristiane R; Brock, Roland; Adjobo-Hermans, Merel J W

    2017-01-01

    To this point, efforts to develop therapeutic peptides for intracellular applications were guided by the perception that unmodified linear peptides are highly unstable and therefore structural modifications are required to reduce proteolytic breakdown. Largely, this concept is a consequence of the fact that most research on intracellular peptides hitherto has focused on peptide degradation in the context of antigen processing, rather than on peptide stability. Interestingly, inside cells, endogenous peptides lacking any chemical modifications to enhance stability escape degradation to the point that they may even modulate intracellular signaling pathways. In addition, many unmodified synthetic peptides designed to interfere with intracellular signaling, following introduction into cells, have the expected activity demonstrating that biologically relevant concentrations can be reached. This review provides an overview of results and techniques relating to the exploration and application of linear, unmodified peptides. After an introduction to intracellular peptide turnover, the review mentions examples for synthetic peptides as modulators of intracellular signaling, introduces endogenous peptides with bioactivity, techniques to measure peptide stability, and peptide delivery. Future experiments should elucidate the rules needed to predict promising peptide candidates. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Targeted nanoparticles containing the proresolving peptide Ac2-26 protect against advanced atherosclerosis in hypercholesterolemic mice.

    PubMed

    Fredman, Gabrielle; Kamaly, Nazila; Spolitu, Stefano; Milton, Jaclyn; Ghorpade, Devram; Chiasson, Raymond; Kuriakose, George; Perretti, Mauro; Farokzhad, Omid; Tabas, Ira

    2015-02-18

    Chronic, nonresolving inflammation is a critical factor in the clinical progression of advanced atherosclerotic lesions. In the normal inflammatory response, resolution is mediated by several agonists, among which is the glucocorticoid-regulated protein called annexin A1. The proresolving actions of annexin A1, which are mediated through its receptor N-formyl peptide receptor 2 (FPR2/ALX), can be mimicked by an amino-terminal peptide encompassing amino acids 2-26 (Ac2-26). Collagen IV (Col IV)-targeted nanoparticles (NPs) containing Ac2-26 were evaluated for their therapeutic effect on chronic, advanced atherosclerosis in fat-fed Ldlr(-/-) mice. When administered to mice with preexisting lesions, Col IV-Ac2-26 NPs were targeted to lesions and led to a marked improvement in key advanced plaque properties, including an increase in the protective collagen layer overlying lesions (which was associated with a decrease in lesional collagenase activity), suppression of oxidative stress, and a decrease in plaque necrosis. In mice lacking FPR2/ALX in myeloid cells, these improvements were not seen. Thus, administration of a resolution-mediating peptide in a targeted NP activates its receptor on myeloid cells to stabilize advanced atherosclerotic lesions. These findings support the concept that defective inflammation resolution plays a role in advanced atherosclerosis, and suggest a new form of therapy. Copyright © 2015, American Association for the Advancement of Science.

  1. Electron transfer in peptides.

    PubMed

    Shah, Afzal; Adhikari, Bimalendu; Martic, Sanela; Munir, Azeema; Shahzad, Suniya; Ahmad, Khurshid; Kraatz, Heinz-Bernhard

    2015-02-21

    In this review, we discuss the factors that influence electron transfer in peptides. We summarize experimental results from solution and surface studies and highlight the ongoing debate on the mechanistic aspects of this fundamental reaction. Here, we provide a balanced approach that remains unbiased and does not favor one mechanistic view over another. Support for a putative hopping mechanism in which an electron transfers in a stepwise manner is contrasted with experimental results that support electron tunneling or even some form of ballistic transfer or a pathway transfer for an electron between donor and acceptor sites. In some cases, experimental evidence suggests that a change in the electron transfer mechanism occurs as a result of donor-acceptor separation. However, this common understanding of the switch between tunneling and hopping as a function of chain length is not sufficient for explaining electron transfer in peptides. Apart from chain length, several other factors such as the extent of the secondary structure, backbone conformation, dipole orientation, the presence of special amino acids, hydrogen bonding, and the dynamic properties of a peptide also influence the rate and mode of electron transfer in peptides. Electron transfer plays a key role in physical, chemical and biological systems, so its control is a fundamental task in bioelectrochemical systems, the design of peptide based sensors and molecular junctions. Therefore, this topic is at the heart of a number of biological and technological processes and thus remains of vital interest.

  2. Cyclization in opioid peptides.

    PubMed

    Piekielna, Justyna; Perlikowska, Renata; Gach, Katarzyna; Janecka, Anna

    2013-06-01

    Endogenous opioid peptides have been studied extensively as potential therapeutics for the treatment of pain. The major problems of using natural opioid peptides as drug candidates are their poor receptor specificity, metabolic instability and inability to reach the brain after systemic administration. A lot of synthetic efforts have been made to opioid analogs with improved pharmacological properties. One important structural modification leading to such analogs is cyclization of linear sequences. Intramolecular cyclization has been shown to improve biological properties of various bioactive peptides. Cyclization reduces conformational freedom responsible for the simultaneous activation of two or more receptors, increases metabolic stability and lipophilicity which may result in a longer half-life and easier penetration across biological membranes. This review deals with various strategies that have been employed to synthesize cyclic analogs of opioid peptides. Discussed are such bridging bonds as amide and amine linkages, sulfur-containing bonds, including monosulfide, disulfide and dithioether bridges, bismethylene bonds, monosulfide bridges of lanthionine and, finally, carbonyl and guanidine linkages. Opioid affinities and activities of cyclic analogs are given and compared with linear opioid peptides. Analgesic activities of analogs evaluated in the in vivo pain tests are also discussed.

  3. Antimicrobial Peptides from Plants

    PubMed Central

    Tam, James P.; Wang, Shujing; Wong, Ka H.; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  4. Peptides in oral diseases.

    PubMed

    Lucchese, Alberta; Guida, Agostino; Petruzzi, Massimo; Capone, Giovanni; Laino, Luigi; Serpico, Rosario

    2012-01-01

    The oral cavity is home to numerous viruses and micro-organisms recognized as having a role in various oral diseases as well as in infections in other parts of the body. Indeed, in general a microbial infection underlies or is believed to underlie the ample spectrum of oral diseases, from tooth enamel decay to periodontal lesions, from candidiasis to virus-induced oral squamous cell carcinomas, and bullous autoimmune oral disorders. This clinico-pathological context stresses the need of targeted therapies to specifically kill infectious agents in a complex environment such as the oral cavity, and explains the current interest in exploring peptide-based therapeutic approaches in oral and dental research. Here, we review the therapeutic potential of antimicrobial peptides such as LL-37, beta defensins, adrenomedullin, histatins, and of various peptides modulating gene expression and immuno-biological interaction(s) in oral diseases.

  5. Molecular modeling of peptides.

    PubMed

    Kuczera, Krzysztof

    2015-01-01

    This article presents a review of the field of molecular modeling of peptides. The main focus is on atomistic modeling with molecular mechanics potentials. The description of peptide conformations and solvation through potentials is discussed. Several important computer simulation methods are briefly introduced, including molecular dynamics, accelerated sampling approaches such as replica-exchange and metadynamics, free energy simulations and kinetic network models like Milestoning. Examples of recent applications for predictions of structure, kinetics, and interactions of peptides with complex environments are described. The reliability of current simulation methods is analyzed by comparison of computational predictions obtained using different models with each other and with experimental data. A brief discussion of coarse-grained modeling and future directions is also presented.

  6. A novel 2-(2-Formyl-4-methyl-phenoxy)-N-phenyl-acetamide-based fluorescence turn-on chemosensor for selenium determination with high selectivity and sensitivity

    NASA Astrophysics Data System (ADS)

    Song, Cairui; Fei, Qiang; Shan, Hongyan; Feng, Guodong; Cui, Minghui; Liu, Yameng; Huan, Yanfu

    2013-12-01

    A novel turn-on fluorescent chemosensor, 2-(2-Formyl-4-methyl-phenoxy)-N-phenyl-acetamide (FMPPA) was designed and synthesized, and its photophysical properties were characterized. Upon coordination with Se (IV), the chemosensor showed incredible fluorescence enhancement (turn-on), other alkali, alkaline earth, transitional metal ions, and common anions including Li+, Na+, K+, Rb+, Cs+, Be2+, Mg2+, Ca2+, Sr2+, Ba2+, Ni2+, Cu2+, Cd2+, Zn2+, Mn2+, As3+, Pt4+, V5+, Fe3+, Mo6+, Al3+, CO32-, Cl-, SCN-, AC-, NO3-, F-, SO42- had no significant interference on Se (IV) determination. The chemosensor exhibits a dynamic response range for Se (IV) from 3.32 × 10-7 to 2.63 × 10-6 M, with a detection limit of 9.38 × 10-9 M (3σ).

  7. An oligodeoxyribonucleotide containing 5-formyl-2'-deoxycytidine (fC) at the CpG site forms a covalent complex with DNA cytosine-5 methyltransferases (DNMTs).

    PubMed

    Sato, Kousuke; Kawamoto, Kyoji; Shimamura, Shintaro; Ichikawa, Satoshi; Matsuda, Akira

    2016-11-15

    5-Methylcytosine (mC) is known to induce epigenetic changes. Ten-eleven translocation (TET) enzymes produce the further oxidized 5-substituted cytosine derivatives, 5-formylcytosine (fC) and 5-carboxylcytosine (caC). However, their roles are unclear thus far. Here, we synthesized oligodeoxyribonucleotides (ODNs) containing 5-formyl-2'-deoxycytidine and examined their interactions with DNA cytosine-5 methyltransferase (DNMT). We found that the ODN sequence containing fCpG formed a covalent complex with both bacterial and mouse recombinant DNMTs in the absence of any cofactors. The covalent bonding with DNMT suggests that the fCpG sequence in DNA may play a role in epigenetic regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Radical-Mediated 1,2-Formyl/Carbonyl Functionalization of Alkenes and Application to the Construction of Medium-Sized Rings.

    PubMed

    Li, Zhong-Liang; Li, Xiao-Hua; Wang, Na; Yang, Ning-Yuan; Liu, Xin-Yuan

    2016-11-21

    A novel radical 1,2-formylfunctionalization of alkenes involving 1,2(4,5)-formyl migration triggered by addition of various carbon- and heteroatom-centered radicals to alkenes has been developed for the first time, thus providing straightforward access to diverse β-functionalized aldehydes with good efficiency, remarkable selectivity, and excellent functional group tolerance. Analogous transformations mediated by a keto-carbonyl migration have also been effected under similar conditions. This method was used to access ring systems including various benzannulated nine-, ten-, and eleven-membered rings, complex 6-5(6,7)-6(5) fused rings, and bridged rings with diverse functionalities. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Novel pathway for N1-acetyl-5-methoxykynuramine: UVB-induced liberation of carbon monoxide from precursor N1-acetyl-N2-formyl-5-methoxykynuramine.

    PubMed

    Seever, Katinka; Hardeland, Rüdiger

    2008-05-01

    Irradiation of the melatonin metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) with UV light of 254 nm causes the release of carbon monoxide (CO) and, thus, deformylation to N(1)-acetyl-5-methoxykynuramine (AMK). Liberation of CO was demonstrated by reduction of PdCl(2) to metallic palladium, under avoidance of actions by other reductants. Photochemical AMK formation was not due to UV-induced hydroxyl radicals, because the reaction also took place with high efficiency in ethanol and 2-propanol. Moreover, AMK was generated from AFMK by UVB on a dry thin layer chromatographic plate. Although AMK seems to be the major primary product generated by UVB radiation, prolonged exposure of AFMK led to various other products, especially formed by destruction of AMK, as shown by irradiation of this latter compound. With regard to the demonstration of melatonin in skin and substantial amounts of AFMK in keratinocytes, these findings may be of dermatologic relevance.

  10. Synthesis and spectroscopic studies of 4-Formyl-4'-N,N-dimethylamino-1,1'-biphenyl: The unusual red edge effect and efficient laser generation.

