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Sample records for peptide penetratin induces

  1. The Homeodomain Derived Peptide Penetratin Induces Curvature of Fluid Membrane Domains

    PubMed Central

    Lamazière, Antonin; Wolf, Claude; Lambert, Olivier; Chassaing, Gérard; Trugnan, Germain; Ayala-Sanmartin, Jesus

    2008-01-01

    Background Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, “physical endocytosis”. Methodology/Principal Findings Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in “raft” microdomains versus disordered fluid “non-raft” domains) in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation) are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like) membrane domains. Conclusions/Significance The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we showed that

  2. Penetratin-induced transdermal delivery from H(II) mesophases of sodium diclofenac.

    PubMed

    Cohen-Avrahami, Marganit; Libster, Dima; Aserin, Abraham; Garti, Nissim

    2012-05-10

    Penetratin, a cell penetrating peptide is embedded within a reversed hexagonal (H(II)) mesophase for improved transdermal delivery of sodium diclofenac (Na-DFC). The H(II) mesophase serves as the solubilization reservoir and gel matrix whereas penetratin is the transdermal penetration enhancer for the drug. The systems were characterized and the interactions between the components were determined by SAXS, ATR-FTIR and SD-NMR. High affinity of Na-DFC to glycerol monooleate (GMO) was revealed, associated with increasing the order within the water channels. This affinity is enhanced upon heating and seems to be associated with GMO dehydration. Penetratin (PEN) is entrapped at the hydrophilic region of the H(II) mesophase, between the GMO headgroups, reducing the order of the system and decreasing the size of the hexagonal domains. The transdermal delivery rate of Na-DFC through porcine skin, from the H(II) mesophases, was enhanced by PEN and so also the cumulative transport crossing the skin. PEN induced accelerated drug diffusion through the stratum corneum, towards the different skin layers. The transdermal delivery enhancement is explained from the results of the ATR-FTIR analysis. It seems that PEN accelerates the structural transition of skin lipids from hexagonal to liquid. The disordering results in enhanced diffusion of Na-DFC through the stratum corneum, followed by enhanced overall penetration of the drug.

  3. Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood-Brain Barrier.

    PubMed

    Bera, Swapna; Kar, Rajiv K; Mondal, Susanta; Pahan, Kalipada; Bhunia, Anirban

    2016-09-01

    Cell-penetrating peptides (CPPs) have shown promise in nonpermeable therapeutic drug delivery, because of their ability to transport a variety of cargo molecules across the cell membranes and their noncytotoxicity. Drosophila antennapedia homeodomain-derived CPP penetratin (RQIKIWFQNRRMKWKK), being rich in positively charged residues, has been increasingly used as a potential drug carrier for various purposes. Penetratin can breach the tight endothelial network known as the blood-brain barrier (BBB), permitting treatment of several neurodegenerative maladies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, a detailed structural understanding of penetratin and its mechanism of action is lacking. This study defines structural features of the penetratin-derived peptide, DK17 (DRQIKIWFQNRRMKWKK), in several model membranes and describes a membrane-induced conformational transition of the DK17 peptide in these environments. A series of biophysical experiments, including high-resolution nuclear magnetic resonance spectroscopy, provides the three-dimensional structure of DK17 in different membranes mimicking the BBB or total brain lipid extract. Molecular dynamics simulations support the experimental results showing preferential binding of DK17 to particular lipids at atomic resolution. The peptide conserves the structure of the subdomain spanning residues Ile6-Arg11, despite considerable conformational variation in different membrane models. In vivo data suggest that the wild type, not a mutated sequence, enters the central nervous system. Together, these data highlight important structural and functional attributes of DK17 that could be utilized in drug delivery for neurodegenerative disorders.

  4. Penetratin-Membrane Association: W48/R52/W56 Shield the Peptide from the Aqueous Phase

    PubMed Central

    Lensink, M. F.; Christiaens, B.; Vandekerckhove, J.; Prochiantz, A.; Rosseneu, M.

    2005-01-01

    Using molecular dynamics simulations, we studied the mode of association of the cell-penetrating peptide penetratin with both a neutral and a charged bilayer. The results show that the initial peptide-lipid association is a fast process driven by electrostatic interactions. The homogeneous distribution of positively charged residues along the axis of the helical peptide, and especially residues K46, R53, and K57, contribute to the association of the peptide with lipids. The bilayer enhances the stability of the penetratin helix. Oriented parallel to the lipid-water interface, the subsequent insertion of the peptide through the bilayer headgroups is significantly slower. The presence of negatively charged lipids considerably enhances peptide binding. Lateral side-chain motion creates an opening for the helix into the hydrophobic core of the membrane. The peptide aromatic residues form a π-stacking cluster through W48/R52/W56 and F49/R53, protecting the peptide from the water phase. Interaction with the penetratin peptide has only limited effect on the overall membrane structure, as it affects mainly the conformation of the lipids which interact directly with the peptide. Charge matching locally increases the concentration of negatively charged lipids, lateral lipid diffusion locally decreases. Lipid disorder increases, through decreased order parameters of the lipids interacting with the penetratin side chains. Penetratin molecules at the membrane surface do not seem to aggregate. PMID:15542560

  5. Cell-Penetrating Peptide Penetratin as a Potential Tool for Developing Effective Nasal Vaccination Systems.

    PubMed

    Muto, Keiya; Kamei, Noriyasu; Yoshida, Mia; Takayama, Kozo; Takeda-Morishita, Mariko

    2016-06-01

    Nasal vaccination is considered an attractive strategy to prevent the infection and spread of viruses. However, the vaccine formulations available on the market remain imperfect on account of their limited effectiveness. In the present study, we hypothesized that the nasal coadministration of antigens with cell-penetrating peptides promotes antigen delivery immune response in the nasal mucosa, thereby enhancing the production of mucosal IgA and systemic IgG. The levels of ovalbumin (OVA)-specific IgG and IgA in plasma and nasal perfusate, respectively, increased after 2 or 4 weeks on nasal coadministration of OVA with l- or d-penetratin, suggesting that OVA antigen was effectively delivered by penetratin to the nasal epithelium. An additional study demonstrated that the production of systemic IgG and nasal mucosal IgA against influenza A virus was specifically promoted by nasal coadministration of influenza A virus with d-penetratin. The results of this study suggested that cell-penetrating peptides are a promising tool for the delivery of vaccines to the nasal mucosa and for the subsequent dual stimulation of systemic and mucosal immune responses. PMID:27155764

  6. Small Peptides Derived from Penetratin as Antibacterial Agents.

    PubMed

    Parravicini, Oscar; Somlai, Csaba; Andujar, Sebastián A; Garro, Adriana D; Lima, Beatriz; Tapia, Alejandro; Feresin, Gabriela; Perczel, Andras; Tóth, Gabor; Cascales, Javier López; Rodríguez, Ana M; Enriz, Ricardo D

    2016-04-01

    The synthesis, in vitro evaluation and conformational study of several small-size peptides acting as antibacterial agents are reported. Among the compounds evaluated, the peptides Arg-Gln-Ile-Lys-Ile-Trp-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2 , Arg-Gln-Ile-Lys-Ile-Arg-Arg-Met-Lys-Trp-Arg-NH2 , and Arg-Gln-Ile-Trp-Trp-Trp-Trp-Gln-Arg-NH2 exhibited significant antibacterial activity. These were found to be very active antibacterial compounds, considering their small molecular size. In order to better understand the antibacterial activity obtained for these peptides, an exhaustive conformational analysis was performed, using both theoretical calculations and experimental measurements. Molecular dynamics simulations using two different media (water and trifluoroethanol/water) were employed. The results of these theoretical calculations were corroborated by experimental circular dichroism measurements. A brief discussion on the possible mechanism of action of these peptides at molecular level is also presented. Some of the peptides reported here constitute very interesting structures to be used as starting compounds for the design of new small-size peptides possessing antibacterial activity. PMID:26972341

  7. Penetratin Peptide-Functionalized Gold Nanostars: Enhanced BBB Permeability and NIR Photothermal Treatment of Alzheimer's Disease Using Ultralow Irradiance.

    PubMed

    Yin, Tiantian; Xie, Wenjie; Sun, Jing; Yang, Licong; Liu, Jie

    2016-08-01

    The structural changes of amyloid-beta (Aβ) from nontoxic monomers into neurotoxic aggregates are implicated with pathogenesis of Alzheimer's disease (AD). Over the past decades, weak disaggregation ability and low permeability to the blood-brain barrier (BBB) may be the main obstacles for major Aβ aggregation blockers. Here, we synthesized penetratin (Pen) peptide loaded poly(ethylene glycol) (PEG)-stabilized gold nanostars (AuNS) modified with ruthenium complex (Ru@Pen@PEG-AuNS), and Ru(II) complex as luminescent probes for tracking drug delivery. We revealed that Ru@Pen@PEG-AuNS could obviously inhibit the formation of Aβ fibrils as well as dissociate preformed fibrous Aβ under the irradiation of near-infrared (NIR) due to the NIR absorption characteristic of AuNS. More importantly, this novel design could be applied in medicine as an appropriate nanovehicle, being highly biocompatible and hemocompatible. In addition, Ru@Pen@PEG-AuNS had excellent neuroprotective effect on the Aβ-induced cellular toxicity by applying NIR irradiation. Meanwhile, Pen peptide could effectively improve the delivery of nanoparticles to the brain in vitro and in vivo, which overcame the major limitation of Aβ aggregation blockers. These consequences illustrated that the enhanced BBB permeability and efficient photothermolysis of Ru@Pen@PEG-AuNS are promising agents in AD therapy. PMID:27411476

  8. The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures.

    PubMed

    Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W

    2014-03-01

    Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to

  9. Penetratin-Mediated Transepithelial Insulin Permeation: Importance of Cationic Residues and pH for Complexation and Permeation.

    PubMed

    Kristensen, Mie; Franzyk, Henrik; Klausen, Mia Thorne; Iversen, Anne; Bahnsen, Jesper Søborg; Skyggebjerg, Rikke Bjerring; Foderà, Vito; Nielsen, Hanne Mørck

    2015-09-01

    Penetratin is a widely used carrier peptide showing promising potential for mucosal delivery of therapeutic proteins. In the present study, the importance of specific penetratin residues and pH was investigated with respect to complexation with insulin and subsequent transepithelial insulin permeation. Besides penetratin, three analogues were studied. The carrier peptide-insulin complexes were characterized in terms of size and morphology at pH 5, 6.5, and 7.4 by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. At pH 7.4 mainly very large complexes were present, while much smaller complexes dominated at pH 5. Presence of arginine residues in the carrier peptide proved to be a prerequisite for complexation with insulin as well as for enhanced transepithelial insulin permeation in vitro. Rearrangement of tryptophan residues resulted in significantly increased insulin permeation as compared to that of the parent penetratin. In general, pre-complexation with penetratin and its analogues at pH 5 gave rise to increased insulin permeation as compared to that observed at pH 7.4; this finding was further supported by a preliminary in vivo study using the parent penetratin.

  10. Monitoring penetratin interactions with lipid membranes and cell internalization using a new hydration-sensitive fluorescent probe.

    PubMed

    Zamotaiev, Oleksandr M; Postupalenko, Viktoriia Y; Shvadchak, Volodymyr V; Pivovarenko, Vasyl G; Klymchenko, Andrey S; Mély, Yves

    2014-09-28

    A new fluorescent label N-[4′-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-β-alanine (7AF) was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The 7AF label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms. PMID:25072870

  11. Bacteriocin Inducer Peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Real-time transmembrane translocation of penetratin driven by light-generated proton pumping.

    PubMed

    Björklund, Jörgen; Biverståhl, Henrik; Gräslund, Astrid; Mäler, Lena; Brzezinski, Peter

    2006-08-15

    Cell penetrating peptides (CPPs) are small peptides that are able to penetrate the plasma membrane of mammalian cells. Because these peptides can also carry large hydrophilic cargos such as proteins, they could potentially be used to transport biologically active drugs across cell membranes to modulate in vivo biology. One characteristic feature of the CPPs is that they typically have a net positive charge. Therefore, a key issue associated with the transport mechanism is the role of the transmembrane electrochemical potential in driving the peptides across the membrane. In this study, we have reconstituted bacteriorhodopsin (bR) in large unilamellar vesicles (LUVs) with fluorescein-labeled CPP penetratin enclosed within the LUVs under conditions when the fluorescence is quenched. Illumination of the bacteriorhodopsin-containing LUVs resulted in creation of a transmembrane proton electrochemical gradient (positive on the inside). Upon generation of this gradient, an increase in fluorescence was observed, which shows that the proton gradient drives the translocation of penetratin. The mechanism most likely can be generalized to other CPPs.