    PubMed

    Chou, P T; Chang, C P; Clements, J H; Meng-Shin, K

    1995-12-01

    The synthesis and photophysics of 4-formyl-4'-N,N-dimethylamino-1,1'-biphenyl are reported. The emission spectrum in various solvent polarities demonstrates solvatochromism, indicating that the fluorescence originates from an electronically excited species with a strong charge transfer character. The change in Δ[Formula: see text] [[Formula: see text] max(absorption) -[Formula: see text] max(emission)] varies from ∼1500 cm(-1) inn-heptane to as much as ∼7500 cm(-1) in acetonitrile. In protic solvents, the unusual excitation energy-dependent steady-state emission (red edge effect), resulting from solvent dielectric relaxation, was observed in media with a low viscosity. The large Stokes-shifted and high-yield fluorescence led to the observation of the efficient lasing action. The frequency tunability of the laser output is strongly solvent dependent, generating a new charge transfer laser dye in the blue-green region.

  11. Synthesis, spectroscopic characterization and biological evaluation studies of Schiff's base derived from naphthofuran-2-carbohydrazide with 8-formyl-7-hydroxy-4-methyl coumarin and its metal complexes

    NASA Astrophysics Data System (ADS)

    Halli, M. B.; Sumathi, R. B.; Kinni, Mallikarjun

    2012-12-01

    Metal complexes of the type ML2, where M = Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Hg(II) and L = Schiff's base derived from the condensation of naphthofuran-2-carbohydrazide with 8-formyl-7-hydroxy-4-methyl coumarin have been synthesized. The chelation of the complexes have been elucidated in the light of analytical, IR, UV-vis, 1H NMR, mass, ESR spectral data, thermal and magnetic studies. The measured molar conductance values indicate that, the complexes are non-electrolytic in nature. The redox behavior of one of the synthesized metal complexes was investigated by cyclic voltammetry. The Schiff's base and its metal complexes have been screened for their in vitro antibacterial and antifungal activities by MIC method. The DNA cleavage activities of all the complexes were studied by agarose gel electrophoresis method. In addition, the free ligand along with its complexes has been studied for their antioxidant activity.

  12. Epitope peptides and immunotherapy.

    PubMed

    Tanabe, Soichi

    2007-02-01

    Allergic diseases affect atopic individuals, who synthesize specific Immunoglobulins E (IgE) to environmental allergens, usually proteins or glycoproteins. These allergens include grass and tree pollens, indoor allergens such as house dust mites and animal dander, and various foods. Because allergen-specific IgE antibodies are the main effector molecules in the immune response to allergens, many studies have focused on the identification of IgE-binding epitopes (called B cell epitopes), specific and minimum regions of allergen molecules that binds to IgE. Our initial studies have provided evidence that only four to five amino acid residues are enough to comprise an epitope, since pentapeptide QQQPP in wheat glutenin is minimally required for IgE binding. Afterwards, various kinds of B cell epitope structures have been clarified. Such information contributes greatly not only to the elucidation of the etiology of allergy, but also to the development of strategies for the treatment and prevention of allergy. Allergen-specific T cells also play an important role in allergy and are obvious targets for intervention in the disease. Currently, the principle approach is to modify B cell epitopes to prevent IgE binding while preserving T cell epitopes to retain the capacity for immunotherapy. There is mounting evidence that the administration of peptide(s) containing immunodominant T cell epitopes from an allergen can induce T cell nonresponsiveness (immunotherapy). There have been clinical studies of peptide immunotherapy performed, the most promising being for bee venom sensitivity. Clinical trials of immunotherapy for cat allergen peptide have also received attention. An alternative strategy for the generation of an effective but hypoallergenic preparation for immunotherapy is to modify T cell epitope peptides by, for example, single amino acid substitution. In this article, I will present an overview of epitopes related to allergic disease, particularly stress on

  13. Kinetics and Molecular Docking Studies of 6-Formyl Umbelliferone Isolated from Angelica decursiva as an Inhibitor of Cholinesterase and BACE1.

    PubMed

    Ali, Md Yousof; Seong, Su Hui; Reddy, Machireddy Rajeshkumar; Seo, Sung Yong; Choi, Jae Sue; Jung, Hyun Ah

    2017-09-24

    Coumarins, which have low toxicity, are present in some natural foods, and are used in various herbal remedies, have attracted interest in recent years because of their potential medicinal properties. In this study, we report the isolation of two natural coumarins, namely umbelliferone (1) and 6-formyl umbelliferone (2), from Angelica decursiva, and the synthesis of 8-formyl umbelliferone (3) from 1. We investigated the anti-Alzheimer disease (anti-AD) potential of these coumarins by assessing their ability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1). Among these coumarins, 2 exhibited poor inhibitory activity against AChE and BChE, and modest activity against BACE1. Structure-activity relationship analysis showed that 2 has an aldehyde group at the C-6 position, and exhibited strong anti-AD activity, whereas the presence or absence of an aldehyde group at the C-8 position reduced the anti-AD activity of 3 and 1, respectively. In addition, 2 exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. A kinetic study revealed that 2 and 3 non-competitively inhibited BACE1. To confirm enzyme inhibition, we predicted the 3D structures of AChE and BACE1, and used AutoDock 4.2 to simulate binding of coumarins to these enzymes. The blind docking studies demonstrated that these molecules could interact with both the catalytic active sites and peripheral anionic sites of AChE and BACE1. Together, our results indicate that 2 has an interesting inhibitory activity in vitro, and can be used in further studies to develop therapeutic modalities for the treatment of AD.

  14. Structure and function of a cyanophage-encoded peptide deformylase

    PubMed Central

    Frank, Jeremy A; Lorimer, Don; Youle, Merry; Witte, Pam; Craig, Tim; Abendroth, Jan; Rohwer, Forest; Edwards, Robert A; Segall, Anca M; Burgin, Alex B

    2013-01-01

    Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 Å resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities. PMID:23407310

  15. Multidimensional signatures in antimicrobial peptides

    PubMed Central

    Yount, Nannette Y.; Yeaman, Michael R.

    2004-01-01

    Conventional analyses distinguish between antimicrobial peptides by differences in amino acid sequence. Yet structural paradigms common to broader classes of these molecules have not been established. The current analyses examined the potential conservation of structural themes in antimicrobial peptides from evolutionarily diverse organisms. Using proteomics, an antimicrobial peptide signature was discovered to integrate stereospecific sequence patterns and a hallmark three-dimensional motif. This striking multidimensional signature is conserved among disulfide-containing antimicrobial peptides spanning biological kingdoms, and it transcends motifs previously limited to defined peptide subclasses. Experimental data validating this model enabled the identification of previously unrecognized antimicrobial activity in peptides of known identity. The multidimensional signature model provides a unifying structural theme in broad classes of antimicrobial peptides, will facilitate discovery of antimicrobial peptides as yet unknown, and offers insights into the evolution of molecular determinants in these and related host defense effector molecules. PMID:15118082

  16. Related impurities in peptide medicines.

    PubMed

    D'Hondt, Matthias; Bracke, Nathalie; Taevernier, Lien; Gevaert, Bert; Verbeke, Frederick; Wynendaele, Evelien; De Spiegeleer, Bart

    2014-12-01

    Peptides are an increasingly important group of pharmaceuticals, positioned between classic small organic molecules and larger bio-molecules such as proteins. Currently, the peptide drug market is growing twice as fast as other drug markets, illustrating the increasing clinical as well as economical impact of this medicine group. Most peptides today are manufactured by solid-phase peptide synthesis (SPPS). This review will provide a structured overview of the most commonly observed peptide-related impurities in peptide medicines, encompassing the active pharmaceutical ingredients (API or drug substance) as well as the finished drug products. Not only is control of these peptide-related impurities and degradants critical for the already approved and clinically used peptide-drugs, these impurities also possess the capability of greatly influencing initial functionality studies during early drug discovery phases, possibly resulting in erroneous conclusions. The first group of peptide-related impurities is SPPS-related: deletion and insertion of amino acids are related to inefficient Fmoc-deprotection and excess use of amino acid reagents, respectively. Fmoc-deprotection can cause racemization of amino acid residues and thus diastereomeric impurities. Inefficient deprotection of amino acid side chains results into peptide-protection adducts. Furthermore, unprotected side chains can react with a variety of reagents used in the synthesis. Oxidation of amino acid side chains and dimeric-to-oligomeric impurities were also observed. Unwanted peptide counter ions such as trifluoroacetate, originating from the SPPS itself or from additional purification treatments, may also be present in the final peptide product. Contamination of the desired peptide product by other unrelated peptides was also seen, pointing out the lack of appropriate GMP. The second impurity group results from typical peptide degradation mechanisms such as β-elimination, diketopiperazine, pyroglutamate

  17. Brain Peptides and Psychopharmacology

    ERIC Educational Resources Information Center

    Arehart-Treichel, Joan

    1976-01-01

    Proteins isolated from the brain and used as drugs can improve and apparently even transfer mental states and behavior. Much of the pioneering work and recent research with humans and animals is reviewed and crucial questions that are being posed about the psychologically active peptides are related. (BT)

  18. Peptide -- Silica Hybrid Networks

    NASA Astrophysics Data System (ADS)

    Altunbas, Aysegul; Sharma, Nikhil; Nagarkar, Radhika; Schneider, Joel; Pochan, Darrin

    2010-03-01

    In this study, a bio-inspired route was used to fabricate scaffolds that display hierarchical organization of an inorganic layer around an organic self-assembled peptide fibril template. The 20 amino acid peptide used in this study intramolecular folds into a beta-hairpin conformation on addition of a desired solution stimulus. This intramolecular folding is followed by intermolecular self-assembly of the peptides into a three dimensional network of entangled fibrils rich in beta-sheet with a high density of lysine groups exposed on the fibril-surfaces. The lysine-rich surface chemistry was utilized to create a silica shell around the fibrils. The mineralization process of the fibrils results in a rigid, porous silica network that retains the microscale and nanoscale structure of the peptide fibril network. Structural characterization via Transmission Electron Microscopy, cryogenic-Scanning Electron Microscopy, mechanical characterization via oscillatory rheology, Small Angle X-ray and Neutron Scattering of the silicified hydrogels will be presented.

  19. Bioinformatic identification of plant peptides.

    PubMed

    Lease, Kevin A; Walker, John C

    2010-01-01

    Plant peptides play a number of important roles in defence, development and many other aspects of plant physiology. Identifying additional peptide sequences provides the starting point to investigate their function using molecular, genetic or biochemical techniques. Due to their small size, identifying peptide sequences may not succeed using the default bioinformatic approaches that work well for average-sized proteins. There are two general scenarios related to bioinformatic identification of peptides to be discussed in this paper. In the first scenario, one already has the sequence of a plant peptide and is trying to find more plant peptides with some sequence similarity to the starting peptide. To do this, the Basic Local Alignment Search Tool (BLAST) is employed, with the parameters adjusted to be more favourable for identifying potential peptide matches. A second scenario involves trying to identify plant peptides without using sequence similarity searches to known plant peptides. In this approach, features such as protein size and the presence of a cleavable amino-terminal signal peptide are used to screen annotated proteins. A variation of this method can be used to screen for unannotated peptides from genomic sequences. Bioinformatic resources related to Arabidopsis thaliana will be used to illustrate these approaches.

  20. Synthesis of peptide analogues using the multipin peptide synthesis method.

    PubMed

    Valerio, R M; Benstead, M; Bray, A M; Campbell, R A; Maeji, N J

    1991-08-15

    Modification of the multipin peptide synthesis method which allows the simultaneous synthesis of large numbers of different peptide analogues is described. Peptides were assembled on polyethylene pins derivatized with a 4-(beta-alanyloxymethyl)benzoate (beta-Ala-HMB) handle. For comparative purposes, peptides were also assembled on the diketopiperazine-forming handle N epsilon-(beta-alanyl)lysylprolyloxylactate. In model studies it was demonstrated that beta-Ala-HMB-linked peptides were cleaved from polyethylene pins with dilute sodium hydroxide or 4% methylamine/water to yield analogues with beta-Ala-free acid (beta-Ala-CO2H) and beta-Ala-methylamide (beta-Ala-CONHCH3), respectively. To assess the suitability of this approach for T-cell determinant analysis, analogues of a known T-cell determinant were synthesized with the various C-terminal endings. Peptides were characterized by amino acid analysis and fast atom bombardment-mass spectrometry. HPLC of the crude cleaved peptides indicated that 22 of the 24 peptides were greater than 95% pure. These crude peptide solutions were nontoxic in sensitive cell culture assays without further purification. All three cleavage procedures gave comparable activities in T-cell proliferation assays. These results demonstrate the potential of the multipin peptide synthesis method for the production of large numbers of different peptide analogues.