  13. Region-Dependent Role of Cell-Penetrating Peptides in Insulin Absorption Across the Rat Small Intestinal Membrane.

    PubMed

    Khafagy, El-Sayed; Iwamae, Ruisha; Kamei, Noriyasu; Takeda-Morishita, Mariko

    2015-11-01

    We have reported that the cell-penetrating peptide (CPP) penetratin acts as a potential absorption enhancer in oral insulin delivery systems and that this action occurs through noncovalent intermolecular interactions. However, the region-dependent role of CPPs in intestinal insulin absorption has not been clarified. To identify the intestinal region where CPPs have the most effect in increasing insulin absorption, the region-dependent action of penetratin was investigated using in situ closed intestinal loops in rats. The order of the insulin area under the insulin concentration-time curve (AUC) increase effect by L-penetratin was ileum > jejunum > duodenum > colon. By contrast, the AUC order after coadministration of insulin with D-penetratin was colon > duodenum ≥ jejunum and ileum. We also compared the effects of the L- and D-forms of penetratin, R8, and PenetraMax on ileal insulin absorption. Along with the CPPs used in this study, L- and D-PenetraMax produced the largest insulin AUCs. An absorption study using ilea pretreated with CPPs showed that PenetraMax had no irreversible effect on the intestinal epithelial membrane. The degradation of insulin in the presence of CPPs was assessed in rat intestinal enzymatic fluid. The half-life (t 1/2) of insulin increased from 14.5 to 23.7 and 184.7 min in the presence of L- and D-PenetraMax, respectively. These enzymatic degradation-resistant effects might contribute partly to the increased ileal absorption of insulin induced by D-PenetraMax. In conclusion, this study demonstrated that the ability of the L- and D-forms of penetratin to increase intestinal insulin absorption was maximal in the ileum and the colon, respectively, and that D-PenetraMax is a powerful but transient enhancer of oral insulin absorption.

  14. Elucidating the Function of Penetratin and a Static Magnetic Field in Cellular Uptake of Magnetic Nanoparticles

    PubMed Central

    Chaudhary, Suman; Smith, Carol Anne; del Pino, Pablo; de la Fuente, Jesus M.; Mullin, Margaret; Hursthouse, Andrew; Stirling, David; Berry, Catherine C.

    2013-01-01

    Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrin- medicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake. Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake. PMID:24275948

  15. Insect inducible antimicrobial peptides and their applications.

    PubMed

    Ezzati-Tabrizi, Reyhaneh; Farrokhi, Naser; Talaei-Hassanloui, Reza; Alavi, Seyed Mehdi; Hosseininaveh, Vahid

    2013-12-01

    Antimicrobial peptides (AMPs) are found as important components of the innate immune system (host defense) of all invertebrates. These peptides can be constitutively expressed or induced in response to microbial infections. Indeed, they vary in their amino acid sequences, potency and antimicrobial activity spectra. The smaller AMPs act greatly by disrupting the structure or function of microbial cell membranes. Here, the insect innate immune system with emphasis on inducible antimicrobial peptide properties against microbial invaders has been discussed.

  16. Hydroxyapatite surface-induced peptide folding.

    PubMed

    Capriotti, Lisa A; Beebe, Thomas P; Schneider, Joel P

    2007-04-25

    Herein, we describe the design and surface-binding characterization of a de novo designed peptide, JAK1, which undergoes surface-induced folding at the hydroxyapatite (HA)-solution interface. JAK1 is designed to be unstructured in buffered saline solution, yet undergo HA-induced folding that is largely governed by the periodic positioning of gamma-carboxyglutamic acid (Gla) residues within the primary sequence of the peptide. Circular dichroism (CD) spectroscopy and analytical ultracentrifugation indicate that the peptide remains unfolded and monomeric in solution under normal physiological conditions; however, CD spectroscopy indicates that in the presence of hydroxyapatite, the peptide avidly binds to the mineral surface adopting a helical structure. Adsorption isotherms indicate nearly quantitative surface coverage and Kd = 310 nM for the peptide-surface binding event. X-ray photoelectron spectroscopy (XPS) coupled with the adsorption isotherm data suggests that JAK1 binds to HA, forming a self-limiting monolayer. This study demonstrates the feasibility of using HA surfaces to trigger the intramolecular folding of designed peptides and represents the initial stages of defining the design rules that allow HA-induced peptide folding.

  17. Hydroxyapatite surface-induced peptide folding.

    PubMed

    Capriotti, Lisa A; Beebe, Thomas P; Schneider, Joel P

    2007-04-25

    Herein, we describe the design and surface-binding characterization of a de novo designed peptide, JAK1, which undergoes surface-induced folding at the hydroxyapatite (HA)-solution interface. JAK1 is designed to be unstructured in buffered saline solution, yet undergo HA-induced folding that is largely governed by the periodic positioning of gamma-carboxyglutamic acid (Gla) residues within the primary sequence of the peptide. Circular dichroism (CD) spectroscopy and analytical ultracentrifugation indicate that the peptide remains unfolded and monomeric in solution under normal physiological conditions; however, CD spectroscopy indicates that in the presence of hydroxyapatite, the peptide avidly binds to the mineral surface adopting a helical structure. Adsorption isotherms indicate nearly quantitative surface coverage and Kd = 310 nM for the peptide-surface binding event. X-ray photoelectron spectroscopy (XPS) coupled with the adsorption isotherm data suggests that JAK1 binds to HA, forming a self-limiting monolayer. This study demonstrates the feasibility of using HA surfaces to trigger the intramolecular folding of designed peptides and represents the initial stages of defining the design rules that allow HA-induced peptide folding. PMID:17397165

  18. The impact of cell-penetrating peptides on membrane bilayer structure during binding and insertion.

    PubMed

    Hirst, Daniel J; Lee, Tzong-Hsien; Kulkarni, Ketav; Wilce, Jacqueline A; Aguilar, Marie-Isabel

    2016-08-01

    We have studied the effect of penetratin and a truncated analogue on the bilayer structure using dual polarisation interferometry, to simultaneously measure changes in mass per unit area and birefringence (an optical parameter representing bilayer order) with high sensitivity during the binding and dissociation from the membrane. Specifically, we studied penetratin (RQIKIWFQNRRMKWKK), along with a shortened and biotinylated version known as R8K-biotin (RRMKWKKK(Biotin)-NH2). Overall both peptides bound only weakly to the neutral DMPC and POPC bilayers, while much higher binding was observed for the anionic DMPC/DMPG and POPC/POPG. The binding of penetratin to gel-phase DMPC/DMPG was adequately represented by a two-state model, whereas on the fluid-phase POPC/POPG it exhibited a distinctly different binding pattern, best represented by a three-state kinetic model. However, R8K-biotin did not bind well to DMPC/DMPG and showed a more transitory and superficial binding to POPC/POPG. Comparing the modelling results for both peptides binding to POPC/POPG suggests an important role for a securely bound intermediate prior to penetratin insertion and translocation. Overall these results further elucidate the mechanism of penetratin, and provide another example of the significance of the ability of DPI to measure structural changes and the use of kinetic analysis to investigate the stages of peptide-membrane interactions. PMID:27163492

  19. Surfactant-induced assembly of enzymatically-stable peptide hydrogels

    SciTech Connect

    Jones, Brad H.; Martinez, Alina M.; Wheeler, Jill S.; Spoerke, Erik D.

    2015-04-07

    The secondary structure of peptides in the presence of interacting additives is an important topic of study, having implications in the application of peptide science to a broad range of modern technologies. Surfactants constitute a class of biologically relevant compounds that are known to influence both peptide conformation and aggregation or assembly. In addition, we have characterized the secondary structure of a linear nonapeptide composed of a hydrophobic alanine/phenylalanine core flanked by hydrophilic acid/amine units. We show that the anionic surfactant sodium dodecyl sulfate (SDS) induces the formation of β-sheets and macroscopic gelation in this otherwise unstructured peptide. Through comparison to related additives, we propose that SDS-induced secondary structure formation is the result of amphiphilicity created by electrostatic binding of SDS to the peptide. In addition, we demonstrate a novel utility of surfactants in manipulating and stabilizing peptide nanostructures. SDS is used to simultaneously induce secondary structure in a peptide and to inhibit the activity of a model enzyme, resulting in a peptide hydrogel that is impervious to enzymatic degradation. These results complement our understanding of the behavior of peptides in the presence of interacting secondary molecules and provide new potential pathways for programmable organization of peptides by the addition of such components.

  20. Surfactant-induced assembly of enzymatically-stable peptide hydrogels

    DOE PAGES

    Jones, Brad H.; Martinez, Alina M.; Wheeler, Jill S.; Spoerke, Erik D.

    2015-04-07

    The secondary structure of peptides in the presence of interacting additives is an important topic of study, having implications in the application of peptide science to a broad range of modern technologies. Surfactants constitute a class of biologically relevant compounds that are known to influence both peptide conformation and aggregation or assembly. In addition, we have characterized the secondary structure of a linear nonapeptide composed of a hydrophobic alanine/phenylalanine core flanked by hydrophilic acid/amine units. We show that the anionic surfactant sodium dodecyl sulfate (SDS) induces the formation of β-sheets and macroscopic gelation in this otherwise unstructured peptide. Through comparisonmore » to related additives, we propose that SDS-induced secondary structure formation is the result of amphiphilicity created by electrostatic binding of SDS to the peptide. In addition, we demonstrate a novel utility of surfactants in manipulating and stabilizing peptide nanostructures. SDS is used to simultaneously induce secondary structure in a peptide and to inhibit the activity of a model enzyme, resulting in a peptide hydrogel that is impervious to enzymatic degradation. These results complement our understanding of the behavior of peptides in the presence of interacting secondary molecules and provide new potential pathways for programmable organization of peptides by the addition of such components.« less

  1. Radiation-induced products of peptides and their enzymatic digestibility

    SciTech Connect

    Gajewski, E.

    1983-01-01

    Chemical characterization of radiation-induced products of peptides and proteins is essential for understanding the effect of ionizing radiation on peptides and proteins. Furthermore, peptides containing radiation-altered amino acid residues might not be completely digestible by proteolytic enzymes. In this work, small homopeptides of Ala, Phe and Met were chosen as model peptides. Lysozyme was used to investigate the effect of ionizing radiation on a small protein. All peptides and lysozyme were irradiated in diluted, oxygen free, N/sub 2/O-saturated aqueous solutions, using a /sup 60/Co-..gamma..-source. HPLC, capillary GC and GC-MS were applied to isolate and characterize the radiation-induced products. The enzymatic digestibility of the products was investigated using aminopeptidase M, leucine aminopeptidase, carboxypeptidase A and carboxypeptidase Y. It was found that irradiation of peptides examined in this work leads to racemization and alteration of amino acid residues and crosslinks between the peptide chains. In addition, it was established that exopeptidases act differently on radiation-induced dimers of peptides composed of aliphatic, aromatic and sulfur-containing amino acids.

  2. Enhanced oral bioavailability of insulin using PLGA nanoparticles co-modified with cell-penetrating peptides and Engrailed secretion peptide (Sec).

    PubMed

    Zhu, Siqi; Chen, Shuangxi; Gao, Yuan; Guo, Feng; Li, Fengying; Xie, Baogang; Zhou, Jianliang; Zhong, Haijun

    2016-07-01

    Biodegradable polymer nanoparticle drug carriers are an attractive strategy for oral delivery of peptide and protein drugs. However, their ability to cross the intestinal epithelium membrane is largely limited. Therefore, in the present study, cell-penetrating peptides (R8, Tat, penetratin) and a secretion peptide (Sec) with N-terminal stearylation were introduced to modify nanoparticles (NPs) on the surface to improve oral bioavailability of peptide and protein drugs. In vitro studies conducted in Caco-2 cells showed the value of the apparent permeability coefficient (Papp) of the nanoparticles co-modified with Sec and penetratin (Sec-Pen-NPs) was about two-times greater than that of the nanoparticles modified with only penetratin (Pen-NPs), while the increase of transcellular transport of nanoparticles modified together with Sec and R8 (Sec-R8-NPs), or Sec and Tat (Sec-Tat-NPs), was not significant compared with nanoparticles modified with only R8 (R8-NPs) or Tat (Tat-NPs). Using insulin as the model drug, in vivo studies performed on rats indicated that compared to Pen-NPs, the relative bioavailability of insulin for Sec-Pen-NPs was 1.71-times increased after ileal segments administration, and stronger hypoglycemic effects was also observed. Therefore, the nanoparticles co-modified with penetratin and Sec could act as attractive carriers for oral delivery of insulin.

  3. Coarse Graining to Investigate Membrane Induced Peptide Folding of Anticancer Peptides

    NASA Astrophysics Data System (ADS)

    Ganesan, Sai; Xu, Hongcheng; Matysiak, Silvina

    Information about membrane induced peptide folding mechanisms using all-atom molecular dynamics simulations is a challenge due to time and length scale issues.We recently developed a low resolution Water Explicit Polarizable PROtein coarse-grained Model by adding oppositely charged dummy particles inside protein backbone beads.These two dummy particles represent a fluctuating dipole,thus introducing structural polarization into the coarse-grained model.With this model,we were able to achieve significant α- β secondary structure content de novo,without any added bias.We extended the model to zwitterionic and anionic lipids,by adding oppositely charged dummy particles inside polar beads, to capture the ability of the head group region to form hydrogen bonds.We use zwitterionic POPC and anionic POPS as our model lipids, and a cationic anticancer peptide,SVS1,as our model peptide.We have characterized the driving forces for SVS1 folding on lipid bilayers with varying anionic and zwitterionic lipid compositions.Based on our results, dipolar interactions between peptide backbone and lipid head groups contribute to stabilize folded conformations.Cooperativity in folding is induced by both intra peptide and membrane-peptide interaction.

  4. Peptide and non-peptide opioid-induced hyperthermia in rabbits

    NASA Technical Reports Server (NTRS)

    Kandasamy, S. B.; Williams, B. A.

    1983-01-01

    The intracerebroventricular administration of prototype nonpeptide opioid receptor (mu, kappa, and sigma) agonists, morphine, ketocyclazocine, and N-allyl-normetazocine was found to induce hyperthermia in rabbits. The similar administration of peptide opioids like beta-endorphin (BE), methionine-enkephalin (ME), and its synthetic analogue D-ala2-methionine-enkephalinamide (DAME) was also found to cause hyperthermia. Results indicate that only the liver-like transport system is important to the ventricular inactivation of BE and DAME. Prostaglandins and norepinephrine were determined not to be involved in peptide and nonpeptide opioid-induced hyperthermia. In addition, cAMP was not required since a phosphodiesterase inhibitor, theophylline, did not accentuate the hyperthermia due to peptide and nonpeptide opioids. Naloxone-sensitive receptors were found to be involved in the induction of hyperthermia by morphine, BE, ME, and DAME since naloxone attenuated them. However, the hyperthermic response to ketocyclazocine and N-allyl-normetazocine was not antagonized by naloxone.

  5. Mucin-like peptides from Echinococcus granulosus induce antitumor activity.

    PubMed

    Noya, Verónica; Bay, Sylvie; Festari, María Florencia; García, Enrique P; Rodriguez, Ernesto; Chiale, Carolina; Ganneau, Christelle; Baleux, Françoise; Astrada, Soledad; Bollati-Fogolín, Mariela; Osinaga, Eduardo; Freire, Teresa

    2013-09-01

    There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.

  6. Steric-electronic effects in malarial peptides inducing sterile immunity

    SciTech Connect

    Moreno-Vranich, Armando; Patarroyo, Manuel E.

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Is it evident that the residues position are relevant regarding of {phi} angular value. Black-Right-Pointing-Pointer The geometry considered for detailing the alterations undergone by HABPs. Black-Right-Pointing-Pointer The inter planar interactions ruled by clashes between the atoms making them up. -- Abstract: Conserved Plasmodium falciparum high activity binding peptides' (HABPs) most relevant proteins involved in malaria parasite invasion are immunologically silent; critical binding residues must therefore be specifically replaced to render them highly immunogenic and protection-inducing. Such changes have a tremendous impact on these peptides' steric-electronic effects, such as modifications to peptide length peptide bonds and electronic orbitals' disposition, to allow a better fit into immune system MHCII molecules and better interaction with the TCR which might account for the final immunological outcome.

  7. PFR peptide, one of the antimicrobial peptides identified from the derivatives of lactoferrin, induces necrosis in leukemia cells

    PubMed Central

    Lu, Yan; Zhang, Teng-Fei; Shi, Yue; Zhou, Han-Wei; Chen, Qi; Wei, Bu-Yun; Wang, Xi; Yang, Tian-Xin; Chinn, Y. Eugene; Kang, Jian; Fu, Cai-Yun

    2016-01-01

    LF11-322 (PFWRIRIRR-NH2) (PFR peptide), a nine amino acid-residue peptide fragment derived from human lactoferricin, possesses potent cytotoxicity against bacteria. We report here the discovery and characterization of its antitumor activity in leukemia cells. PFR peptide inhibited the proliferation of MEL and HL-60 leukemia cells by inducing cell death in the absence of the classical features of apoptosis, including chromatin condensation, Annexin V staining, Caspase activation and increase of abundance of pro-apoptotic proteins. Instead, necrotic cell death as evidenced by increasing intracellular PI staining and LDH release, inducing membrane disruption and up-regulating intracellular calcium level, was observed following PFR peptide treatment. In addition to necrotic cell death, PFR peptide also induced G0/G1 cell cycle arrest. Moreover, PFR peptide exhibited favorable antitumor activity and tolerability in vivo. These findings thus provide a new clue of antimicrobial peptides as a potential novel therapy for leukemia. PMID:26860588

  8. A lipocentric view of peptide-induced pores.

    PubMed

    Fuertes, Gustavo; Giménez, Diana; Esteban-Martín, Santi; Sánchez-Muñoz, Orlando L; Salgado, Jesús

    2011-04-01

    Although lipid membranes serve as effective sealing barriers for the passage of most polar solutes, nonmediated leakage is not completely improbable. A high activation energy normally keeps unassisted bilayer permeation at a very low frequency, but lipids are able to self-organize as pores even in peptide-free and protein-free membranes. The probability of leakage phenomena increases under conditions such as phase coexistence, external stress or perturbation associated to binding of nonlipidic molecules. Here, we argue that pore formation can be viewed as an intrinsic property of lipid bilayers, with strong similarities in the structure and mechanism between pores formed with participation of peptides, lipidic pores induced by different types of stress, and spontaneous transient bilayer defects driven by thermal fluctuations. Within such a lipocentric framework, amphipathic peptides are best described as pore-inducing rather than pore-forming elements. Active peptides bound to membranes can be understood as a source of internal surface tension which facilitates pore formation by diminishing the high activation energy barrier. This first or immediate action of the peptide has some resemblance to catalysis. However, the presence of membrane-active peptides has the additional effect of displacing the equilibrium towards the pore-open state, which is then maintained over long times, and reducing the size of initial individual pores. Thus, pore-inducing peptides, regardless of their sequence and oligomeric organization, can be assigned a double role of increasing the probability of pore formation in membranes to high levels as well as stabilizing these pores after they appear.

  9. Assembly of naphthalenediimide conjugated peptides: aggregation induced changes in fluorescence.

    PubMed

    Basak, Shibaji; Nanda, Jayanta; Banerjee, Arindam

    2013-08-01

    Naphthalenediimide appended peptide based self-assembly was studied. Interestingly, an aggregation induced drastic change in the fluorescence property and gel formation were observed depending on the solvent composition (chloroform : methylcyclohexane) at a fixed concentration of 1.6 mM at room temperature. PMID:23799544

  10. Pseudoprolines as removable turn inducers: tools for the cyclization of small peptides.

    PubMed

    Skropeta, Danielle; Jolliffe, Katrina A; Turner, Peter

    2004-12-10

    The cyclization of small peptides which do not incorporate turn inducers is often difficult. We have developed a method involving the use of removable turn inducers, in the form of pseudoprolines, for the cyclization of difficult peptide sequences. The pseudoprolines induce a cisoid amide bond in the peptide backbone which facilitates cyclization. They are then readily removed to yield a cyclic peptide that does not contain any turn inducers.

  11. Peptide hydrogelation triggered by enzymatic induced pH switch

    NASA Astrophysics Data System (ADS)

    Cheng, Wei; Li, Ying

    2016-07-01

    It remains challenging to develop methods that can precisely control the self-assembling kinetics and thermodynamics of peptide hydrogelators to achieve hydrogels with optimal properties. Here we report the hydrogelation of peptide hydrogelators by an enzymatically induced pH switch, which involves the combination of glucose oxidase and catalase with D-glucose as the substrate, in which both the gelation kinetics and thermodynamics can be controlled by the concentrations of D-glucose. This novel hydrogelation method could result in hydrogels with higher mechanical stability and lower hydrogelation concentrations. We further illustrate the application of this hydrogelation method to differentiate different D-glucose levels.

  12. Immunotherapy for pulmonary TB: antimicrobial peptides and their inducers.

    PubMed

    Rivas-Santiago, Cesar Enrique; Hernández-Pando, Rogelio; Rivas-Santiago, Bruno

    2013-10-01

    TB is an infectious disease that still has an enormous impact on public health worldwide. With the continuous increasing epidemic of multidrug-resistant TB, new drugs and vaccines are urgently needed. In the last decade there has been a broad advance in the knowledge of innate immunity in TB. Together with the growing research regarding immunomodulators, new promising insights have been developed that can contribute in the control of TB. This is the case of antimicrobial peptides, which can be potential therapeutic or adjuvant agents. The current high cost of antimicrobial peptide synthesis may be a current deterrent for treatment; antimicrobial peptide-inducers can be an alternative for low-cost treatment and/or adjuvants.

  13. Peptide-induced patterning of gold nanoparticle thin films

    NASA Astrophysics Data System (ADS)

    Borteh, Hassan M.; Ferrell, Nicholas J.; Butler, Randall T.; Olesik, Susan V.; Hansford, Derek J.

    2011-10-01

    In this work, the use of patterned proteins and peptides for the deposition of gold nanoparticles on several substrates with different surface chemistries is presented. The patterned biomolecule on the surface acts as a catalyst to precipitate gold nanoparticles from a precursor solution of HAuCl 4 onto the substrate. The peptide patterning on the surfaces was accomplished by physical adsorption or covalent attachment. It was shown that by using covalent attachment with a linker molecule, the influence of the surface properties from the different substrates on the biomolecule adsorption and subsequent nanoparticle deposition could be avoided. By adjusting the reaction conditions such as pH or HAuCl 4 concentration, the sizes and morphologies of deposited gold nanoparticle agglomerates could be controlled. Two biomolecules were used for this experiment, 3XFLAG peptide and bovine serum albumin (BSA). A micro-transfer molding technique was used to pattern the peptides on the substrates, in which a pre-patterned poly(dimethylsiloxane) (PDMS) mold was used to deposit a lift-off pattern of polypropylmethacrylate (PPMA) on the various substrates. The proteins were either physically adsorbed or covalently attached to the substrates, and an aqueous HAuCl 4 solution was applied on the substrates with the protein micropatterns, causing the precipitation of gold nanoparticles onto the patterns. SEM, AFM, and Electron Beam Induced Current (EBIC) were used for characterization.

  14. Surface-Induced Dissociation of Peptide Ions: Kinetics and Dynamics

    SciTech Connect

    Laskin, Julia; Futrell, Jean H.; Shukla, Anil K.

    2003-12-01

    Kinetics and dynamics studies have been carried out for the surface-induced dissociation (SID) of a set of model peptides utilizing a specially designed electrospray ionization Fourier Transform ion cyclotron resonance mass spectrometer in which mass-selected and vibrationally relaxed ions are collided on a orthogonally-mounted fluorinated self-assembled monolayer on Au{l_brace}111{r_brace} crystal. The sampling time in this apparatus can be varied from hundreds of microseconds to tens of seconds, enabling the investigation of kinetics of ion decomposition over an extended range of decomposition rates. RRKM-based modeling of these reactions for a set of polyalanines demonstrates that SID kinetics of these simple peptides is very similar to slow, multiple-collision activation and that the distribution of internal energies following collisional activation is indistinguishable from a thermal distribution. For more complex peptides comprised of several amino acids and with internal degrees of freedom ( DOF) of the order of 350 there is a dramatic change in kinetics in which RRKM kinetics is no longer capable of describing the decomposition of these complex ions. A combination of RRKM kinetics and the sudden death approximation, according to which decomposition occurs instantaneously, is a satisfactory description. This implies that a population of ions-which is dependant on the nature of the peptide, kinetic energy and sampling time-decomposes on or very near the surface. The shattering transition is described quantitatively for the limited set of molecules examined to date

  15. Surface-Induced Dissociation of Peptide Ions: Kinetics and Dynamics

    SciTech Connect

    Laskin, Julia; Futrell, Jean H.