  1. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  2. A Peptide Filtering Relation Quantifies MHC Class I Peptide Optimization

    PubMed Central

    Goldstein, Leonard D.; Howarth, Mark; Cardelli, Luca; Emmott, Stephen; Elliott, Tim; Werner, Joern M.

    2011-01-01

    Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human

  3. High-resolution mono- and multidimensional magic angle spinning 1H nuclear magnetic resonance of membrane peptides in nondeuterated lipid membranes and H2O.

    PubMed Central

    Le Guernevé, C; Seigneuret, M

    1996-01-01

    High-speed (14 kHz) solid-state magic angle spinning (MAS) 1H NMR has been applied to several membrane peptides incorporated into nondeuterated dilauroyl or dimyristoylphosphatidylcholine membranes suspended in H2O. It is shown that solvent suppression methods derived from solution NMR, such as presaturation or jump-return, can be used to reduce water resonance, even at relatively high water content. In addition, regioselective excitation of 1H peptide resonances promotes an efficient suppression of lipid resonances, even in cases where these are initially two orders of magnitude more intense. As a consequence, 1H MAS spectra of the peptide low-field region are obtained without interference from water and lipid signals. These display resonances from amide and other exchangeable 1H as well as from aromatic nonexchangeable 1H. The spectral resolution depends on the specific types of resonance and membrane peptide. For small amphiphilic or hydrophobic oligopeptides, resolution of most individual amide resonance is achieved, whereas for the transmembrane peptide gramicidin A, an unresolved amide spectrum is obtained. Partial resolution of aromatic 1H occurs in all cases. Multidimensional 1H-MAS spectra of membrane peptides can also be obtained by using water suppression and regioselective excitation. For gramicidin A, F2-regioselective 2D nuclear Overhauser effect spectroscopy (NOESY) spectra are dominated by intermolecular through-space connectivities between peptide aromatic or formyl 1H and lipid 1H. These appear to be compatible with the known structure and topography of the gramicidin pore. On the other hand, for the amphiphilic peptide leucine-enkephalin, F2-regioselective NOESY spectra mostly display cross-peaks originating from though-space proximities of amide or aromatic 1H with themselves and with aliphatic 1H. F3-regioselective 3D NOESY-NOESY spectra can be used to obtain through-space correlations within aliphatic 1H. Such intrapeptide proximities should

  4. Concepts for Biologically Active Peptides

    PubMed Central

    Kastin, Abba J.; Pan, Weihong

    2012-01-01

    Here we review a unique aspect of CNS research on biologically active peptides that started against a background of prevalent dogmas but ended by exerting considerable influence on the field. During the course of refuting some doctrines, we introduced several concepts that were unconventional and paradigm-shifting at the time. We showed that (1) hypothalamic peptides can act ‘up’ on the brain as well as ‘down’ on the pituitary, (2) peripheral peptides can affect the brain, (3) peptides can cross the blood-brain barrier, (4) the actions of peptides can persist longer than their half-lives in blood, (5) perinatal administration of peptides can exert actions persisting into adulthood, (6) a single peptide can have more than one action, (7) dose-response relationships of peptides need not be linear, (8) the brain produces antiopiate as well as opiate peptides, (9) there is a selective high affinity endogenous peptide ligand for the mu-opiate receptor, (10) a peptide’s name does not restrict its effects, and (11) astrocytes assume an active role in response to metabolic disturbance and hyperleptinemia. The evolving questions in our laboratories reflect the diligent effort of the neuropeptide community to identify the roles of peptides in the CNS. The next decade is expected to see greater progress in the following areas: (a) interactions of peptides with other molecules in the CNS; (b) peptide involvement in cell-cell interactions; and (c) peptides in neuropsychiatric, autoimmune, and neurodegenerative diseases. The development of peptidomics and gene silencing approaches will expedite the formation of many new concepts in a new era. PMID:20726835

  5. Crystal structure of methyl (2Z)-2-{[N-(2-formyl-phen-yl)-4-methyl-benzene-sulfonamido]-meth-yl}-3-(4-meth-oxy-phen-yl)prop-2-enoate.

    PubMed

    Trigunait, Ankur; Damodharan, Kannan; Manickam, Bakthadoss; Krishnasamy, Gunasekaran

    2015-12-01

    In the title compound, C26H25NO6S, the S atom shows a distorted tetra-hedral geometry, with O-S-O [119.46 (9)°] and N-S-C [107.16 (7)°] angles deviating from ideal tetra-hedral values, a fact attributed to the Thorpe-Ingold effect. The sulfonyl-bound phenyl ring forms dihedral angles of 41.1 (1) and 83.3 (1)°, respectively, with the formyl-phenyl and phenyl rings. The dihedral angle between formyl-phenyl and phenyl rings is 47.6 (1)°. The crystal packing features C-H⋯O hydrogen-bond inter-actions.

  6. Crystal structure of methyl (2Z)-2-{[N-(2-formyl­phen­yl)-4-methyl­benzene­sulfonamido]­meth­yl}-3-(4-meth­oxy­phen­yl)prop-2-enoate

    PubMed Central

    Trigunait, Ankur; Damodharan, Kannan; Manickam, Bakthadoss; Krishnasamy, Gunasekaran

    2015-01-01

    In the title compound, C26H25NO6S, the S atom shows a distorted tetra­hedral geometry, with O—S—O [119.46 (9)°] and N—S—C [107.16 (7)°] angles deviating from ideal tetra­hedral values, a fact attributed to the Thorpe–Ingold effect. The sulfonyl-bound phenyl ring forms dihedral angles of 41.1 (1) and 83.3 (1)°, respectively, with the formyl­phenyl and phenyl rings. The dihedral angle between formyl­phenyl and phenyl rings is 47.6 (1)°. The crystal packing features C—H⋯O hydrogen-bond inter­actions. PMID:26870512

  7. Avian host defense peptides.

    PubMed

    Cuperus, Tryntsje; Coorens, Maarten; van Dijk, Albert; Haagsman, Henk P

    2013-11-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.

  8. Peptide mass fingerprinting.

    PubMed

    Thiede, Bernd; Höhenwarter, Wolfgang; Krah, Alexander; Mattow, Jens; Schmid, Monika; Schmidt, Frank; Jungblut, Peter R

    2005-03-01

    Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.

  9. Antimicrobial Peptides and Colitis

    PubMed Central

    Ho, Samantha; Pothoulakis, Charalabos; Koon, Hon Wai

    2013-01-01

    Antimicrobial peptides (AMPs) are important components of innate immunity. They are often expressed in response to colonic inflammation and infection. Over the last several years, the roles of several antimicrobial peptides have been explored. Gene expression of many AMPs (beta defensin HBD2-4 and cathelicidin) is induced in response to invasion of gut microbes into the mucosal barrier. Some AMPs are expressed in a constitutive manner (alpha defensin HD 5-6 and beta defensin HBD1), while others (defensin and bactericidal/permeability increasing protein BPI) are particularly associated with Inflammatory Bowel Disease (IBD) due to altered defensin expression or development of autoantibodies against Bactericidal/permeability increasing protein (BPI). Various AMPs have different spectrum and strength of antimicrobial effects. Some may play important roles in modulating the colitis (cathelicidin) while others (lactoferrin, hepcidin) may represent biomarkers of disease activity. The use of AMPs for therapeutic purposes is still at an early stage of development. A few natural AMPs were shown to be able to modulate colitis when delivered intravenously or intracolonically (cathelicidin, elafin and SLPI) in mouse colitis models. New AMPs (synthetic or artificial non-human peptides) are being developed and may represent new therapeutic approaches against colitis. This review discusses the latest research developments in the AMP field with emphasis in innate immunity and pathophysiology of colitis. PMID:22950497

  10. Synthesis, characterisation, conformational preferences, dynamic NMR studies and antimicrobial evaluation of N-nitroso- and N-formyl-c-3,t-3-dimethyl-r-2,c-7-diphenyl-1,4-diazepan-5-ones

    NASA Astrophysics Data System (ADS)

    Ponnuswamy, S.; Akila, A.; Kiruthiga devi, D.; Maheshwaran, V.; Ponnuswamy, M. N.

    2016-04-01

    The stereochemical preferences of N-nitroso- and N-formyl-c-3,t-3-dimethyl-r-2,c-7-diphenyl-1,4-diazepan-5-ones 3 & 4, respectively, have been determined using 1D and 2D NMR spectral techniques. Interestingly, the N-nitroso compound 3 is found to prefer an equilibrium between alternate chair conformations with diaxial phenyl groups, while the N-formyl compound 4 prefers flattened boat conformation. This is stereochemically a novel report on the flexible rings adopting a chair conformation with diaxial phenyl groups. The X-ray crystal structure of N-nitroso-c-3,t-3-dimethyl-r-2,c-7-diphenyl-1,4-diazepan-5-one (3) also supports the chair conformation with diaxial phenyl groups. Dynamic 1H NMR spectral studies have been carried out on the N-nitroso and N-formyl diazepan-5-ones 3 &4 and the energy barriers for N-NO and N-CO rotations are found to be 88.7 and 75.7 kJ/mol, respectively. The antimicrobial activities have been determined for the compounds 2-4 and it is found that all the compounds exhibit significant antibacterial and antifungal activities when compared to the standard chloramphenicol.

  11. Gold(III) chloride catalyzed synthesis of chiral substituted 3-formyl furans from carbohydrates: application in the synthesis of 1,5-dicarbonyl derivatives and furo[3,2-c]pyridine.

    PubMed

    Mal, Kanchan; Sharma, Abhinandan; Das, Indrajit

    2014-09-08

    This report describes a gold(III)-catalyzed efficient general route to densely substituted chiral 3-formyl furans under extremely mild conditions from suitably protected 5-(1-alkynyl)-2,3-dihydropyran-4-one using H2 O as a nucleophile. The reaction proceeds through the initial formation of an activated alkyne-gold(III) complex intermediate, followed by either a domino nucleophilic attack/anti-endo-dig cyclization, or the formation of a cyclic oxonium ion with subsequent attack by H2 O. To confirm the proposed mechanistic pathway, we employed MeOH as a nucleophile instead of H2 O to result in a substituted furo[3,2-c]pyran derivative, as anticipated. The similar furo[3,2-c]pyran skeleton with a hybrid carbohydrate-furan derivative has also been achieved through pyridinium dichromate (PDC) oxidation of a substituted chiral 3-formyl furan. The corresponding protected 5-(1-alkynyl)-2,3-dihydropyran-4-one can be synthesized from the monosaccharides (both hexoses and pentose) following oxidation, iodination, and Sonogashira coupling sequences. Furthermore, to demonstrate the potentiality of chiral 3-formyl furan derivatives, a TiBr4 -catalyzed reaction of these derivatives has been shown to offer efficient access to 1,5-dicarbonyl compounds, which on treatment with NH4 OAc in slightly acidic conditions afforded substituted furo[3,2-c]pyridine.

  12. Solid-phase peptide synthesis and solid-state NMR spectroscopy of (Ala/sup 3/-/sup 15/N)(Val/sup 1/)gramicidin A

    SciTech Connect

    Fields, G.B.; Fields, C.G.; Petefish, J.; Van Wart, H.E.; Cross, T.A.

    1988-03-01

    (Ala/sup 3-15/N)(Val/sup 1/)Gramicidin A has been prepared by solid-phase peptide synthesis and studied by solid-state /sup 15/N nuclear magnetic resonance spectroscopy. The synthesis of desformyl(Ala/sup 3-15/N)(Val/sup 1/)gramicidin A employed N-hydroxysuccinimide esters of 9-fluorenylmethoxycarbonyl-N/sup ..cap alpha../-amino acids and completely avoided the use of acid. Since deblocking was done with piperidine and the peptide was removed from the resin by treatment with ethanolamine, this synthetic protocol prevented oxidation of the indole rings of this tryptophan-rich peptide and reduced truncations produced by acid hydrolysis. After formylation and purification by anion-exchange and high-pressure liquid chromatography, the peptide was obtained in an overall yield of 30%. Solid-state /sup 15/N nuclear magnetic resonance spectra of this peptide and uniformly labeled (/sup 15/N)gramicidin A' oriented in hydrated lipid bilayers have been obtained, allowing unambiguous assignment of the (/sup 15/N)Ala/sup 3/ resonance in the latter. The solid-state /sup 15/N nuclear magnetic resonance experiments provide evidence that (Val/sup 1/)gramicidin A is rotating about an axis that is perpendicular to the plane of the lipid bilayer and that the N-H axis is nearly parallel with the rotational axis. This study demonstrates that site-specifically labeled (/sup 15/N)gramicidin A analogs prepared by solid-phase peptide synthesis are valuable tools in the study of the solid-state nuclear magnetic resonance spectra of samples in oriented lipid bilayers.

  13. [Hydrolysis of peptides by immobilized bacterial peptide hydrolases].

    PubMed

    Nekliudov, A D; Deniakina, E K

    2004-01-01

    The feasibility of hydrolysis of a mixture of peptides with an enzyme from the bacterium Xanthomonas rubrilineans, displaying a peptidase activity and immobilized on aluminum oxide, was studied. Kinetic schemes and equations allowing for approaching quantitative description of peptide hydrolysis in complex mixtures containing free amino acids and peptides were obtained. It was demonstrated that as a result of hydrolysis, the content of free amino acids in hydrolysates decreased 2.5- to 3-fold and the molecular weight of the constituent peptides, 2-fold.

  14. The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells

    PubMed Central

    Ingangi, Vincenzo; Bifulco, Katia; Yousif, Ali Munaim; Ragone, Concetta; Motti, Maria Letizia; Rea, Domenica; Minopoli, Michele; Botti, Giovanni; Scognamiglio, Giuseppe; Fazioli, Flavio; Gallo, Michele; De Chiara, Annarosaria; Arra, Claudio; Grieco, Paolo; Carriero, Maria Vincenza

    2016-01-01

    The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88–92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88–92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream. PMID:27323409

  15. Mobile protons versus mobile radicals: gas-phase unimolecular chemistry of radical cations of cysteine-containing peptides.

    PubMed

    Lam, Adrian K Y; Ryzhov, Victor; O'Hair, Richard A J

    2010-08-01

    A combination of electrospray ionization (ESI), multistage, and high-resolution mass spectrometry experiments are used to examine the gas-phase fragmentation reactions of radical cations of cysteine containing di- and tripeptides. Two different chemical methods were used to form initial populations of radical cations in which the radical sites were located at different positions: (1) sulfur-centered cysteinyl radicals via bond homolysis of protonated S-nitrosocysteine containing peptides; and (2) alpha-carbon backbone-centered radicals via Siu's sequence of reactions (J. Am. Chem. Soc.2008, 130, 7862). Comparison of the fragmentation reactions of these regiospecifically generated radicals suggests that hydrogen atom transfer (HAT) between the alpha C-H of adjacent residues and the cysteinyl radical can occur. In addition, using accurate mass measurements, deuterium labeling, and comparison with an authentic sample, a novel loss of part of the N-terminal cysteine residue was shown to give rise to the protonated, truncated N-formyl peptide (an even-electron x(n) ion). DFT calculations were performed on the radical cation [GCG]*(+) to examine: the relative stabilities of isomers with different radical and protonation sites; the barriers associated with radical migration between four possible radical sites, [G*CG](+), [GC*G](+), [GCG*](+), and [GC(S*)G](+); and for dissociation from these sites to yield b(2)-type ions. Copyright 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

  16. The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells.

    PubMed

    Ingangi, Vincenzo; Bifulco, Katia; Yousif, Ali Munaim; Ragone, Concetta; Motti, Maria Letizia; Rea, Domenica; Minopoli, Michele; Botti, Giovanni; Scognamiglio, Giuseppe; Fazioli, Flavio; Gallo, Michele; De Chiara, Annarosaria; Arra, Claudio; Grieco, Paolo; Carriero, Maria Vincenza

    2016-08-23

    The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88-92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.

  17. Formation of the First Peptide Bond: The Structure of EF-P Bound to the 70S Ribosome

    SciTech Connect

    Blaha, Gregor; Stanley, Robin E.; Steitz, Thomas A.; Yale

    2009-10-21

    Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. Here we report the crystal structure of EF-P bound to the Thermus thermophilus 70S ribosome along with the initiator transfer RNA N-formyl-methionyl-tRNAi (fMet-tRNA{sub i}{sup fMet}) and a short piece of messenger RNA (mRNA) at a resolution of 3.5 angstroms. EF-P binds to a site located between the binding site for the peptidyl tRNA (P site) and the exiting tRNA (E site). It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site-bound initiator tRNA. Domain II of EF-P interacts with the ribosomal protein L1, which results in the largest movement of the L1 stalk that has been observed in the absence of ratcheting of the ribosomal subunits. EF-P facilitates the proper positioning of the fMet-tRNA{sub i}{sup fMet} for the formation of the first peptide bond during translation initiation.

  18. Formation of the First Peptid Bond: the Structure of EF-P Bound to the 70S Ribosome

    SciTech Connect

    Blaha, G.; Stanley, R; Steitz, T

    2009-01-01

    Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. Here we report the crystal structure of EF-P bound to the Thermus thermophilus 70S ribosome along with the initiator transfer RNA N-formyl-methionyl-tRNAi (fMet-tRNAifMet) and a short piece of messenger RNA (mRNA) at a resolution of 3.5 angstroms. EF-P binds to a site located between the binding site for the peptidyl tRNA (P site) and the exiting tRNA (E site). It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site-bound initiator tRNA. Domain II of EF-P interacts with the ribosomal protein L1, which results in the largest movement of the L1 stalk that has been observed in the absence of ratcheting of the ribosomal subunits. EF-P facilitates the proper positioning of the fMet-tRNAifMet for the formation of the first peptide bond during translation initiation.

  19. Macrocyclization of Unprotected Peptide Isocyanates.

    PubMed

    Vinogradov, Alexander A; Choo, Zi-Ning; Totaro, Kyle A; Pentelute, Bradley L

    2016-03-18

    A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities.

  20. Antitumor Peptides from Marine Organisms

    PubMed Central

    Zheng, Lan-Hong; Wang, Yue-Jun; Sheng, Jun; Wang, Fang; Zheng, Yuan; Lin, Xiu-Kun; Sun, Mi

    2011-01-01

    The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides. PMID:22072999

  1. Improving Peptide Applications Using Nanotechnology.

    PubMed

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

  2. Peptides that influence membrane topology

    NASA Astrophysics Data System (ADS)

    Wong, Gerard C. L.

    2014-03-01

    We examine the mechanism of a range of polypeptides that influence membrane topology, including antimicrobial peptides, cell penetrating peptides, viral fusion peptides, and apoptosis proteins, and show how a combination of geometry, coordination chemistry, and soft matter physics can be used to approach a unified understanding. We will also show how such peptides can impact biomedical problems such as auto-immune diseases (psoriasis, lupus), infectious diseases (viral and bacterial infections), and mitochondrial pathologies (under-regulated apoptosis leads to neurodegenerative diseases whereas over-regulated apoptosis leads to cancer.)

  3. The PeptideAtlas Project.

    PubMed

    Deutsch, Eric W

    2010-01-01

    PeptideAtlas is a multi-species compendium of peptides observed with tandem mass spectrometry methods. Raw mass spectrometer output files are collected from the community and reprocessed through a uniform analysis and validation pipeline that continues to advance. The results are loaded into a database and the information derived from the raw data is returned to the community via several web-based data exploration tools. The PeptideAtlas resource is useful for experiment planning, improving genome annotation, and other data mining projects. PeptideAtlas has become especially useful for planning targeted proteomics experiments.

  4. Antitumor peptides from marine organisms.

    PubMed

    Zheng, Lan-Hong; Wang, Yue-Jun; Sheng, Jun; Wang, Fang; Zheng, Yuan; Lin, Xiu-Kun; Sun, Mi

    2011-01-01

    The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides.

  5. Biodiscovery of aluminum binding peptides

    NASA Astrophysics Data System (ADS)

    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.

  6. Ligand and Structure-Based Approaches for the Identification of Peptide Deformylase Inhibitors as Antibacterial Drugs

    PubMed Central

    Gao, Jian; Liang, Li; Zhu, Yasheng; Qiu, Shengzhi; Wang, Tao; Zhang, Ling

    2016-01-01

    Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins, which makes it an important antibacterial drug target. Given the importance of PDF inhibitors like actinonin in antibacterial drug discovery, several reported potent PDF inhibitors were used to develop pharmacophore models using the Galahad module of Sybyl 7.1 software. Generated pharmacophore models were composed of two donor atom centers, four acceptor atom centers and two hydrophobic groups. Model-1 was screened against the Zinc database and several compounds were retrieved as hits. Compounds with Qfit values of more than 60 were employed to perform a molecular docking study with the receptor Escherichia coli PDF, then compounds with docking score values of more than 6 were used to predict the in silico pharmacokinetic and toxicity risk via OSIRIS property explorer. Two known PDF inhibitors were also used to perform a molecular docking study with E. coli PDF as reference molecules. The results of the molecular docking study were validated by reproducing the crystal structure of actinonin. Molecular docking and in silico pharmacokinetic and toxicity prediction studies suggested that ZINC08740166 has a relatively high docking score of 7.44 and a drug score of 0.78. PMID:27428963

  7. Macrocyclic inhibitors for peptide deformylase: a structure-activity relationship study of the ring size.

    PubMed

    Hu, Xubo; Nguyen, Kiet T; Jiang, Vernon C; Lofland, Denene; Moser, Heinz E; Pei, Dehua

    2004-09-23

    Peptide deformylase (PDF) catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria. Its essential role in bacterial cells but not in mammalian cells makes it an attractive target for antibacterial drug design. We have previously reported an N-formylhydroxylamine-based, metal-chelating macrocyclic PDF inhibitor, in which the P(1)' and P(3)' side chains are covalently joined. In this work, we have carried out a structure-activity relationship study on the size of the macrocycle and found that 15-17-membered macrocycles are optimal for binding to the PDF active site. Unlike the acyclic compounds, which are simple competitive inhibitors, the cyclic compounds all act as slow-binding inhibitors. As compared to their acyclic counterparts, the cyclic inhibitors displayed 20-50-fold higher potency against the PDF active site (K(I) as low as 70 pM), improved selectivity toward PDF, and improved the metabolic stability in rat plasma. Some of the macrocyclic inhibitors had potent, broad spectrum antibacterial activity against clinically significant Gram-positive and Gram-negative pathogens. These results suggest that the macrocyclic scaffold provides an excellent lead for the development of a new class of antibiotics.

  8. Vasoactive intestinal peptide inhibits fMLP-induced respiratory burst in human lymphocytes.

    PubMed

    Bellido, L; López-González, M A; Pedrera, C; Lucas, M

    1994-01-01

    N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) induced in lymphocytes the production of reactive oxygen intermediates in a process which was inhibited by the presence of Vasoactive Intestinal Peptide (VIP) in a dose-dependent response at VIP concentrations in the range 10(-10)-10(-7) M. The dissociation constant for the high-affinity receptors of VIP agrees with the ID50 of the activation of adenylate cyclase which are close to 0.2 nM VIP, whereas the ID50 for the inhibition by VIP of fMLP-induced chemiluminescence approaches to 5 nM VIP. Both IBMX and Forskolin produced in lymphocytes an inhibition of fMLP-induced chemiluminescence. The degree of inhibition was ascertained to be additive in the presence of the above indicated agents and suboptimal concentrations of VIP. The saturation by cAMP of its putative target, the regulatory subunit of protein kinase A, appears to be required for the onset of the inhibitory effect of VIP. This study provides evidence of the molecular signal, namely cAMP, which provokes an inhibitory effect on chemoatractant-stimulated human lymphocytes and further support a role for VIP as a mediator in the neuroimmune system.