    2003-12-01

    Kinetics and dynamics studies have been carried out for the surface-induced dissociation (SID) of a set of model peptides utilizing a specially designed electro spray ionization Fourier Transform Ion cyclotron resonance mass spectrometer in which mass-selected and vibrationally relaxed ions are collided on an orthogonally-mounted fluorinated self-assembled monolayer on Au {111} crystal. The sampling time in this apparatus can be varied from hundreds of microseconds to tens of seconds, enabling the investigation of kinetics of ion decomposition over an extended range of decomposition rates. RRKM-based modeling of these reactions for a set of polyalanines demonstrates that kinetics of these simple peptides is very similar to slow, multiple-collision activation and that the distribution of internal energies following collisional activation is indistinguishable from a thermal distribution. For more complex peptides comprised of several amino acids and with internal degrees of freedom (DOF) of the order of 350 there is a dramatic change in kinetics in which RRKM kinetics is no longer capable of describing the decomposition of these complex ions. A combination of RRKM kinetics and the “sudden death” approximation, according to which decomposition occurs instantaneously, is a satisfactory description. This implies that a population of ions – which is dependant on the nature of the peptide, kinetic energy and sampling time – decomposes on or very near the surface. The shattering transition is described quantitatively for the limited set of molecules examined to date.

  16. Amyloid Beta Peptide Slows Down Sensory-Induced Hippocampal Oscillations

    PubMed Central

    Peña-Ortega, Fernando; Bernal-Pedraza, Ramón

    2012-01-01

    Alzheimer's disease (AD) progresses with a deterioration of hippocampal function that is likely induced by amyloid beta (Aβ) oligomers. Hippocampal function is strongly dependent on theta rhythm, and disruptions in this rhythm have been related to the reduction of cognitive performance in AD. Accordingly, both AD patients and AD-transgenic mice show an increase in theta rhythm at rest but a reduction in cognitive-induced theta rhythm. We have previously found that monomers of the short sequence of Aβ (peptide 25–35) reduce sensory-induced theta oscillations. However, considering on the one hand that different Aβ sequences differentially affect hippocampal oscillations and on the other hand that Aβ oligomers seem to be responsible for the cognitive decline observed in AD, here we aimed to explore the effect of Aβ oligomers on sensory-induced theta rhythm. Our results show that intracisternal injection of Aβ1–42 oligomers, which has no significant effect on spontaneous hippocampal activity, disrupts the induction of theta rhythm upon sensory stimulation. Instead of increasing the power in the theta band, the hippocampus of Aβ-treated animals responds to sensory stimulation (tail pinch) with an increase in lower frequencies. These findings demonstrate that Aβ alters induced theta rhythm, providing an in vivo model to test for therapeutic approaches to overcome Aβ-induced hippocampal and cognitive dysfunctions. PMID:22611415

  17. 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. PMID:26516201

  18. Peptide Fragmentation by Corona Discharge Induced Electrochemical Ionization

    PubMed Central

    Lloyd, John R.; Hess, Sonja

    2010-01-01

    Fundamental studies have greatly improved our understanding of electrospray, including the underlying electrochemical reactions. Generally regarded as disadvantageous, we have recently shown that corona discharge (CD) can be used as an effective method to create a radical cation species [M]+•, thus optimizing the electrochemical reactions that occur on the surface of the stainless steel (SS) electrospray capillary tip. This technique is known as CD initiated electrochemical ionization (CD-ECI). Here, we report on the fundamental studies using CD-ECI to induce analytically useful in-source fragmentation of a range of molecules that complex transition metals. Compounds that have been selectively fragmented using CD-ECI include enolate forming phenylglycine containing peptides, glycopeptides, nucleosides and phosphopeptides. Collision induced dissociation (CID) or other activation techniques were not necessary for CD-ECI fragmentation. A four step mechanism was proposed: 1. Complexation using either Fe in the SS capillary tip material or Cu(II) as an offline complexation reagent; 2. Electrochemical oxidation of the complexed metal and thus formation of a radical cation (e.g.; Fe - e− → Fe +•); 3. Radical fragmentation of the complexed compound. 4. Electrospray ionization of the fragmented neutrals. Fragmentation patterns resembling b- and y-type ions were observed and allowed the localization of the phosphorylation sites. PMID:20869880

  19. Euler buckling, membrane corrugation and pore formation induced by antimicrobial peptide

    NASA Astrophysics Data System (ADS)

    Golubovic, Leonardo; Gao, Lianghui; Chen, Licui; Jia, Nana; Fang, Weihai

    2014-03-01

    Antimicrobial peptides serve as defense weapons against bacteria. They are secreted by organisms of plants and animals and have a wide variety in composition and structure. In this study, we theoretically explore the effects of the antimicrobial peptides on the lipid bilayer membrane by using analytic arguments and the coarse grained dissipative particle dynamics simulations. We study peptide/lipid membrane complexes by considering peptides with various structure, hydrophobicity and peptide/lipid interaction strength. The role of lipid/water interaction is also discussed. We discuss a rich variety of membrane morphological changes induced by peptides, such as pore formation, membrane corrugation and Euler buckling. Such buckled membrane states have been indeed seen in a number of experiments with bacteria affected by peptide, yet this is the first theoretical study addressing these phenomena more deeply.

  20. CTL responses induced by a single immunization with peptide encapsulated in biodegradable microparticles.

    PubMed

    Partidos, C D; Vohra, P; Jones, D; Farrar, G; Steward, M W

    1997-08-01

    A synthetic peptide representing a measles virus (MV) cytotoxic T cell epitope (CTL) when encapsulated in poly (D,L-lactide co-glycolide) (PLG) 50:50 microparticles induced a strong CTL response after a single intraperitoneal immunization of mice which was greater than that following administration of the peptide in Freund's complete adjuvant. A 100 micrograms dose of encapsulated peptide was shown to be more effective for CTL priming than 50 and 25 micrograms doses. A vaccine formulation prepared by simply mixing empty 50:50 PLG microparticles with the peptide resulted in the induction of CTL responses comparable to those induced by the encapsulated peptide. Moreover, a CTL response against MV-infected target cells was observed. These findings highlight the potential immunostimulatory effect of PLG microparticles for the induction of MV and peptide-specific CTL responses.

  1. Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays.

    PubMed

    Pai, Jaeyoung; Hyun, Soonsil; Hyun, Ji Young; Park, Seong-Hyun; Kim, Won-Je; Bae, Sung-Hun; Kim, Nak-Kyoon; Yu, Jaehoon; Shin, Injae

    2016-01-27

    MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing. PMID:26771315

  2. Protective Effect of Wheat Peptides against Indomethacin-Induced Oxidative Stress in IEC-6 Cells

    PubMed Central

    Yin, Hong; Pan, Xingchang; Song, Zhixiu; Wang, Shaokang; Yang, Ligang; Sun, Guiju

    2014-01-01

    Recent studies have demonstrated that wheat peptides protected rats against non-steroidal anti-inflammatory drugs-induced small intestinal epithelial cells damage, but the mechanism of action is unclear. In the present study, an indomethacin-induced oxidative stress model was used to investigate the effect of wheat peptides on the nuclear factor-κB(NF-κB)-inducible nitric oxide synthase-nitric oxide signal pathway in intestinal epithelial cells-6 cells. IEC-6 cells were treated with wheat peptides (0, 125, 500 and 2000 mg/L) for 24 h, followed by 90 mg/L indomethacin for 12 h. Wheat peptides significantly attenuated the indomethacin-induced decrease in superoxide dismutase and glutathione peroxidase activity. Wheat peptides at 2000 mg/L markedly decreased the expression of the NF-κB in response to indomethacin-induced oxidative stress. This study demonstrated that the addition of wheat peptides to a culture medium significantly inhibited the indomethacin-induced release of malondialdehyde and nitrogen monoxide, and increased antioxidant enzyme activity in IEC-6 cells, thereby providing a possible explanation for the protective effect proposed for wheat peptides in the prevention of indomethacin-induced oxidative stress in small intestinal epithelial cells. PMID:24481130

  3. [Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila].

    PubMed

    Sarantseva, S V; Bol'shakova, O I; Timoshenko, S I; Kolobov, A A; Vitek, M P; Shvartsman, A L

    2009-01-01

    Protein transduction domain (PTD)-peptides greatly facilitate the delivery of high molecular weight macromolecules across the blood-brain barrier (BBB). This BBB-transport function is highly desirable and helps to enable the development of new therapeutics for treatment of brain disorders. However, the drug discovery process is limited by the generation of a simple and reliable BBB model that is amenable to testing of large number of samples and simultaneously, reproduces the physiological and functional characteristics of the human BBB. To address these challenges, we have studied whether the PTD-peptide penetratin, derived from a Drosophila Antennapedia homeodomain protein, is capable of crossing the BBB in Drosophila while carrying a cargo into the fly brain. An initial in vivo experiment in Drosophila showed that abdominal injection of biotin-tagged penetratin permeated the BBB. The same effect was observed for biotin-tagged penetratin fused with apoE mimetic peptide with demonstrated anti-inflammatory and neuroprotective activities. PMID:19351032

  4. A multicomponent conjugation strategy to unique N-steroidal peptides: first evidence of the steroidal nucleus as a β-turn inducer in acyclic peptides.

    PubMed

    Rivera, Daniel G; Vasco, Aldrin V; Echemendía, Radell; Concepción, Odette; Pérez, Carlos S; Gavín, José A; Wessjohann, Ludger A

    2014-10-01

    Constraining small peptides into specific secondary structures has been a major challenge in peptide ligand design. So far, the major solution for decreasing the conformational flexibility in small peptides has been cyclization. An alternative is the use of topological templates, which are able to induce and/or stabilize peptide secondary structures by means of covalent attachment to the peptide. Herein a multicomponent strategy and structural analysis of a new type of peptidosteroid architecture having the steroid as N-substituent of an internal amide bond is reported. The approach comprises the one-pot conjugation of two peptide chains (or amino acid derivatives) to aminosteroids by means of the Ugi reaction to give a unique family of N-steroidal peptides. The conjugation efficiency of a variety of peptide sequences and steroidal amines, as well as their consecutive head-to-tail cyclization to produce chimeric cyclopeptide-steroid conjugates, that is, macrocyclic lipopeptides, was assessed. Determination of the three-dimensional structure of an acyclic N-steroidal peptide in solution proved that the bulky, rigid steroidal template is capable of both increasing significantly the conformational rigidity, even in a peptide sequence as short as five amino acid residues, and inducing a β-turn secondary structure even in the all-s-trans isomer. This report provides the first evidence of the steroid skeleton as β-turn inducer in linear peptide sequences.

  5. The prelamin A pre-peptide induces cardiac and skeletal myoblast differentiation

    SciTech Connect

    Brodsky, Gary L. . E-mail: Gary.Brodsky@uchsc.edu; Bowersox, Jeffrey A.; Fitzgerald-Miller, Lisa; Miller, Leslie A.; Maclean, Kenneth N.

    2007-05-18

    Prelamin A processing is unique amongst mammalian proteins and results in the production of a farnesylated and carboxymethylated peptide. We examined the effect of pathogenic LMNA mutations on prelamin A processing, and of the covalently modified peptide on cardiac and skeletal myoblast differentiation. Here we report a mutation associated with dilated cardiomyopathy prevents prelamin A peptide production. In addition, topical application of the covalently modified C-terminal peptide to proliferating skeletal and cardiac myoblasts induced myotube and striated tissue formation, respectively. Western blot analysis revealed that skeletal and cardiac myoblasts are the first cell lines examined to contain unprocessed prelamin A, and immunostaining of peptide-treated cells revealed a previously unidentified role for prelamin A in cytoskeleton formation and intercellular organization. These results demonstrate a direct role for prelamin A in myoblast differentiation and indicate the prelamin A peptide may have therapeutic potential.