  9. Peptide Amyloid Surface Display

    PubMed Central

    2015-01-01

    Homomeric self-assembly of peptides into amyloid fibers is a feature of many diseases. A central role has been suggested for the lateral fiber surface affecting gains of toxic function. To investigate this, a protein scaffold that presents a discrete, parallel β-sheet surface for amyloid subdomains up to eight residues in length has been designed. Scaffolds that present the fiber surface of islet amyloid polypeptide (IAPP) were prepared. The designs show sequence-specific surface effects apparent in that they gain the capacity to attenuate rates of IAPP self-assembly in solution and affect IAPP-induced toxicity in insulin-secreting cells. PMID:25541905

  10. Peptides and food intake.

    PubMed

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  11. Plant antimicrobial peptides.

    PubMed

    Nawrot, Robert; Barylski, Jakub; Nowicki, Grzegorz; Broniarczyk, Justyna; Buchwald, Waldemar; Goździcka-Józefiak, Anna

    2014-05-01

    Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.

  12. Peptides and Food Intake

    PubMed Central

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  13. Synthesis, spectroscopy (vibrational, NMR and UV-vis) studies, HOMO-LUMO and NBO analysis of 8-formyl-7-hydroxy-4-methylcoumarin by ab initio calculations

    NASA Astrophysics Data System (ADS)

    Moghanian, Hassan; Mobinikhaledi, Akbar; Monjezi, Roya

    2013-11-01

    In this work, 8-formyl-7-hydroxy-4-methylcoumarin has been synthesized and characterized by elemental analysis, FT-IR, FT Raman, 1H NMR, 13C NMR and UV-vis spectra. The molecular geometry, harmonic vibrational frequencies and gauge including atomic orbital (GIAO) 1H and 13C chemical shift values of the title compound in the ground state have been calculated by using Hartree-Fock (HF) and density functional methods (B3LYP) with 6-311++G(d,p) as basis set. The vibrational assignments of wave numbers were interpreted in terms of potential energy distribution (PED) analysis and the scaled B3LYP/6-311++G(d,p) results show the good agreement with the experimental values. The UV spectra of investigated compound were recorded in the region of 230-500 nm in chloroform solution. The energy and oscillator strength calculated by Time-Dependent Density Functional Theory (TD-DFT) in gas and CHCl3 theoretically and results were compared with experimental observations. The molecular stability arising from hyperconjugative interactions and charge delocalization have been analyzed using natural bond orbital (NBO) analysis. In addition, Frontier Molecular Orbitals (FMO), Molecular Electrostatic Potential (MEP) and thermodynamic properties of the studied compound such as heat capacity (C), entropy (S) and enthalpy changes (H) at different temperatures have been calculated.

  14. Luminol-dependent chemiluminescence is related to the extracellularly released reactive oxygen intermediates in the case of rat neutrophils activated by formyl-methionyl-leucyl-phenylalanine.

    PubMed

    Németh, Krisztina; Fûrész, József; Csikor, Katalin; Schweitzer, Katalin; Lakatos, Susan

    2002-01-01

    Luminol is a non-radical-specific amplifying molecule which produces light upon interaction with various reactive oxygen intermediates (ROIs). ROI production of rat peritoneal polymorphonuclear leukocytes (PMNLs) elicited by 2.3 microM formyl-methionyl-leucyl-phenylalanine (fMLP) results in a biphasic luminol-dependent chemiluminescence (LDCL) signal. Whereas ROIs are also produced intracellularly, as judged by flow cytometry, addition of non-membrane-permeable catalase reduces the first and second phases of the LDCL signal to around 3% and less than 3%, respectively. This suggests that in the case of fMLP-stimulated rat PMNLs, the LDCL signal is related to the ROIs in the extracellular medium and hydrogen peroxide has a key role in the formation of the LDCL signal. In the presence of the non-specific myeloperoxidase inhibitor Na-azide, the first phase of the LDCL signal decreases slightly (87+/-8%), while the second phase almost disappears (< 3%), indicating the myeloperoxidase dependence of the second phase. The hydroxyl radical scavenger histidine results in an 84+/-4% and a 71+/-4% decrease in the intensity of the first and second phases, respectively. Based on these data, it is concluded that hydrogen peroxide might be the source of hydroxyl radicals directly oxidizing luminol in the first phase of the LDCL signal, while in the second phase it serves as a substrate of myeloperoxidase in the peroxidation reaction of the luminol.

  15. Analysis of N1-acetyl-N2-formyl-5-methoxykynuramine/N1-acetyl-5-methoxy-kynuramine formation from melatonin in mice

    PubMed Central

    Niu, Sida; Li, Feng; Tan, Dun-Xian; Zhang, Lirong; Idle, Jeffrey R.; Gonzalez, Frank J.; Ma, Xiaochao

    2015-01-01

    The interactions of melatonin, a potent endogenous antioxidant, with reactive oxygen species generate several products that include N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK). The physiological or pathological significance of AFMK/AMK formation during the process of melatonin metabolism in mammals has not been clarified. Using a metabolomic approach in the current study, the AFMK/AMK pathway was thoroughly investigated both in mice and humans. Unexpectedly, AFMK and AMK were not identified in the urine of humans nor in the urine, feces or tissues (including liver, brain, and eyes) in mice under the current experimental conditions. Metabolomic analysis did identify novel metabolites of AMK, i.e. hydroxy-AMK and glucuronide-conjugated hydroxy-AMK. These two newly identified metabolites were, however, not found in the urine of humans. In addition, oxidative stress induced by acetaminophen in the mouse model did not boost AFMK/AMK formation. These data suggest that AFMK/AMK formation is not a significant pathway of melatonin disposition in mice, even under conditions of oxidative stress. PMID:20545825

  16. Melatonin enhances photo-oxidation of 2',7'-dichlorodihydrofluorescein by an antioxidant reaction that renders N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK).

    PubMed

    Hevia, David; Mayo, Juan C; Tan, Dun-Xian; Rodriguez-Garcia, Aida; Sainz, Rosa M

    2014-01-01

    The indolamine melatonin (MEL) is described as an antioxidant and a free radical scavenger. However occasionally, the indoleamine has been reported to increase free radicals with insufficient mechanistic explanation. In an attempt to find a reason for those controversial results, a potential mechanism that explains MEL prooxidant activity is investigated. The current controversy about redox detection methods has prompted us to search a possible interaction between MEL and dichlorodihydrofluorescein (DCFH2), perhaps the most widely fluorescence probe employed for free radicals detection in cellular models. Here, it is demonstrated that melatonin potentiates the photooxidation of DCFH2 in a cell-free system, increasing the production of its fluorescent metabolite. Indeed, MEL works as an antioxidant scavenging hydroxyl radicals in this system. Thus, this reaction between MEL and DCFH2 produces N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a biogenic amine with antioxidant properties too. This reaction is O2 and light dependent and it is prevented by antioxidants such as N-acetylcysteine or ascorbic acid. Furthermore, when DCFH2 has been employed to evaluate antioxidant or prooxidant activities of MEL in cellular models it is confirmed that it works as an antioxidant but these results can be modulated by light misleading to a prooxidant conclusion. In conclusion, here is demonstrated that DCFH2, light and melatonin interact and results obtained using these fluorescence probes in studies with melatonin have to be carefully interpreted.

  17. Novel rhythms of N1-acetyl-N2-formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation.

    PubMed

    Tan, Dun-Xian; Manchester, Lucien C; Di Mascio, Paolo; Martinez, Glaucia R; Prado, Fernanda M; Reiter, Russel J

    2007-06-01

    N1-acetyl-N2-formyl-5-methoxykynuramine (AMFK) is a major metabolite of melatonin in mammals. To investigate whether AFMK exists in plants, an aquatic plant, water hyacinth, was used. To achieve this, LC/MS/MS with a deuterated standard was employed. AFMK was identified in any plant for the first time. Both it and its precursor, melatonin, were rhythmic with peaks during the late light phase. These novel rhythms indicate that these molecules do not serve as the chemical signal of darkness as in animals but may relate to processes of photosynthesis or photoprotection. These possibilities are supported by higher production of melatonin and AFMK in plants grown in sunlight (10,000-15,000 microW/cm2) compared to those grown under artificial light (400-450 microW/cm2). Melatonin and AFMK, as potent free radical scavengers, may assist plants in coping with harsh environmental insults, including soil and water pollutants. High levels of melatonin and AFMK in water hyacinth may explain why this plant more easily tolerates environmental pollutants, including toxic chemicals and heavy metals and is successfully used in phytoremediation. These novel findings could lead to improvements in the phytoremediative capacity of plants by either stimulating endogenous melatonin synthesis or by adding melatonin to water/soil in which they are grown.

  18. Inhibition of 5-aminolevulinic acid-induced DNA damage by melatonin, N1-acetyl-N2-formyl-5-methoxykynuramine, quercetin or resveratrol.

    PubMed

    Onuki, Janice; Almeida, Eduardo A; Medeiros, Marisa H G; Di Mascio, Paolo

    2005-03-01

    Porphyrias are defined as either inborn or acquired diseases related to enzymatic deficiencies in the heme biosynthetic pathway. Lead poisoning, hereditary tyrosinemia, and acute intermittent porphyria (AIP) are characterized by the absence of photosensitivity and the accumulation of 5-aminolevulinic acid (ALA) together with its increased urinary excretion. The main clinical manifestations of AIP are intermittent attacks of abdominal pain, neuromuscular weaknesses and neuropsychiatry alterations, and also an association with primary liver cancer, in which may be involved the oxidative potential of ALA which is able to cause DNA damage. The use of antioxidants in the treatment of ALA-induced oxidative stress is not well established. In the current work, we show the antioxidant efficacy of several compounds including melatonin, quercetin, resveratrol and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a melatonin oxidation product, in terms of their ability to limit DNA damage induced by ALA/Fe2+ in an in vitro system. Damage was measured by plasmid DNA strand breaks and detection of 8-oxo, 7-8-dihydro,2'-deoxyguanosine (8-oxodGuo) by high-performance liquid chromatography coupled with electrochemical detection. All compounds tested showed a dose-dependent protective action against free radical damage. These results could be the first step toward studies of the possible use of these antioxidants in oxidative stress promoted by ALA or other pro-oxidants.

  19. Melatonin Enhances Photo-Oxidation of 2′,7′-Dichlorodihydrofluorescein by an Antioxidant Reaction That Renders N1-Acetyl-N2-Formyl-5-Methoxykynuramine (AFMK)

    PubMed Central

    Hevia, David; Mayo, Juan C.; Tan, Dun-Xian; Rodriguez-Garcia, Aida; Sainz, Rosa M.

    2014-01-01

    The indolamine melatonin (MEL) is described as an antioxidant and a free radical scavenger. However occasionally, the indoleamine has been reported to increase free radicals with insufficient mechanistic explanation. In an attempt to find a reason for those controversial results, a potential mechanism that explains MEL prooxidant activity is investigated. The current controversy about redox detection methods has prompted us to search a possible interaction between MEL and dichlorodihydrofluorescein (DCFH2), perhaps the most widely fluorescence probe employed for free radicals detection in cellular models. Here, it is demonstrated that melatonin potentiates the photooxidation of DCFH2 in a cell-free system, increasing the production of its fluorescent metabolite. Indeed, MEL works as an antioxidant scavenging hydroxyl radicals in this system. Thus, this reaction between MEL and DCFH2 produces N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a biogenic amine with antioxidant properties too. This reaction is O2 and light dependent and it is prevented by antioxidants such as N-acetylcysteine or ascorbic acid. Furthermore, when DCFH2 has been employed to evaluate antioxidant or prooxidant activities of MEL in cellular models it is confirmed that it works as an antioxidant but these results can be modulated by light misleading to a prooxidant conclusion. In conclusion, here is demonstrated that DCFH2, light and melatonin interact and results obtained using these fluorescence probes in studies with melatonin have to be carefully interpreted. PMID:25275380

  20. Formyl-coenzyme A (CoA):oxalate CoA-transferase from the acidophile Acetobacter aceti has a distinctive electrostatic surface and inherent acid stability

    PubMed Central

    Mullins, Elwood A; Starks, Courtney M; Francois, Julie A; Sael, Lee; Kihara, Daisuke; Kappock, T Joseph

    2012-01-01

    Bacterial formyl-CoA:oxalate CoA-transferase (FCOCT) and oxalyl-CoA decarboxylase work in tandem to perform a proton-consuming decarboxylation that has been suggested to have a role in generalized acid resistance. FCOCT is the product of uctB in the acidophilic acetic acid bacterium Acetobacter aceti. As expected for an acid-resistance factor, UctB remains folded at the low pH values encountered in the A. aceti cytoplasm. A comparison of crystal structures of FCOCTs and related proteins revealed few features in UctB that would distinguish it from nonacidophilic proteins and thereby account for its acid stability properties, other than a strikingly featureless electrostatic surface. The apparently neutral surface is a result of a “speckled” charge decoration, in which charged surface residues are surrounded by compensating charges but do not form salt bridges. A quantitative comparison among orthologs identified a pattern of residue substitution in UctB that may be a consequence of selection for protein stability by constant exposure to acetic acid. We suggest that this surface charge pattern, which is a distinctive feature of A. aceti proteins, creates a stabilizing electrostatic network without stiffening the protein or compromising protein–solvent interactions. PMID:22374910

  1. Altered postreceptor signal transduction of formyl-Met-Leu-Phe receptors in polymorphonuclear leukocytes of patients with non-insulin-dependent diabetes mellitus.

    PubMed

    Fóris, G; Paragh, G; Dezsõ, B; Keresztes, T; Balogh, Z; Szabó, J

    1998-01-01

    The signal transduction of the formyl-Met-Leu-Phe (FMLP) receptor in polymorphonuclear leukocytes (PMNLs) from patients with non-insulin-dependent diabetes mellitus (NIDDM) was compared to that of PMNLs obtained from healthy volunteers. According to our previous studies in this group of patients neither the decrease in insulin binding capacity nor the enhanced insulin-degrading enzyme activity was involved. In control PMNLs, 10 nM FMLP induced a pertussis toxin-sensitive increase in phosphatidyl inositol (PI) cleavage and a subsequent Ca2+ signaling from the intracellular pools. On the other hand, the FMLP-induced protein kinase C (PKC) activation and translocation into the membrane could not be detected in these cells via the measurement of 32P incorporation into histone. In contrast, in PMNLs of this special group of patients suffering from NIDDM the FMLP stimulus produced a significantly low increase in PI cleavage and Ca2+ signaling from the intracellular pools. Moreover, in resting PMNLs of these patients with NIDDM, not only the [Ca2+]i but also the membrane-bound PKC activity was found to be significantly increased. In addition, PKC translocation into the cell membrane of diabetic PMNLs could be further triggered with FMLP as judged by the measurement of 32P incorporation into histone. Based on these results, it appears that the signaling of FMLP receptors in PMNLs of some NIDDM patients may have an alternative pathway through Ca2+ influx from extracellular medium, arachidonic acid cascade, and PKC activation.

  2. The co-catalytic effect of chlorpromazine on peroxidase-mediated oxidation of melatonin: enhanced production of N1-acetyl-N2-formyl-5-methoxykynuramine.

    PubMed

    Ximenes, Valdecir F; Rodrigues, Ana Paula; Cabello, Cláudio; de Menezes, Manoel L; Fernandes, João Roberto

    2008-03-01

    Accumulating evidence points to relationships between increased production of reactive oxygen or decreased antioxidant protection in schizophrenic patients. Chlorpromazine (CPZ), which remains a benchmark treatment for people with schizophrenia, has been described as a pro-oxidant compound. Because the antioxidant compound melatonin exerts protective effects against CPZ-induced liver disease in rats, in this investigation, our main objective was to study the effect of CPZ as a co-catalyst of peroxidase-mediated oxidation of melatonin. We found that melatonin was an excellent reductor agent of preformed CPZ cation radical (CPZ(*+)). The addition of CPZ during the horseradish peroxidase (HRP)-catalyzed oxidation of melatonin provoked a significant increase in the rate of oxidation and production of N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). Similar results were obtained using myeloperoxidase. The effect of CPZ on melatonin oxidation was rather higher at alkaline pH. At pH 9.0, the efficiency of oxidation of melatonin was 15 times higher and the production of AFMK was 30 times higher as compared with the assays in the absence of CPZ. We suggest that CPZ is able to exacerbate the rate of oxidation of melatonin by an electron transfer mechanism where CPZ(*+), generated during the peroxidase-catalyzed oxidation, is able to efficiently oxidize melatonin.

  3. Oxidation of melatonin by singlet molecular oxygen (O2(1deltag)) produces N1-acetyl-N2-formyl-5-methoxykynurenine.

    PubMed

    de Almeida, Eduardo A; Martinez, Glaucia R; Klitzke, Clécio F; de Medeiros, Marisa H G; Di Mascio, Paolo

    2003-09-01

    It has been shown that melatonin exhibits antioxidant properties. Chemical structures of some of the products formed by the interaction of melatonin with reactive oxygen and nitrogen species have been elucidated. Despite some evidence that the reaction of melatonin with singlet molecular oxygen (O2(1deltag)) produces N1-acetyl-N2-formyl-5-methoxykynurenine (AFMK), it has not been fully documented. In this investigation, melatonin was oxidized by photosensitization with methylene blue or by a clean chemical source of O2(1deltag), the thermodecomposition of N,N'-di(2,3-dihydroxypropyl)-1,4-naphtalenedipropanamide (DHPNO2). The resulting product was characterized by high performance liquid chromatography, coupled to electrospray ionization mass spectrometry and also by 1H, 13C and dept135 nuclear magnetic resonance spectroscopy. An isotopically labeled DHPN18O2 was also prepared and used as a chemical source of labeled 18[O2(1deltag)] to unequivocally characterize the end product. The results uncovered by this work confirm the hypothesis that oxidation of melatonin by O2(1deltag) produces AFMK.

  4. Quantum chemical studies and vibrational analysis of 4-acetyl benzonitrile, 4-formyl benzonitrile and 4-hydroxy benzonitrile--a comparative study.

    PubMed

    Arjunan, V; Carthigayan, K; Periandy, S; Balamurugan, K; Mohan, S

    2012-12-01

    The FTIR and FT-Raman vibrational spectra of 4-actetyl benzonitrile, 4-formyl benzonitrile and 4-hydroxy benzonitrile molecules have been recorded in the range 4000-400 and 4000-100 cm(-1), respectively. The complete vibrational assignment and analysis of the fundamental modes of the most stable geometry of the compounds were carried out using the experimental FTIR and FT-Raman data on the basis of peak positions, relative intensities and quantum chemical studies. The observed vibrational frequencies were compared with the theoretical wavenumbers of the optimised geometry of the compounds obtained from the DFT-B3LYP gradient calculations employing the standard 6-31G**, high level 6-311++G** and cc-pVDZ basis sets. The structural parameters and vibrational wavenumbers obtained from the DFT methods are in good agreement with the experimental data. With hope of providing more and effective information on the fundamental vibrations, total energy distributions of the fundamental modes have been performed by assuming C(s) point group symmetry. The effect of substituents -COCH(3), -CHO and -OH in the benzonitrile moiety have been analysed and compared. The kinetic and thermodynamic stability and chemical hardness of the molecule have been determined. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Urinary Peptides in Rett Syndrome.

    ERIC Educational Resources Information Center

    Solaas, K. M.; Skjeldal, O.; Gardner, M. L. G.; Kase, B. F.; Reichelt, K. L.

    2002-01-01

    A study found a significantly higher level of peptides in the urine of 53 girls with Rett syndrome compared with controls. The elevation was similar to that in 35 girls with infantile autism. Levels of peptides were lower in girls with classic Rett syndrome than those with congenital Rett syndrome. (Contains references.) (Author/CR)

  6. Clinical uses of gut peptides.

    PubMed Central

    Geoghegan, J; Pappas, T N

    1997-01-01

    OBJECTIVE: The authors review clinical applications of gut-derived peptides as diagnostic and therapeutic agents. SUMMARY BACKGROUND DATA: An increasing number of gut peptides have been evaluated for clinical use. Earlier uses as diagnostic agents have been complemented more recently by increasing application of gut peptides as therapeutic agents. METHOD: The authors conducted a literature review. RESULTS: Current experience with clinical use of gut peptides is described. Initial clinical applications focused on using secretomotor effects of gut peptides in diagnostic tests, many of which have now fallen into disuse. More recently, attention has been directed toward harnessing these secretomotor effects for therapeutic use in a variety of disorders, and also using the trophic effects of gut peptides to modulate gut mucosal growth in benign and malignant disease. Gut peptides have been evaluated in a variety of other clinical situations including use as adjuncts to imaging techniques, and modification of behaviors such as feeding and panic disorder. CONCLUSIONS: Gut peptides have been used successfully in an increasing variety of clinical conditions. Further refinements in analogue and antagonist design are likely to lead to even more selective agents that may have important clinical applications. Further studies are needed to identity and evaluate these new agents. PMID:9065291

  7. Urinary Peptides in Rett Syndrome.

    ERIC Educational Resources Information Center

    Solaas, K. M.; Skjeldal, O.; Gardner, M. L. G.; Kase, B. F.; Reichelt, K. L.

    2002-01-01

    A study found a significantly higher level of peptides in the urine of 53 girls with Rett syndrome compared with controls. The elevation was similar to that in 35 girls with infantile autism. Levels of peptides were lower in girls with classic Rett syndrome than those with congenital Rett syndrome. (Contains references.) (Author/CR)

  8. Systematic screening for bioactive peptides.

    PubMed

    Maeji, N J; Bray, A M; Valerio, R M; Seldon, M A; Wang, J X; Geysen, H M

    1991-01-01

    Using simultaneous multiple peptide synthesis by the multipin approach, the feasibility of systematic large-scale pharmacological screening of peptide ligands was investigated. The method involves the assembly of small quantities of peptides (ca. 50 nmol) on plastic pins derivatized with an ester linker based on glycolate and 4-(hydroxymethyl)benzoate. These esters are stable under peptide synthesis and side-chain deprotection conditions but cleave under relatively mild basic conditions to generate peptides having C-terminal acid, amide and methylamide. A two-step approach to side-chain deprotection and cleavage from the solid support allows potentially toxic reagents to be removed (washed) from the peptide prior to cleavage. Consequently, the resulting peptide solutions can be used in bioassays with minimal processing. A series of angiotensin II and substance P analogs were synthesized and evaluated in an in vivo rat model and in vitro radioreceptor assay, respectively. Structure-activity studies on analogs of these bioactive peptides are well documented. The data obtained were consistent with that reported in the literature.

  9. Microscopic characterization of peptide nanostructures.

    PubMed

    Mammadov, Rashad; Tekinay, Ayse B; Dana, Aykutlu; Guler, Mustafa O

    2012-02-01

    Peptide-based nanomaterials have been utilized for various applications from regenerative medicine to electronics since they provide several advantages including easy synthesis methods, numerous routes for functionalization and biomimicry of secondary structures of proteins which leads to design of self-assembling peptide molecules to form nanostructures. Microscopic characterization at nanoscale is critical to understand processes directing peptide molecules to self-assemble and identify structure-function relationship of the nanostructures. Here, fundamental studies in microscopic characterization of peptide nanostructures are discussed to provide insights in widely used microscopy tools. In this review, we will encompass characterization studies of peptide nanostructures with modern microscopes, such as TEM, SEM, AFM, and advanced optical microscopy techniques. We will also mention specimen preparation methods and describe interpretation of the images.

  10. Bioactive peptides derived from food.

    PubMed

    Rutherfurd-Markwick, Kay J; Moughan, Paul J

    2005-01-01

    As interest in the ability of functional foods to impact on human health has grown over the past decade, so has the volume of knowledge detailing the beneficial roles of food-derived bioactive peptides. Bioactive peptides from both plant and animal proteins have been discovered, with to date, by far the most being isolated from milk-based products. A wide range of activities has been described, including antimicrobial and antifungal properties, blood pressure-lowering effects, cholesterol-lowering ability, antithrombotic effects, enhancement of mineral absorption, immunomodulatory effects, and localized effects on the gut. Although there is still considerable research to be performed in the area of food-derived bioactive peptides, it is clear that the generation of bioactive peptides from dietary proteins during the normal digestive process is of importance. Therefore, it will become necessary when determining dietary protein quality to consider the potential effects of latent bioactive peptides that are released during digestion of the protein.