  6. Sodium diclofenac and cell-penetrating peptides embedded in H(II) mesophases: physical characterization and delivery.

    PubMed

    Cohen-Avrahami, Marganit; Libster, Dima; Aserin, Abraham; Garti, Nissim

    2011-09-01

    Glycerol monooleate (GMO)-based mesophases offer extensive prospects for incorporation of various bioactive molecules. This work deals with the solubilization of selected cell-penetrating peptides (CPPs) together with sodium diclofenac (Na-DFC) within the H(II) mesophase for transdermal applications. The effect of CPPs such as RALA (an amphipatic CPP), penetratin (PEN), and oligoarginine (NONA) on Na-DFC skin permeation kinetics to provide controlled release and tune the drug transdermal diffusion was studied. The location of the drug and the CPPs within the mesophase was probed by DSC and FTIR. Na-DFC was found to be located at the interfacial region between the surfactant chains, leading to denser H(II) mesophase. The hydrophilic NONA was intercalated into the aqueous cylinders and caused their swelling. It induced a significant decrease in the hydrogen binding between the GMO carbonyls and their surrounding. The amphiphilic PEN was entrapped within two different regions, depending on its concentration. PEN and NONA improved Na-DFC permeation by 100%, whereas RALA enhanced permeation by 50%. When estimating Na-DFC migration rate out of the mesophase toward surrounding aqueous media, it appeared to be slower with the CPPs. The peptides were not involved at this diffusion-controlled step. It seems that their effect on skin permeation is based on their specific interaction with the skin.

  7. T cell priming versus T cell tolerance induced by synthetic peptides

    PubMed Central

    1995-01-01

    It is well known that synthetic peptides are able to both induce and tolerize T cells. We have examined the parameters leading either to priming or tolerance of CD8+ cytotoxic T lymphocytes (CTL) in vivo with a major histocompatibility complex class I (H-2 Db) binding peptide derived from the glycoprotein (GP aa33-41) of lymphocytic choriomeningitis virus (LCMV). By varying dose, route, and frequency of LCMV GP peptide application, we found that a single local subcutaneous injection of 50-500 micrograms peptide emulsified in incomplete Freund's adjuvant protected mice against LCMV infection, whereas repetitive and systemic intraperitoneal application of the same dose caused tolerance of LCMV-specific CTL. The peptide-induced tolerance was transient in euthymic mice but permanent in thymectomized mice. These findings are relevant for a selective use of peptides as a therapeutic approach: peptide-induced priming of T cells for vaccination and peptide-mediated T cell tolerance for intervention in immunopathologies and autoimmune diseases. PMID:7540654

  8. Epitope spreading induced by immunization with synthetic SSB peptides

    PubMed Central

    DING, MIN; ZHANG, JIANZHONG

    2016-01-01

    Sjogren's syndrome type B (SSB)/La antibody is an autoantibody generally observed in connective tissue diseases whereas double-stranded deoxyribonucleic acid (dsDNA) antibodies are the most characteristic autoantibodies found in systemic lupus erythematosus (SLE) patients. The relationship of these autoantibodies remains unclear. The aim of the study was to determine the profile of antibody production in rabbits immunized with synthetic SSB peptides alone or with dsDNA. For this purpose, 214–225aa peptide of SSB antigen was synthesized based on the organic chemistry solid-phase peptide synthesis. Rabbits were immunized with the following antigens: i) synthetic SSB peptides linked with keyhole limpet hemocyanin (KLH); ii) dsDNA; iii) SSB plus dsDNA; iv) KLH; and v) phosphate-buffered saline. SSB peptide antibody was measured using the enzyme-linked immunosorbent assay while extractable nuclear antigens (ENA) antibody and dsDNA antibody were measured by immunoblotting and immunofluorescence, respectively. The results showed that a specific anti-SSB peptide antibody was produced following immunization with SSB epitope alone or with dsDNA. The SSB peptide antibody titer in the coimmunization group was higher than that of the SSB alone group. In addition, antibodies against ribonucleoprotein (RNP), Smith and/or dsDNA were detected in rabbits of the coimmunization group. The presence of anti-dsDNA antibodies in the rabbits immunized with SSB peptide suggested the induction of epitope spreading. In conclusions, SSB antibodies were produced in rabbits immunized with SSB peptide or SSB+dsDNA, whereas SSB antibody titers were higher in the coimmunization group. Furthermore, coimmunization was associated with epitope spreading. PMID:27347030

  9. Mutual Amino Acid Catalysis in Salt-Induced Peptide Formation Supports this Mechanism's Role in Prebiotic Peptide Evolution

    NASA Astrophysics Data System (ADS)

    Suwannachot, Yuttana; Rode, Bernd M.

    1999-10-01

    The presence of some amino acids and dipeptides under the conditions of the salt-induced peptide formation reaction (aqueous solution at 85 °C, Cu(II) and NaCl) has been found to catalyze the formation of homopeptides of other amino acids, which are otherwise produced only in traces or not at all by this reaction. The condensation of Val, Leu and Lys to form their homodipeptides can occur to a considerable extent due to catalytic effects of other amino acids and related compounds, among which glycine, histidine, diglycine and diketopiperazine exhibit the most remarkable activity. These findings also lead to a modification of the table of amino acid sequences preferentially formed by the salt-induced peptide formation (SIPF) reaction, previously used for a comparison with the sequence preferences in membrane proteins of primitive organisms

  10. Requirements of the engineered leader peptide of nisin for inducing modification, export, and cleavage.

    PubMed

    Plat, Annechien; Kluskens, Leon D; Kuipers, Anneke; Rink, Rick; Moll, Gert N

    2011-01-01

    Nisin A is a pentacyclic peptide antibiotic produced by Lactococcus lactis. The leader peptide of prenisin keeps nisin inactive and has a role in inducing NisB- and NisC-catalyzed modifications of the propeptide and NisT-mediated export. The highly specific NisP cleaves off the leader peptide from fully modified and exported prenisin. We present here a detailed mutagenesis analysis of the nisin leader peptide. For alternative cleavage, we successfully introduced a putative NisP autocleavage site and sites for thrombin, enterokinase, Glu-C, and factor Xa in the C-terminal part of the leader peptide. Replacing residue F-18 with Trp or Thr strongly reduced production. On the other hand, D-19A, F-18H, F-18M, L-16D, L-16K, and L-16A enhanced production. Substitutions within and outside the FNLD box enhanced or reduced the transport efficiency. None of the above substitutions nor even an internal 6His tag from positions -13 to -8 had any effect on the capacity of the leader peptide to induce NisB and NisC modifications. Therefore, these data demonstrate a large mutational freedom. However, simultaneous replacement of the FNLD amino acids by four alanines strongly reduced export and even led to a complete loss of the capacity to induce modifications. Reducing the leader peptide to MSTKDFNLDLR led to 3- or 4-fold dehydration. Taken together, the FNLD box is crucial for inducing posttranslational modifications. PMID:21097596

  11. A novel system enhancing the endosomal escapes of peptides promotes Bak BH3 peptide inducing apoptosis in lung cancer A549 cells.

    PubMed

    Lin, Nanjing; Zheng, Wenyun; Li, Linfeng; Liu, Hui; Wang, Tianwen; Wang, Ping; Ma, Xingyuan

    2014-06-01

    Therapeutic peptides have been proven useful for treating various diseases. However, it is difficult for therapeutic peptides to reach their target sites with an effective concentration due to inefficient intracellular delivery. Although Tat transduction peptide is a promising tool to deliver therapeutic peptides into cells, the entrapment within endosomes and the nuclear localization of Tat transduction peptide severely limited the biological effects of Tat-linked cargos. In this study, we designed a novel peptide delivering system, Tat-INF7-ubiquitin (TIU), which consisted of Tat transduction peptide, endosomal escape enhancer peptide INF7, and ubiquitin protein. We found that the TIU system was able to efficiently deliver the mCherry fluorescent proteins and the apoptosis-inducing Bak BH3 peptide into the cytosol. The Bak BH3 peptide transported into the cells by the TIU system increased the apoptotic rate to 46.15 ± 4.86% (p < 0.001) in A549 cells, while Tat-BH3 could result in only 20.45 ± 2.89%. These results demonstrated that the TIU system could enhance the effects of therapeutic peptides by facilitating the transmembrane delivery of peptides into the cells and the escape of target proteins from the endosome efficiently.

  12. Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides

    PubMed Central

    Bilichak, Andriy; Luu, Justin; Eudes, François

    2015-01-01

    Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells. PMID:26379691

  13. Effect of Curcumin on the metal ion induced fibrillization of Amyloid-β peptide

    NASA Astrophysics Data System (ADS)

    Banerjee, Rona

    2014-01-01

    The effect of Curcumin on Cu(II) and Zn(II) induced oligomerization and protofibrillization of the amyloid-beta (Aβ) peptide has been studied by spectroscopic and microscopic methods. Curcumin could significantly reduce the β-sheet content of the peptide in a time dependent manner. It also plays an antagonistic role in β-sheet formation that is promoted by metal ions like Cu(II) and Zn(II) as observed by Circular Dichroism (CD) spectroscopy. Atomic force microscopic (AFM) images show that spontaneous fibrillization of the peptide occurs in presence of Cu(II) and Zn(II) but is inhibited on incubation of the peptide with Curcumin indicating the beneficial role of Curcumin in preventing the aggregation of Aβ peptide.

  14. Tailor-made designer helical peptides that induce mitochondrion-mediated cell death without necrosis.

    PubMed

    Nogami, Kagayaki; Takahama, Kentaro; Okushima, Ayako; Oyoshi, Takanori; Fujimoto, Kazuhisa; Inouye, Masahiko

    2014-11-24

    Managing protein-protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side-chain-crosslinked helical peptides based on the binding domain of a pro-apoptotic protein (Bad) that induces programmed cell death. The peptide showed high helical content and bound to its target, Bcl-XL, more strongly than its non-crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl-XL localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70% of HeLa cells died by the apoptosis pathway (without evidence of necrosis).

  15. KL4 Peptide Induces Reversible Collapse Structures on Multiple Length Scales in Model Lung Surfactant

    PubMed Central

    Holten-Andersen, Niels; Michael Henderson, J.; Walther, Frans J.; Waring, Alan J.; Ruchala, Piotr; Notter, Robert H.; Lee, Ka Yee C.

    2011-01-01

    We investigated the effects of KL4, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL4 improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds). PMID:22208194

  16. Evaporation cycle experiments — A simulation of salt-induced peptide synthesis under possible prebiotic conditions

    NASA Astrophysics Data System (ADS)

    Saetia, Somporn; Liedl, Klaus R.; Eder, Artur H.; Rode, Bernd M.

    1993-06-01

    Evaporation cycles applied to dilute solutions of amino acids, Cu(II) and NaCl lead to peptides within 1 3 days. This simulation of possible coastal or laguna processes in a primitive earth environment gives further indications towards the relevance of the salt-induced peptide formation reaction in chemical evolution. The experiments were successfully applied to glycine, alanine, aspartic and glutamic acid. Besides isolated amino acids, also their mixtures with glycine as reaction partner were studied, leading to peptides for all of the aforementioned substances, as well as for valine and proline, which do not dimerize alone. Sequence preferences and some conservation of optical purity were observed.

  17. Protein-peptide interaction: study of heat-induced aggregation and gelation of β-lactoglobulin in the presence of two peptides from its own hydrolysate.

    PubMed

    Kosters, Hans A; Wierenga, Peter A; de Vries, Renko; Gruppen, Harry

    2013-05-01

    Two peptides, [f135-158] and [f135-162]-SH, were used to study the binding of the peptides to native β-lactolobulin, as well as the subsequent effects on aggregation and gelation of β-lactoglobulin. The binding of the peptide [f135-158] to β-lactoglobulin at room temperature was confirmed by SELDI-TOF-MS. It was further illustrated by increased turbidity of mixed solutions of peptide and protein (at pH 7), indicating association of proteins and peptides in larger complexes. At pH below the isoelectric point of the protein, the presence of peptides did not lead to an increased turbidity, showing the absence of complexation. The protein-peptide complexes formed at pH 7 were found to dissociate directly upon heating. After prolonged heating, extensive aggregation was observed, whereas no aggregation was seen for the pure protein or pure peptide solutions. The presence of the free sulfhydryl group in [f135-162]-SH resulted in a 10 times increase in the amount of aggregation of β-lactoglobulin upon heating, illustrating the additional effect of the free sulfhydryl group. Subsequent studies on the gel strength of heat-induced gels also showed a clear difference between these two peptides. The replacement of additional β-lactoglobulin by [f135-158] resulted in a decrease in gel strength, whereas replacement by peptide [f135-162]-SH increased gel strength.

  18. A Unified Conformational Selection and Induced Fit Approach to Protein-Peptide Docking

    PubMed Central

    Trellet, Mikael; Melquiond, Adrien S. J.; Bonvin, Alexandre M. J. J.