  11. Peptides and peptidomimetics as immunomodulators

    PubMed Central

    Gokhale, Ameya S; Satyanarayanajois, Seetharama

    2014-01-01

    Peptides and peptidomimetics can function as immunomodulating agents by either blocking the immune response or stimulating the immune response to generate tolerance. Knowledge of B- or T-cell epitopes along with conformational constraints is important in the design of peptide-based immunomodulating agents. Work on the conformational aspects of peptides, synthesis and modified amino acid side chains have contributed to the development of a new generation of therapeutic agents for autoimmune diseases and cancer. The design of peptides/peptidomimetics for immunomodulation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus and HIV infection is reviewed. In cancer therapy, peptide epitopes are used in such a way that the body is trained to recognize and fight the cancer cells locally as well as systemically. PMID:25186605

  12. Antimicrobial peptides: therapeutic potentials.

    PubMed

    Kang, Su-Jin; Park, Sung Jean; Mishig-Ochir, Tsogbadrakh; Lee, Bong-Jin

    2014-12-01

    The increasing appearance of multidrug-resistant pathogens has created an urgent need for suitable alternatives to current antibiotics. Antimicrobial peptides (AMPs), which act as defensive weapons against microbes, have received great attention because of broad-spectrum activities, unique action mechanisms and rare antibiotic-resistant variants. Despite desirable characteristics, they have shown limitations in pharmaceutical development due to toxicity, stability and manufacturing costs. Because of these drawbacks, only a few AMPs have been tested in Phase III clinical trials and no AMPs have been approved by the US FDA yet. However, these obstacles could be overcome by well-known methods such as changing physicochemical characteristics and introducing nonnatural amino acids, acetylation or amidation, as well as modern techniques like molecular targeted AMPs, liposomal formulations and drug delivery systems. Thus, the current challenge in this field is to develop therapeutic AMPs at a reasonable cost as well as to overcome the limitations.

  13. Asymmetric Peptide Nanoribbons.

    PubMed

    Yu, Zhilin; Tantakitti, Faifan; Palmer, Liam C; Stupp, Samuel I

    2016-11-09

    Asymmetry in chemical structure or shape at molecular, nanoscale, or microscopic levels is essential to a vast number of functionalities in both natural and artificial systems. Bottom-up approaches to create asymmetric supramolecular nanostructures are considered promising but this strategy suffers from the potentially dynamic nature of noncovalent interactions. We report here on supramolecular self-assembly of asymmetric peptide amphiphiles consisting of two different molecularly linked domains. We found that strong noncovalent interactions and a high degree of internal order among the asymmetric amphiphiles lead to nanoribbons with asymmetric faces due to the preferential self-association of the two domains. The capture of gold nanoparticles on only one face of the nanoribbons demonstrates symmetry breaking in these supramolecular structures.

  14. Short Antimicrobial Peptides and Peptide Scaffolds as Promising Antibacterial Agents.

    PubMed

    Domalaon, Ronald; Zhanel, George G; Schweizer, Frank

    2016-01-01

    Antimicrobial peptides have recently garnered significant attention as an emerging source of potential antibiotics, due to the swift emergence of multidrug-resistant bacteria and a dwindling antibiotic pipeline. The vast majority of antimicrobial peptides are long, comprised of more than 10 amino acids, resulting in significant production costs for their synthesis while simultaneously displaying metabolic instability and relatively poor pharmacological profiles. To counter these problems, efforts have been shifted to shorter molecules and the development of new peptidomimetic approaches. In this paper, we review promising short, naturally-isolated or synthetic, antimicrobial peptides, along with their mimics, and discuss their merits as potential antibacterial agents.

  15. Peptide-formation on cysteine-containing peptide scaffolds

    NASA Technical Reports Server (NTRS)

    Chu, B. C.; Orgel, L. E.

    1999-01-01

    Monomeric cysteine residues attached to cysteine-containing peptides by disulfide bonds can be activated by carbonyldiimidazole. If two monomeric cysteine residues, attached to a 'scaffold' peptide Gly-Cys-Glyn-Cys-Glu10, (n = 0, 1, 2, 3) are activated, they react to form the dipeptide Cys-Cys. in 25-65% yield. Similarly, the activation of a cysteine residue attached to the 'scaffold' peptide Gly-Cys-Gly-Glu10 in the presence of Arg5 leads to the formation of Cys-Arg5 in 50% yield. The significance of these results for prebiotic chemistry is discussed.

  16. Epimerization in peptide thioester condensation.

    PubMed

    Teruya, Kenta; Tanaka, Takeyuki; Kawakami, Toru; Akaji, Kenichi; Aimoto, Saburo

    2012-11-01

    Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C-terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid-phase method, no substantial epimerization of the C-terminal amino acid was detected. Epimerization was, however, observed during a thioester-thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester-thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively.

  17. Activation of Toll-like receptor 2 on microglia promotes cell uptake of Alzheimer disease-associated amyloid beta peptide.

    PubMed

    Chen, Keqiang; Iribarren, Pablo; Hu, Jinyue; Chen, Jianhong; Gong, Wanghua; Cho, Edward H; Lockett, Stephen; Dunlop, Nancy M; Wang, Ji Ming

    2006-02-10

    The human G-protein-coupled formyl peptide receptor-like 1 (FPRL1) and its mouse homologue mFPR2 mediate the chemotactic activity of a variety of polypeptides associated with inflammation and bacterial infection, including the 42-amino acid form of amyloid beta peptide (Abeta42), a pathogenic factor in Alzheimer disease. Because mFPR2 was inducible in mouse microglial cells by proinflammatory stimulants, such as bacterial lipopolysaccharide, a ligand for the Toll-like receptor 4 (TLR4), we investigated the role of TLR2 in the regulation of mFPR2. We found that a TLR2 agonist, peptidoglycan (PGN) derived from Gram-positive bacterium Staphylococcus aureus, induced considerable mFpr2 mRNA expression in a mouse microglial cell line and primary microglial cells. This was associated with a markedly increased chemotaxis of the cells in response to mFPR2 agonist peptides. In addition, activation of TLR2 markedly enhanced mFPR2-mediated uptake of Abeta42 by microglia. Studies of the mechanistic basis showed that PGN activates MAPK and IkappaBalpha, and the effect of PGN on induction of mFPR2 was dependent on signaling pathways via ERK1/2 and p38 MAPKs. The use of TLR2 on microglial cells by PGN was supported by the fact that N9 cells transfected with short interfering RNA targeting mouse TLR2 failed to show increased expression of functional mFPR2 after stimulation with PGN. Our results demonstrated a potentially important role for TLR2 in microglial cells of promoting cell responses to chemoattractants produced in lesions of inflammatory and neurodegenerative diseases in the brain.

  18. The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells

    PubMed Central

    Coffelt, Seth B.; Marini, Frank C.; Watson, Keri; Zwezdaryk, Kevin J.; Dembinski, Jennifer L.; LaMarca, Heather L.; Tomchuck, Suzanne L.; zu Bentrup, Kerstin Honer; Danka, Elizabeth S.; Henkle, Sarah L.; Scandurro, Aline B.

    2009-01-01

    Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells. PMID:19234121

  19. The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells.

    PubMed

    Coffelt, Seth B; Marini, Frank C; Watson, Keri; Zwezdaryk, Kevin J; Dembinski, Jennifer L; LaMarca, Heather L; Tomchuck, Suzanne L; Honer zu Bentrup, Kerstin; Danka, Elizabeth S; Henkle, Sarah L; Scandurro, Aline B

    2009-03-10

    Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

  20. Multidimensional peptide separations in proteomics.

    PubMed

    Link, Andrew J

    2002-12-01

    Multidimensional peptide separation will play an increasingly important role in the drive to identify and quantitate the proteome. By increasing the peak and load capacity, multidimensional approaches increase the number and dynamic range of peptides that can be analyzed in a complex biological organism. Separation methods using different physical properties of peptides have been combined with varying degrees of success. The ultimate goal is a rapid separation strategy that can be coupled with analytical methods, such as mass spectrometry, to provide comprehensive monitoring of the changing concentration, interactions, and structures of proteins in the proteome.

  1. Anti-inflammatory actions of melatonin and its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in macrophages.

    PubMed

    Mayo, Juan C; Sainz, Rosa M; Tan, Dun-Xian; Hardeland, Rüdiger; Leon, Josefa; Rodriguez, Carmen; Reiter, Russel J

    2005-08-01

    Inflammation is a complex phenomenon involving multiple cellular and molecular interactions which must be tightly regulated. Cyclooxygenase-2 (COX) is the key enzyme that catalyzes the two sequential steps in the biosynthesis of PGs from arachidonic acid. The inducible isoform of COX, namely COX-2, plays a critical role in the inflammatory response and its over-expression has been associated with several pathologies including neurodegenerative diseases and cancer. Melatonin is the main product of the pineal gland with well documented antioxidant and immuno-modulatory effects. Since the action of the indole on COX-2 has not been previously described, the goal of the present report was to test the effect of melatonin on the activities of COX-2 and inducible nitric oxide synthase (iNOS), using lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as a model. Melatonin and its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), prevented COX-2 activation induced by LPS, without affecting COX-1 protein levels. The structurally related compound 6-methoxy-melatonin only partially prevented the increase in COX-2 protein levels induced by the toxin. Likewise melatonin prevented iNOS activation and reduced the concentration of products from both enzymes, PGE(2) and nitric oxide. Another endogenous antioxidant like N-acetyl-cysteine (NAC) did not reduced COX-2 significantly. The current finding corroborates a role of melatonin as an anti-inflammatory agent and, for the first time, COX-2 and iNOS as molecular targets for either melatonin or its metabolites AFMK and AMK. These anti-inflammatory actions seem not to be exclusively mediated by the free radical scavenging properties of melatonin. As a consequence, the present work suggests these substances as a new class of potential anti-inflammatory agents without the classical side effects due to COX-1 inhibition.

  2. Melatonin metabolite, N(1)-acetyl-N(1)-formyl-5-methoxykynuramine (AFMK), attenuates acute pancreatitis in the rat: in vivo and in vitro studies.

    PubMed

    Jaworek, J; Szklarczyk, J; Bonior, J; Kot, M; Goralska, M; Pierzchalski, P; Reiter, R J; Czech, U; Tomaszewska, R

    2016-06-01

    Melatonin protects the pancreas from inflammation and free radical damage but the effect of the melatonin metabolite: N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on acute pancreatitis is unknown. This study assessed the effects of AFMK on acute pancreatitis (AP) in the rats in vivo and on pancreatic cell line AR42J in vitro. AFMK (5, 10 or 20 mg/kg) was given intraperitoneally to the rats 30 min prior to the induction of AP by subcutaneous caerulein infusion (25 μg/kg). Lipid peroxidation products (MDA + 4-HNE) and the activity of an antioxidant enzyme glutathione peroxidase (GPx) were measured in pancreatic tissue. Blood samples were taken for evaluation of amylase activity and TNF-α concentration. GPx, TNF-α, proapoptotic Bax protein, antiapoptotic Bcl-2 protein and the executor of apoptosis, caspase-3, were determined by Western blot in AR42J cells subjected to AFMK or to melatonin (both used at 10(-12), 10(-10), or 10(-8)M), without or with addition of caerulein (10(-8)M). AP was confirmed by histological examination and by serum increases of amylase and TNF-α (by 800% and 300%, respectively). In AP rats, pancreatic MDA + 4-HNE levels were increased by 300%, whereas GPx was reduced by 50%. AFMK significantly diminished histological manifestations of AP, decreased serum amylase activity and TNF-α concentrations, reduced MDA + 4-HNE levels and augmented GPx in the pancreas of AP rats. In AR42J cells, AFMK combined with caerulein markedly increased protein signals for GPx, Bax, caspase-3 and reduced these for TNF-α and Bcl-2. In conclusion, AFMK significantly attenuated acute pancreatitis in the rat. This may relate to the antioxidative and anti-inflammatory effects of this molecule and possibly to the stimulation of proapoptotic signal transduction pathway.