    2013-01-01

    Protein-peptide interactions are vital for the cell. They mediate, inhibit or serve as structural components in nearly 40% of all macromolecular interactions, and are often associated with diseases, making them interesting leads for protein drug design. In recent years, large-scale technologies have enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. Yet, the paucity of data regarding their molecular binding mechanisms together with their inherent flexibility makes the structural prediction of protein-peptide interactions very challenging. This leaves flexible docking as one of the few amenable computational techniques to model these complexes. We present here an ensemble, flexible protein-peptide docking protocol that combines conformational selection and induced fit mechanisms. Starting from an ensemble of three peptide conformations (extended, a-helix, polyproline-II), flexible docking with HADDOCK generates 79.4% of high quality models for bound/unbound and 69.4% for unbound/unbound docking when tested against the largest protein-peptide complexes benchmark dataset available to date. Conformational selection at the rigid-body docking stage successfully recovers the most relevant conformation for a given protein-peptide complex and the subsequent flexible refinement further improves the interface by up to 4.5 Å interface RMSD. Cluster-based scoring of the models results in a selection of near-native solutions in the top three for ∼75% of the successfully predicted cases. This unified conformational selection and induced fit approach to protein-peptide docking should open the route to the modeling of challenging systems such as disorder-order transitions taking place upon binding, significantly expanding the applicability limit of biomolecular interaction modeling by docking. PMID:23516555

  19. An analogue peptide from the Cancer/Testis antigen PASD1 induces CD8+ T cell responses against naturally processed peptide

    PubMed Central

    Hardwick, Nicola; Buchan, Sarah; Ingram, Wendy; Khan, Ghazala; Vittes, Gisella; Rice, Jason; Pulford, Karen; Mufti, Ghulam; Stevenson, Freda; Guinn, Barbara-ann

    2013-01-01

    We have previously identified the novel Cancer/Testis antigen PASD1 by immunoscreening a testis library with pooled acute myeloid leukemia (AML) patient sera. To develop a cytotoxic T lymphocyte (CTL)-inducing vaccine, we have now investigated the carboxy-terminal region, known to contain serological determinants, for MHC class I (HLA-A⋆0201)-binding peptides. Algorithm-selected natural peptides failed to show detectable HLA-A⋆0201 binding in T2 assays. However, anchor-modified analogue peptides showed enhanced binding, with decreased off-rates. Analogue peptide-loaded antigen-presenting cells (APCs) induced IFN-γ production by T cells from normal donors and patients. In addition, peptide-specific T cells could be expanded from cancer patients by stimulation with the PASD1 analogue peptide Pa14. For clinical application, a DNA fusion gene vaccine encoding Pa14 was designed and tested in “humanized” mice. Splenocytes from vaccinated mice showed in vitro cytotoxicity against tumour cells, either exogenously loaded with the corresponding wild-type peptide (Pw8) or expressing endogenously processed PASD1 protein. We show for the first time that a DNA vaccine encoding an altered PASD1 epitope can induce CTLs to target the natural peptide expressed by human tumour cells. PMID:23882161

  20. A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides*

    PubMed Central

    Giese, Sven H.; Fischer, Lutz; Rappsilber, Juri

    2016-01-01

    Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers. PMID:26719564

  1. Amnesia induced in mice by centrally administered beta-amyloid peptides involves cholinergic dysfunction.

    PubMed

    Maurice, T; Lockhart, B P; Privat, A

    1996-01-15

    Substantial evidences suggest that the increased cerebral deposition, and neurotoxic action of the beta-amyloid peptide, the major constituent of senile plaques, may represent the underlying cause of the cognitive deficits observed in Alzheimer's disease. Herein, we attempted to verify this hypothesis by inducing a potential Alzheimer's-type amnesia after direct intracerebroventricular administration of aggregated beta 25-35-amyloid peptide in mice. In this aim, mnesic capacities were evaluated after 6-13 days, using spontaneous alternation in the Y-maze, step-down type passive avoidance and place learning in a water-maze. Pretraining administration of aggregated beta 25-35 peptide induced dose-dependent decreases in both alternation behaviour and passive avoidance, at doses of 3 and 9 nmol/mouse. A reduced but still significant impairment was observed when the peptide was not aggregated, or 'aged', by preincubation for 4 days at 37 degrees C. The beta 1-28 peptide, at 3 nmol/mouse, also induced a marked decrease in step-down latency. Posttraining, but not preretention, administration of beta 25-35 peptide also significantly impaired learning. The beneficial effects of cholinergic agents on beta 25-35-induced amnesia was examined using the cholinesterase inhibitor tacrine (THA, 1.3 and 4.3 mumol/kg i.p.) and the nicotinic receptor agonist (-)-nicotine (NIC, 0.06 and 0.2 mumol/kg i.p.). Both drugs induced a dose-dependent abrogation of the beta 25-35-induced decreases in alternation behaviour and passive avoidance. Furthermore, THA, at 1.3 mumol/kg, and NIC, at 0.2 mumol/kg, also reversed the beta 25-35-induced impairment of place learning and retention in the water-maze. Histological examination of Cresyl violet-stained brain sections indicated a moderate but significant cell loss within the frontoparietal cortex and the hippocampal formation of mice treated with aged beta 25-35 peptide (9 nmol). Examination of Congo red-stained sections in the same animals

  2. Evidence for membrane thinning effect as the mechanism for peptide-induced pore formation.

    PubMed

    Chen, Fang-Yu; Lee, Ming-Tao; Huang, Huey W

    2003-06-01

    Antimicrobial peptides have two binding states in a lipid bilayer, a surface state S and a pore-forming state I. The transition from the S state to the I state has a sigmoidal peptide-concentration dependence indicating cooperativity in the peptide-membrane interactions. In a previous paper, we reported the transition of alamethicin measured in three bilayer conditions. The data were explained by a free energy that took into account the membrane thinning effect induced by the peptides. In this paper, the full implications of the free energy were tested by including another type of peptide, melittin, that forms toroidal pores, instead of barrel-stave pores as in the case of alamethicin. The S-to-I transitions were measured by oriented circular dichroism. The membrane thinning effect was measured by x-ray diffraction. All data were in good agreement with the theory, indicating that the membrane thinning effect is a plausible mechanism for the peptide-induced pore formations.

  3. Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide.

    PubMed

    Hanson, Joshua M; Gettel, Douglas L; Tabaei, Seyed R; Jackman, Joshua; Kim, Min Chul; Sasaki, Darryl Y; Groves, Jay T; Liedberg, Bo; Cho, Nam-Joon; Parikh, Atul N

    2016-01-01

    The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of ∼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity.

  4. Potassium ions are more effective than sodium ions in salt induced peptide formation.

    PubMed

    Dubina, Michael V; Vyazmin, Sergey Yu; Boitsov, Vitali M; Nikolaev, Eugene N; Popov, Igor A; Kononikhin, Alexey S; Eliseev, Igor E; Natochin, Yuri V

    2013-04-01

    Prebiotic peptide formation under aqueous conditions in the presence of metal ions is one of the plausible triggers of the emergence of life. The salt-induced peptide formation reaction has been suggested as being prebiotically relevant and was examined for the formation of peptides in NaCl solutions. In previous work we have argued that the first protocell could have emerged in KCl solution. Using HPLC-MS/MS analysis, we found that K(+) is more than an order of magnitude more effective in the L-glutamic acid oligomerization with 1,1'-carbonyldiimidazole in aqueous solutions than the same concentration of Na(+), which is consistent with the diffusion theory calculations. We anticipate that prebiotic peptides could have formed with K(+) as the driving force, not Na(+), as commonly believed. PMID:23536046

  5. Potassium Ions are More Effective than Sodium Ions in Salt Induced Peptide Formation

    NASA Astrophysics Data System (ADS)

    Dubina, Michael V.; Vyazmin, Sergey Yu.; Boitsov, Vitali M.; Nikolaev, Eugene N.; Popov, Igor A.; Kononikhin, Alexey S.; Eliseev, Igor E.; Natochin, Yuri V.

    2013-04-01

    Prebiotic peptide formation under aqueous conditions in the presence of metal ions is one of the plausible triggers of the emergence of life. The salt-induced peptide formation reaction has been suggested as being prebiotically relevant and was examined for the formation of peptides in NaCl solutions. In previous work we have argued that the first protocell could have emerged in KCl solution. Using HPLC-MS/MS analysis, we found that K+ is more than an order of magnitude more effective in the L-glutamic acid oligomerization with 1,1'-carbonyldiimidazole in aqueous solutions than the same concentration of Na+, which is consistent with the diffusion theory calculations. We anticipate that prebiotic peptides could have formed with K+ as the driving force, not Na+, as commonly believed.

  6. Investigations on the mechanism of the salt-induced peptide formation

    NASA Astrophysics Data System (ADS)

    Schwendinger, Michael G.; Rode, Bernd M.

    1992-11-01

    The applicability of the salt-induced peptide formation in aqueous solution — the simplest model so far for peptide synthesis under primitive earth conditions — is demonstrated for valine as another amino acid, and the formation of mixed peptides in systems containing glycine, alanine and valine is investigated. The dominant dipeptides formed are Gly-Gly, Gly-Ala and Gly-Val, at longer reaction times sequence inversion produces Ala-Gly and, considerably slower, Val-Gly. Ala-Ala is also produced and the relative amounts of the diastereomers prove the high conservation of optical purity of the original amino acids over a considerable time. The results lead to some further conclusions about the reaction mechanism and the possible dominance of peptide sequences in primordial dipeptides.

  7. Established theory of radiation-induced decay is not generalizable to Bolton-Hunter labeled peptides.

    PubMed

    Doran, Amanda C; Wan, Yieh-Ping; Kopin, Alan S; Beinborn, Martin

    2003-05-01

    Peptide hormones radiolabeled with 125I are widely used for the pharmacological characterization of cognate receptors. As a prerequisite for calculating ligand affinities from competition binding assays, and for estimating receptor densities from such studies, it is necessary to know the concentration of bioactive radioligand that is used in respective experiments. It has been demonstrated previously that radioiodinated peptides undergo decay catastrophe, i.e. disintegration of the radioactive label leads to the concomitant destruction of the carrier peptide. Here, we demonstrate that decay catastrophe does not apply to two peptide hormones that are iodinated by Bolton-Hunter conjugation: cholecystokinin octapeptide and glucagon-like peptide 2. The function of aged samples of these radioligands at corresponding recombinantly expressed receptors was assessed by measuring ligand-induced inositol phosphate production or generation of cyclic AMP, respectively. Both of the tested compounds, although predicted by decay catastrophe to contain little or subthreshold remaining bioactivity, stimulated an unexpectedly high level of receptor-mediated second messenger signaling. Quantitative comparison of observed functions with those of corresponding unlabeled peptides suggested that the bioactivity of each radioligand had been largely conserved despite the radioactive decay of the iodine label. Consistent with an apparent absence of decay catastrophe, we noted that the specific radioactivity, when determined immediately following peptide iodination, was close to the theoretical maximum but exponentially decreased over time. These findings raise the possibility that attachment of a Bolton-Hunter conjugate may shield labeled peptides from radiation-induced damage, a scenario that should be considered when performing radioligand binding experiments.

  8. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    SciTech Connect

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

  9. Inhibitory Effects of Antimicrobial Peptides on Lipopolysaccharide-Induced Inflammation

    PubMed Central

    Sun, Yue; Shang, Dejing

    2015-01-01

    Antimicrobial peptides (AMPs) are usually small molecule peptides, which display broad-spectrum antimicrobial activity, high efficiency, and stability. For the multiple-antibiotic-resistant strains, AMPs play a significant role in the development of novel antibiotics because of their broad-spectrum antimicrobial activities and specific antimicrobial mechanism. Besides broad-spectrum antibacterial activity, AMPs also have anti-inflammatory activity. The neutralization of lipopolysaccharides (LPS) plays a key role in anti-inflammatory action of AMPs. On the one hand, AMPs can readily penetrate the cell wall barrier by neutralizing LPS to remove Gram-negative bacteria that can lead to infection. On the contrary, AMPs can also inhibit the production of biological inflammatory cytokines to reduce the inflammatory response through neutralizing circulating LPS. In addition, AMPs also modulate the host immune system by chemotaxis of leukocytes, to promote immune cell proliferation, epithelialization, and angiogenesis and thus play a protective role. This review summarizes some recent researches about anti-inflammatory AMPs, with a focus on the interaction of AMPs and LPS on the past decade. PMID:26612970

  10. Inhibition of Hepatocyte Apoptosis: An Important Mechanism of Corn Peptides Attenuating Liver Injury Induced by Ethanol.

    PubMed

    Ma, Zhili; Hou, Tao; Shi, Wen; Liu, Weiwei; He, Hui

    2015-09-11

    In this study, the effects of mixed corn peptides and synthetic pentapeptide (QLLPF) on hepatocyte apoptosis induced by ethanol were investigated in vivo. QLLPF, was previously characterized from corn protein hydrolysis, which had been shown to exert good facilitating alcohol metabolism activity. Mice were pre-treated with the mixed corn peptides and the pentapeptide for 1 week and then treated with ethanol. After treatment of three weeks, the biochemical indices and the key ethanol metabolizing enzymes, the serum TNF-α, liver TGF-β1 concentrations and the protein expressions related to apoptosis were determined. We found that the Bcl-2, Bax and cytochrome c expressions in the intrinsic pathway and the Fas, FasL and NF-κB expressions in the extrinsic pathway together with higher TNF-α and TGF-β1 concentrations were reversed compared with the model group by both the mixed corn peptides and the pentapeptide. The activation of caspase3 was also suppressed. Additionally, apoptosis was further confirmed with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the TUNEL assay demonstrated peptides suppressed hepatocyte apoptosis. Our results suggest that apoptosis induced by ethanol is alleviated in response to the treatment of corn peptides, potentially due to reversing the related protein expression.

  11. Peptide-lipid interactions of the stress-response peptide TisB that induces bacterial persistence.

    PubMed

    Steinbrecher, Thomas; Prock, Sebastian; Reichert, Johannes; Wadhwani, Parvesh; Zimpfer, Benjamin; Bürck, Jochen; Berditsch, Marina; Elstner, Marcus; Ulrich, Anne S

    2012-10-01

    The bacterial stress-response peptide TisB in Escherichia coli has been suggested to dissipate the transmembrane potential, such that the depletion of ATP levels induces the formation of dormant persister cells which can eventually form biofilms. We studied the structure and membrane interactions of TisB to find out whether it forms pores or other proton-selective channels. Circular dichroism revealed an amphiphilic α-helical structure when reconstituted in lipid vesicles, and oriented circular dichroism showed that the helix assumes a transmembrane alignment. The addition of TisB to dye-loaded vesicles caused leakage only at very high peptide concentration, notably with a Hill coefficient of 2, which suggests that dimers must be involved. Coarse-grained molecular dynamics simulations showed that membrane binding of monomeric TisB is rapid and spontaneous, and transmembrane insertion is energetically feasible. When TisB oligomers are assembled as transmembrane pores, these channels collapse during the simulations, but transmembrane dimers are found to be stable. Given the pattern of charges on the amphiphilic TisB helix, we postulate that antiparallel dimers could be assembled via a ladder of salt bridges. This electrostatic charge-zipper could enable protons to pass along a wire of trapped water molecules across the hydrophobic membrane. PMID:23062338

  12. Peptide-Lipid Interactions of the Stress-Response Peptide TisB That Induces Bacterial Persistence

    PubMed Central

    Steinbrecher, Thomas; Prock, Sebastian; Reichert, Johannes; Wadhwani, Parvesh; Zimpfer, Benjamin; Bürck, Jochen; Berditsch, Marina; Elstner, Marcus; Ulrich, Anne S.

    2012-01-01

    The bacterial stress-response peptide TisB in Escherichia coli has been suggested to dissipate the transmembrane potential, such that the depletion of ATP levels induces the formation of dormant persister cells which can eventually form biofilms. We studied the structure and membrane interactions of TisB to find out whether it forms pores or other proton-selective channels. Circular dichroism revealed an amphiphilic α-helical structure when reconstituted in lipid vesicles, and oriented circular dichroism showed that the helix assumes a transmembrane alignment. The addition of TisB to dye-loaded vesicles caused leakage only at very high peptide concentration, notably with a Hill coefficient of 2, which suggests that dimers must be involved. Coarse-grained molecular dynamics simulations showed that membrane binding of monomeric TisB is rapid and spontaneous, and transmembrane insertion is energetically feasible. When TisB oligomers are assembled as transmembrane pores, these channels collapse during the simulations, but transmembrane dimers are found to be stable. Given the pattern of charges on the amphiphilic TisB helix, we postulate that antiparallel dimers could be assembled via a ladder of salt bridges. This electrostatic charge-zipper could enable protons to pass along a wire of trapped water molecules across the hydrophobic membrane. PMID:23062338

  13. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

    PubMed Central

    Bahou, W F; Coller, B S; Potter, C L; Norton, K J; Kutok, J L; Goligorsky, M S

    1993-01-01

    A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin-induced

  14. Nuclear Localization Signal Peptides Induce Molecular Delivery along Microtubules

    PubMed Central

    Salman, Hanna; Abu-Arish, Asmahan; Oliel, Shachar; Loyter, Avraham; Klafter, Joseph; Granek, Rony; Elbaum, Michael

    2005-01-01

    Many essential processes in eukaryotic cells depend on regulated molecular exchange between its two major compartments, the cytoplasm and the nucleus. In general, nuclear import of macromolecular complexes is dependent on specific peptide signals and their recognition by receptors that mediate translocation through the nuclear pores. Here we address the question of how protein products bearing such nuclear localization signals arrive at the nuclear membrane before import, i.e., by simple diffusion or perhaps with assistance of cytoskeletal elements or cytoskeleton-associated motor proteins. Using direct single-particle tracking and detailed statistical analysis, we show that the presence of nuclear localization signals invokes active transport along microtubules in a cell-free Xenopus egg extract. Chemical and antibody inhibition of minus-end directed cytoplasmic dynein blocks this active movement. In the intact cell, where microtubules project radially from the centrosome, such an interaction would effectively deliver nuclear-targeted cargo to the nuclear envelope in preparation for import. PMID:16040740

  15. Peptide-induced emesis in dogs: possible relevance to radiation-induced emesis. Final report Oct 80-Sep 81

    SciTech Connect

    Carpenter, D.O.

    1982-09-01

    Results of earlier investigators indicate that radioemesis is mediated by some humoral agent(s). Peptides are likely candiates since they exert a number of biological effects and are released from storage sites by various stimuli, including radiation. Peptides at various concentrations were injected singly intravenously into conscious dogs, and the dog's emetic response was observed. Of the peptides tested, neurotensin, angiotensin II, vasopressin, oxytocin, and TRH produced consistent emetic responses. Inhibition of drug-induced emesis was studied both centrally (chlorpromazine) and peripherally (domperidone) acting dopamine antagonists. Results indicate inhibition by chlorpromazine, which crosses the blood brain barrier, but only partial blockade by domperidone, which does not cross the blood brain barrier. Preliminary studies were conducted attempting to characterize types of receptors on area postrema neurons. Single-cell recordings from these neurons, challenged by iontophoretic administration of various neurotransmitters, show stimulation by glutamic acid and serotonin and inhibiiton by norepinephrine.

  16. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides.

    PubMed

    Grage, Stephan L; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state (2)H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of

  17. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides

    PubMed Central

    Grage, Stephan L.; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S.

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state 2H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of a

  18. Vv-AMP1, a ripening induced peptide from Vitis vinifera shows strong antifungal activity

    PubMed Central

    de Beer, Abré; Vivier, Melané A

    2008-01-01

    Background Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors. Results A berry specific cDNA sequence designated Vv-AMP1, Vitis vinifera antimicrobial peptide 1, was isolated from Vitis vinifera. Vv-AMP1 encodes for a 77 amino acid peptide that shows sequence homology to the family of plant defensins. Vv-AMP1 is expressed in a tissue specific, developmentally regulated manner, being only expressed in berry tissue at the onset of berry ripening and onwards. Treatment of leaf and berry tissue with biotic or abiotic factors did not lead to increased expression of Vv-AMP1 under the conditions tested. The predicted signal peptide of Vv-AMP1, fused to the green fluorescent protein (GFP), showed that the signal peptide allowed accumulation of its product in the apoplast. Vv-AMP1 peptide, produced in Escherichia coli, had a molecular mass of 5.495 kDa as determined by mass spectrometry. Recombinant Vv-AMP1 was extremely heat-stable and showed strong antifungal activity against a broad spectrum of plant pathogenic fungi, with very high levels of activity against the wilting disease causing pathogens Fusarium oxysporum and Verticillium dahliae. The Vv-AMP1 peptide did not induce morphological changes on the treated fungal hyphae, but instead strongly inhibited hyphal elongation. A propidium iodide uptake assay suggested that the inhibitory activity of Vv-AMP1 might be associated with altering the membrane permeability of the fungal

  19. 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.

  20. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

    PubMed Central

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-01-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  1. Development of a novel therapy for Lipo-oligosaccharide-induced experimental neuritis: use of peptide glycomimics

    PubMed Central

    Usuki, Seigo; Taguchi, Kyoji; Gu, Yi-Hua; Thompson, Stuart A.; Yu, Robert K.

    2010-01-01

    Recent etiological studies have revealed that molecular mimicry between the lipo-oligosaccharide (LOS) component of Campylobacter jejuni and gangliosides of peripheral nervous system plays an important role in the pathogenesis of Guillain-Barré syndrome (GBS). Previously, we demonstrated GD3 ganglioside molecular mimicry in a model of GBS in Lewis rats by sensitization with GD3-like LOS (LOSGD3) from C. jejuni. Since the neuropathophysiological consequences were due largely to the anti-GD3-like antibodies, we subsequently focused our effort upon eliminating the pathogenic antibodies using several strategies to mimic GD3 in this model. Here, we have validated this strategy by the use of peptide glycomimics based on epitopic mimicry between carbohydrates and peptides. We treated rats by i.p. administration of phage-displayed GD3-like peptides. One GD3-like peptide (PGD3-4; RHAYRSMAEWGFLYS) induced in treated rats a remarkable restoration of motor nerve functions, as evidenced by improved histopathology, rotarod performance, and motor nerve conduction velocity. PGD3-4 effectively decreased the titer of anti-GD3/anti-LOSGD3 antibodies and ameliorated peripheral nerve dysfunction in the sera of treated rats. The data suggest that peptide glycomimics of ganglioside may be potential powerful reagents for therapeutic intervention in GBS by neutralizing specific pathogenic anti-ganglioside antibodies. PMID:20132479

  2. The VGF-derived peptide TLQP-21 contributes to inflammatory and nerve injury-induced hypersensitivity

    PubMed Central

    Fairbanks, Carolyn A.; Peterson, Cristina D.; Speltz, Rebecca H.; Riedl, Maureen S.; Kitto, Kelley F.; Dykstra, Jaclyn A.; Braun, Patrick D.; Sadahiro, Masato; Salton, Stephen R.; Vulchanova, Lucy

    2014-01-01

    VGF (non-acronymic) is a granin-like protein that is packaged and proteolytically processed within the regulated secretory pathway. VGF and peptides derived from its processing have been implicated in neuroplasticity associated with learning, memory, depression, and chronic pain. In sensory neurons, VGF is rapidly increased following peripheral nerve injury and inflammation. Several bioactive peptides generated from the C-terminus of VGF have pro-nociceptive spinal effects. The goal of the present study was to examine the spinal effects of the peptide TLQP-21 and determine whether it participates in spinal mechanisms of persistent pain. Application of exogenous TLQP-21 induced dose-dependent thermal hyperalgesia in the warm water immersion tail withdrawal test. This hyperalgesia was inhibited by a p38 MAPK inhibitor as well as inhibitors of cyclooxygenase and lipoxygenase. We used immunoneutralization of TLQP-21 to determine the function of the endogenous peptide in mechanisms underlying persistent pain. In mice injected intradermally with complete Freund’s adjuvant, intrathecal treatment with anti-TLQP21 IgG immediately prior to or 5 h after induction of inflammation dose-dependently inhibited tactile hypersensitivity and thermal hyperalgesia. Intrathecal anti-TL21 administration also attenuated the development and maintenance of tactile hypersensitivity in the spared nerve injury model of neuropathic pain. These results provide evidence that endogenous TLQP-21 peptide contributes to the mechanisms of spinal neuroplasticity after inflammation and nerve injury. PMID:24657450

  3. A health food high-peptide meal alleviates immunosuppression induced by hydrocortisone and cyclophosphamide in mice.

    PubMed

    Yingjian, Lv; Junming, Huang; Min, Cai; Chenyue, Li; Dachao, Zhang; Yuanhua, Hu; Zhi, Li

    2013-09-01

    This study set out to determine the immunomodulatory effects of a health food high-peptide meal in immunodeficient mice that was induced by either hydrocortisone (HY) or cyclophosphamide (CP). Five separate trials were performed in which animals were randomly divided into 5 groups of 12 mice for each experimental trial. Group 1 served as the vehicle control. Animals assigned to groups 3-5 (dose groups), were each administered once daily with 1.67, 3.33 or 6.67 g kg(-1) body weight of a high-peptide meal, respectively, for 30 consecutive days. Animals from groups 2 to 5 that were included in trials 1 to 4, each received an intramuscular administration of HY at 40 mg kg(-1) body weight on days 22, 24, 26, 28, 30. Animals from groups 2-5, in trial 5, each received an intraperitoneal administration of CP at 50 mg kg(-1) body weight, on days 26 and 27. On day 31, all groups of mice were differentially screened for immunomodulatory activity following the conclusion of the above experiments. In HY-treated mice, the high-peptide meal accelerated the recovery of the phagocytic function of both macrophages and the reticuloendothelial system, and restored NK cell activity. In CP-treated mice, the high-peptide meal promoted a humoral immune response to sheep red blood cells (SRBCs). These results demonstrated the immunomodulatory effects of a high-peptide meal.