  3. Tumor necrosis factor-alpha modulates the selective interference of hypnotics and sedatives to suppress N-formyl-methionyl-leucyl-phenylalanine-induced oxidative burst formation in neutrophils.

    PubMed

    Weiss, M; Buhl, R; Medve, M; Schneider, E M

    1997-01-01

    To clarify whether tumor necrosis factor (TNF)-alpha modulates the inhibitory effect of clinically applied hypnotics and sedatives on neutrophil function. Prospective, randomized, controlled, dose response, in vitro study. A university research laboratory. Neutrophils from healthy human volunteers. Neutrophils were primed by incubation with TNF-alpha (25 ng/mL) for 15 mins. Subsequently, TNF-alpha-primed neutrophils were incubated with two concentrations of commercially available drug preparations and drug-free solutions, respectively. The following commercially available preparations of hypnotics and sedatives as well as their corresponding drug-free solutions were tested: methohexital, thiopental, midazolam, diazepam, etomidate, and propofol. The production of oxygen radicals was initiated by adding N-formyl-methionyl-leucyl-phenylalanine (FMLP) 10(-7) mol/L and detected by luminol-enhanced chemiluminescence measurements for 15 mins. Within the range of therapeutic plasma concentrations, only thiopental, diazepam, and propofol suppressed chemiluminescence of unprimed neutrophils. Additionally, propofol alone suppressed TNF-alpha-primed neutrophils. Hypnotics and sedatives were unable to suppress oxygen radical production of TNF-alpha-primed neutrophils below the level of their control activity, measured in FMLP-induced unprimed neutrophils in the absence of the respective drug or drug-free solution. However, the effect of etomidate could not be evaluated secondarily to effects mediated by its drug-free solution. In a cell-free chemiluminescence system, thiopental and propofol demonstrated scavenging of oxygen free radicals. Priming with TNF-alpha counteracts the inhibitory effect by certain drugs for oxygen radical formation by FMLP-stimulated neutrophils. Thus, TNF-alpha plus FMLP mediate additive effects in stimulating oxygen radical formation in neutrophils. The following drugs dose-dependently interfere with these activating pathways: thiopental, diazepam

  4. Transcriptional and functional analysis of oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes from Lactobacillus acidophilus.

    PubMed

    Azcarate-Peril, M Andrea; Bruno-Bárcena, Jose M; Hassan, Hosni M; Klaenhammer, Todd R

    2006-03-01

    Oxalic acid is found in dietary sources (such as coffee, tea, and chocolate) or is produced by the intestinal microflora from metabolic precursors, like ascorbic acid. In the human intestine, oxalate may combine with calcium, sodium, magnesium, or potassium to form less soluble salts, which can cause pathological disorders such as hyperoxaluria, urolithiasis, and renal failure in humans. In this study, an operon containing genes homologous to a formyl coenzyme A transferase gene (frc) and an oxalyl coenzyme A decarboxylase gene (oxc) was identified in the genome of the probiotic bacterium Lactobacillus acidophilus. Physiological analysis of a mutant harboring a deleted version of the frc gene confirmed that frc expression specifically improves survival in the presence of oxalic acid at pH 3.5 compared with the survival of the wild-type strain. Moreover, the frc mutant was unable to degrade oxalate. These genes, which have not previously been described in lactobacilli, appear to be responsible for oxalate degradation in this organism. Transcriptional analysis using cDNA microarrays and reverse transcription-quantitative PCR revealed that mildly acidic conditions were a prerequisite for frc and oxc transcription. As a consequence, oxalate-dependent induction of these genes occurred only in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 5.5. Where genome information was available, other lactic acid bacteria were screened for frc and oxc genes. With the exception of Lactobacillus gasseri and Bifidobacterium lactis, none of the other strains harbored genes for oxalate utilization.

  5. catena-Poly[[aqua-bis-(4-formyl-benzoato-κ(2) O (1),O (1'))cadmium]-μ-pyrazine-κ(2) N:N'].

    PubMed

    Celik, Fatih; Dilek, Nefise; Caylak Delibaş, Nagihan; Necefoğlu, Hacali; Hökelek, Tuncer

    2014-02-01

    The polymeric title compound, [Cd(C8H5O3)2(C4H4N2)(H2O)] n , contains two 4-formyl-benzoate (FB) anions, one pyrazine mol-ecule and one coordinating water mol-ecule; the FB anions act as bidentate ligands. The O atom, the aldehyde H atom and the benzene ring of one of the FB anions are disordered over two positions. The O atoms were freely refined [refined occupancy ratio 0.79 (2):0.21 (2)], while the aldehyde H atoms and the benzene ring atoms were refined with fixed occupancy ratios of 0.8:0.2 and 0.5:0.5, respectively. In the ordered FB anion, the carboxyl-ate group is twisted away from the attached benzene ring (A) by 22.7 (8)°. In the disordered FB anion, the corresponding angles are 15.6 (10) and 11.4 (11)° for rings B and B', respectively. Benzene rings A and B are oriented at a dihedral angle of 24.2 (7), A and B' at 43.0 (8)°. The pyrazine ring makes dihedral angles of 67.5 (4), 89.6 (7) and 86.2 (7)°, respectively, with benzene rings A, B and B'. The pyrazine ligands bridge the Cd(II) cations, forming polymeric chains running along the b-axis direction. In the crystal, O-Hwater ⋯ Ocarboxyl-ate hydrogen bonds link adjacent chains into layers parallel to the bc plane. These layers are linked via C-Hpyrazine ⋯ Oform-yl hydrogen bonds, forming a three-dimensional network. π-π interactions [centroid-centroid distances = 3.870 (11)-3.951 (5) Å] further stabilize the crystal structure. There is also a weak C-H⋯π inter-action present.

  6. Early peptidic enzymes

    NASA Astrophysics Data System (ADS)

    Brack, André; Barbier, Bernard

    Oligopeptides supposed to be essential to primitive living cells could not be obtained by a prebiotic organic chemistry working mainly at random. Selection pathways were required. Experimental evidence is given for selective condensation of amino-acids in water as well as for selective resistance to degradation. Polycationic polypeptides containing lysyl (or arginyl) and hydrophobic residues strongly accelerate the hydrolysis of oligoribonucleotides. A ionic complex is first formed and the polypeptides are particularly active when they adopt a stable conformation, β-sheet or α-helix, in the complex. Well-defined short peptides were synthesized in order to determine the critical chain-length required for chemical activity. In a contemporary cell, proteins represent about 40 % of the dry weight. They fulfil a structural role and they are particularly helpful as chemical catalysts (enzymes). They can be represented as long chains made of twenty different building blocks, the amino-acids NH2-CHR-COOH, which differ by the side-chain R. Proteins are remarkable in the sense that they use amino-acids having only one carbon atom between the -NH2 and -COOH functions. The central carbon atom has always the same spatial asymmetry (chirality) and always bears a hydrogen atom. When the side-chain R is a hydrocarbon, it is branched. When R contains a chemical function, the side functions do not participate to the peptide bond construction. The protein chain results from the condensation of amino-acids, i.e. water molecules are removed between molecules in a medium which is mainly aqueous (the cell contains 75 % of water). The protein chains adopt rigid asymmetric conformations (α-helices, β-sheet structures) which are essential for the protein functions. Proteins, even the smallest ones, are too sophisticated entities to be considered as the products of an organic chemistry working at random, without any chemical selection. The chemist has therefore to understand, with simple

  7. Biomimetic synthesis and antioxidant evaluation of 3,4-DHPEA-EDA [2-(3,4-hydroxyphenyl) ethyl (3S,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate].

    PubMed

    Nardi, Monica; Bonacci, Sonia; De Luca, Giuseppina; Maiuolo, Jessica; Oliverio, Manuela; Sindona, Giovanni; Procopio, Antonio

    2014-11-01

    Phenolic compounds present in extra virgin olive oil have attracted considerable recent attention. Many of them, show specific anti-inflammatory and anti-tumor activities. In this work we describe the biomimetic synthesis of 3,4-DHPEA-EDA [2-(3,4-hydroxyphenyl) ethyl (3S,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate], starting from natural demethyloleuropein present in olive tissues. A comparison between 3,4-DHPEA-EDA (6) and oleuropein (1), oleuropein aglycone (4) and hydroxytyrosol ORACFL values was undertaken. Published by Elsevier Ltd.

  8. Nickel-Catalyzed Aminoxylation of Inert Aliphatic C(sp(3))-H Bonds with Stable Nitroxyl Radicals under Air: One-Pot Route to α-Formyl Acid Derivatives.

    PubMed

    Wang, Chunxia; Zhang, Luoqiang; You, Jingsong

    2017-04-07

    Nickel-catalyzed aminoxylation of an unactivated C(sp(3))-H bond with a stable nitroxyl radical has been accomplished for the first time to offer various N-alkoxyamine derivatives, which further enables a one-pot approach to α-formyl acid derivatives. The aminoxylation process reported also provides direct evidence for the oxidative addition of a cyclometallic intermediate with a free radical, which is helpful for the reaction-mechanism study in transition-metal-catalyzed functionalization of inert C(sp(3))-H bonds.

  9. Plant-mediated green synthesis of ZnO nanoparticles using Garcinia gummi-gutta seed extract: Photoluminescence, screening of their catalytic activity in antioxidant, formylation and biodiesel production

    NASA Astrophysics Data System (ADS)

    Raghavendra, M.; Yatish, K. V.; Lalithamba, H. S.

    2017-08-01

    The green synthesis of multifunctional ZnO nanoparticles (NPs) was prepared by treatment of zinc nitrate with an extract of Garcinia gummi-gutta seed by the combustion method. The ZnO NPs were characterized by XRD, scanning electron microscopy, UV-visible, FTIR spectroscopic techniques. The prepared ZnO NPs were evaluated for photoluminescence (PL), antioxidant properties and also utilized as a catalyst for the formylation of aromatic amines and biodiesel production. The study reveals that the reaction is simple, mild and environmental friendly. Furthermore, the reaction results in excellent yield of products.

  10. Marine Peptides: Bioactivities and Applications

    PubMed Central

    Cheung, Randy Chi Fai; Ng, Tzi Bun; Wong, Jack Ho

    2015-01-01

    Peptides are important bioactive natural products which are present in many marine species. These marine peptides have high potential nutraceutical and medicinal values because of their broad spectra of bioactivities. Their antimicrobial, antiviral, antitumor, antioxidative, cardioprotective (antihypertensive, antiatherosclerotic and anticoagulant), immunomodulatory, analgesic, anxiolytic anti-diabetic, appetite suppressing and neuroprotective activities have attracted the attention of the pharmaceutical industry, which attempts to design them for use in the treatment or prevention of various diseases. Some marine peptides or their derivatives have high commercial values and had reached the pharmaceutical and nutraceutical markets. A large number of them are already in different phases of the clinical and preclinical pipeline. This review highlights the recent research in marine peptides and the trends and prospects for the future, with special emphasis on nutraceutical and pharmaceutical development into marketed products. PMID:26132844

  11. Moonlighting Peptides with Emerging Function

    PubMed Central

    Rodríguez Plaza, Jonathan G.; Villalón Rojas, Amanda; Herrera, Sur; Garza-Ramos, Georgina; Torres Larios, Alfredo; Amero, Carlos; Zarraga Granados, Gabriela; Gutiérrez Aguilar, Manuel; Lara Ortiz, María Teresa; Polanco Gonzalez, Carlos; Uribe Carvajal, Salvador; Coria, Roberto; Peña Díaz, Antonio; Bredesen, Dale E.; Castro-Obregon, Susana; del Rio, Gabriel

    2012-01-01

    Hunter-killer peptides combine two activities in a single polypeptide that work in an independent fashion like many other multi-functional, multi-domain proteins. We hypothesize that emergent functions may result from the combination of two or more activities in a single protein domain and that could be a mechanism selected in nature to form moonlighting proteins. We designed moonlighting peptides using the two mechanisms proposed to be involved in the evolution of such molecules (i.e., to mutate non-functional residues and the use of natively unfolded peptides). We observed that our moonlighting peptides exhibited two activities that together rendered a n