  4. Synergistic effects of C-peptide and insulin on low O2-induced ATP release from human erythrocytes

    PubMed Central

    Stephenson, Alan H.; Ellsworth, Mary L.; Sprague, Randy S.

    2013-01-01

    Erythrocytes participate in the matching of oxygen (O2) delivery with local need in skeletal muscle via the release of O2 and the vasodilator, ATP. It was reported that a concentration of insulin found in humans with insulin resistance inhibits low O2-induced ATP release. However, in vivo, insulin is coreleased with connecting peptide (C-peptide) at equimolar concentrations, but because of the shorter insulin half-life, the peptides circulate at ratios of C-peptide to insulin ranging from 1:1 to 6:1. Here, we investigate the hypothesis that C-peptide and insulin work synergistically to maintain low O2-induced ATP release from human erythrocytes. Using a thin-film tonometer to alter O2 tension, we determined that either C-peptide or insulin alone inhibits low O2-induced ATP release in a concentration-dependent manner; however, coadministration of the peptides at a 1:1 ratio does not (n = 5; P < 0.05). Because this ratio of C-peptide to insulin is not present in vivo for extended periods, we also investigated the effect of additional physiological ratios on ATP release. In the presence of insulin concentrations that would be found in fasting humans (0.05 nM), C-peptide to insulin ratios of 4:1 and 6:1 did not adversely affect low O2-induced ATP release. However, at a concentration of insulin found in the peripheral circulation of humans under postprandial conditions (0.5 nM), a ratio of C-peptide to insulin of 6:1 inhibited low O2-induced ATP release (n = 5). These findings demonstrate a heretofore unrecognized synergism between C-peptide and insulin that could have physiological importance in the regulation of perfusion distribution in skeletal muscle. PMID:24089376

  5. Peptide-induced membrane curvature in edge-stabilized open bilayers: A theoretical and molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Pannuzzo, Martina; Raudino, Antonio; Böckmann, Rainer A.

    2014-07-01

    Peptide- or protein-induced curvatures of lipid membranes may be studied in molecular dynamics (MD) simulations. In these, membranes are usually modeled as infinitely extended bilayers by using periodic boundary conditions. However, the enforced periodicity results in an underestimation of the bending power of peptides, unless the patch size is much larger than the induced curvature radii. In this letter, we propose a novel approach to evaluate the bending power of a given distribution and/or density of peptides based on the use of flat open-edged lipid patches. To ensure long-lived metastable structures, the patch rim is stabilized in MD simulations by a local enrichment with short-chain lipids. By combining the theory of continuum elastic media with MD simulations, we prove that open-edged patches evolve from a planar state to a closed vesicle, with a transition rate that strongly depends on the concentration of lipid soluble peptides. For close-to-critical values for the patch size and edge energy, the response to even small changes in peptide concentration adopts a transition-like behavior (buckling instability). The usage of open-edged membrane patches amplifies the bending power of peptides, thereby enabling the analysis of the structural properties of membrane-peptide systems. We applied the presented method to investigate the curvature induced by aggregating β -amyloid peptides, unraveling a strong sensitivity of membrane deformation to the peptide concentration.

  6. Anti-apoptotic peptides protect against radiation-induced cell death

    SciTech Connect

    McConnell, Kevin W.; Muenzer, Jared T.; Chang, Kathy C.; Davis, Chris G.; McDunn, Jonathan E.; Coopersmith, Craig M.; Hilliard, Carolyn A.; Hotchkiss, Richard S.; Grigsby, Perry W.; Hunt, Clayton R. . E-mail: chunt@radonc.wustl.edu

    2007-04-06

    The risk of terrorist attacks utilizing either nuclear or radiological weapons has raised concerns about the current lack of effective radioprotectants. Here it is demonstrated that the BH4 peptide domain of the anti-apoptotic protein Bcl-xL can be delivered to cells by covalent attachment to the TAT peptide transduction domain (TAT-BH4) and provide protection in vitro and in vivo from radiation-induced apoptotic cell death. Isolated human lymphocytes treated with TAT-BH4 were protected against apoptosis following exposure to 15 Gy radiation. In mice exposed to 5 Gy radiation, TAT-BH4 treatment protected splenocytes and thymocytes from radiation-induced apoptotic cell death. Most importantly, in vivo radiation protection was observed in mice whether TAT-BH4 treatment was given prior to or after irradiation. Thus, by targeting steps within the apoptosis signaling pathway it is possible to develop post-exposure treatments to protect radio-sensitive tissues.

  7. Hydroxymethyl-phytochelatins [(gamma-glutamylcysteine)n-serine] are metal-induced peptides of the Poaceae.

    PubMed

    Klapheck, S; Fliegner, W; Zimmer, I

    1994-04-01

    Exposure of several species of the family Poaceae to cadmium results in the formation of metal-induced peptides of the general structure (gamma-Glu-Cys)n-Ser (n=2-4). They are assumed to be formed from hydroxymethyl-glutathione (gamma-Glu-Cys-Ser) and are termed hydroxymethyl-phytochelatins (hm-PCs) in analogy to the homo-phytochelatins [(gamma-Glu-Cys)n-beta-Ala], discovered in legumes, and the phytochelatins [PCs, (gamma-Glu-Cys)n-Gly] found in most other plants and many fungi. The hm-PCs were isolated from the roots of cadmium-exposed rice (Oryza sativa L. cv Strella), and their structure was confirmed by amino acid analysis after total and enzymic hydrolysis and by tandem mass spectrometry. The hm-PCs probably play a significant role in heavy metal detoxication in rice. In addition to this new form of gamma-Glu-Cys (gamma EC) peptide, PCs and gamma EC peptides without C-terminal Ser or Gly are found. All gamma EC peptides are synthesized without delay after incubation of rice plants in 100 microM CdCl2 in the roots as well as in the shoots. Incubation times exceeding 24 h or higher concentrations of cadmium result in a selective enrichment of gamma EC peptides with higher chain length and an increased ratio of PCs to hm-PCs. gamma EC peptide synthesis is accompanied by a decrease of the glutathione content and an increase of the hydroxymethyl-glutathione content in roots and shoots of rice plants. PMID:8016264

  8. The Combination of Salt Induced Peptide Formation Reaction and Clay Catalysis: A Way to Higher Peptides under Primitive Earth Conditions

    NASA Astrophysics Data System (ADS)

    Rode, Bernd M.; Son, Hoang L.; Suwannachot, Yuttana; Bujdak, Juraj

    1999-05-01

    Two reactions with suggested prebiotic relevance for peptide evolution, the saltinduced peptide formation reaction and the peptide chain elongation/stabilization on clay minerals have been combined in experimental series starting from dipeptides and dipeptide/amino acid mixtures. The results show that both reactions can take place simultaneously in the same reaction environment and that the presence of mineral catalysts favours the formation of higher oligopeptides. These findings lend further support to the relevance of these reactions for peptide evolution on the primitive earth. The detailed effects of the specific clay mineral depend both on the nature of the mineral and the reactants in solution.

  9. Heterotypic passive protection induced by synthetic peptides corresponding to VP7 and VP4 of bovine rotavirus.

    PubMed Central

    Ijaz, M K; Attah-Poku, S K; Redmond, M J; Parker, M D; Sabara, M I; Frenchick, P; Babiuk, L A

    1991-01-01

    We have evaluated the potential of two peptides derived from highly conserved regions of rotavirus outer capsid proteins (VP7 and VP4) to act as a rotavirus vaccine. The capacity of peptides coupled to rotavirus VP6 spherical particles to provide passive protection in a murine model was compared with the protection induced by peptide-keyhole limpet hemocyanin (KLH) conjugates. Female mice were immunized a total of three times before and during pregnancy. Suckling mouse pups were challenged at 7 days of age with either homologous or heterologous rotavirus serotypes. The efficacy of vaccination was determined by analyzing the clinical symptoms and measuring xylose adsorption in the intestine. In this model the VP4 peptide-VP6 conjugate provided protection equal to that obtained using bovine rotavirus (BRV) as the immunogen. The VP7 peptide-VP6 conjugate provided slightly less protection than the VP4 peptide-VP6 conjugate. A mixture of the VP4 peptide-VP6 and VP7 peptide-VP6 conjugates provided better heterologous protection than immunization with BRV. In contrast, KLH-conjugated peptides provided only partial protection. The significance of a synthetic-peptide-based rotavirus vaccine in the prevention of rotavirus infections is discussed. Images PMID:1851871

  10. Granulosa Cell Apoptosis Induced by a Novel FSH Binding Inhibitory Peptide From Human Ovarian Follicular Fluid

    PubMed Central

    Chitnis, Swati S.; Navlakhe, Rajshri M.; Shinde, Gayatri C.; Barve, Sharmila J.; D'Souza, Serena; Mahale, Smita D.; Nandedkar, Tarala D.

    2008-01-01

    Pituitary gonadotropins, follicle-stimulating hormone and luteinizing hormone, are the key regulators of ovarian folliculogenesis; these are known to be directly or indirectly modulated by many intraovarian factors. Our group has identified and studied one such novel peptide from human ovarian follicular fluid. Its partial N-terminal eight amino acid sequence has been deduced, referred to as octapeptide (OP). OP induces follicular atresia in mice and interferes with normal ovarian function in non-human primates, this action being similar to the native peptide. Thus, in this study, an attempt has been made to elucidate the mechanism of action of the synthetic OP by studying the pathway of follicular atresia in mouse ovary. Changes in granulosa cells were studied using various apoptotic markers by flow cytometry and immunohistochemistry. An increase in apoptotic cell population in atretic- and peptide-treated groups was observed compared with normal controls. Interestingly, both these groups exhibited differences in the apoptotic pathway. Results showed that the mitochondrial pathway was predominant in the atretic group, whereas the Fas-FasL pathway was predominant in the peptide-treated groups. The ultrastructural study also showed apoptotic changes in the OP-treated and atretic groups; the pattern of apoptosis differed at the subcellular level. (J Histochem Cytochem 56:961–968, 2008) PMID:18645207

  11. Identification of salt-inducible peptide with putative kinase activity in halophilic bacterium Virgibacillus halodenitrificans.

    PubMed

    Rafiee, Mahmoud-Reza; Sokhansanj, Ashrafaddin; Yoosefi, Mitra; Naghizadeh, Mohammad-Ali

    2007-09-01

    Strain XII, a moderately halophilic bacterium, expressed a peptide in response to saline media. This peptide was designated as salt-inducible factor (Sif-A). The purpose of this study is to describe Sif-A, which might be involved in the osmoresistance mechanism of strain XII. The complete sequence of sif-A was determined using PCR. sif-A codes for a polypeptide of 20.518 kDa. The polypeptide has a putative signal peptide of 27 amino acids (2.667 kDa) preceding the mature protein (17.869 kDa). Motif analysis of the deduced amino acid sequence indicated that there is a p-loop NTPase domain on the C-terminal of the peptide, which might correlate with its function. The sequence of the 16S rRNA gene was analyzed phylogenetically to classify strain XII. This organism was found to have the closest association with Virgibacillus halodenitrificans, which was proven by its phenotypic characteristics. PMID:17964480

  12. Acaloleptins A: inducible antibacterial peptides from larvae of the beetle, Acalolepta luxuriosa.

    PubMed

    Imamura, M; Wada, S; Koizumi, N; Kadotani, T; Yaoi, K; Sato, R; Iwahana, H

    1999-01-01

    We purified and characterized three structurally related antibacterial peptides with a molecular mass of 8 kDa (acaloleptins A1, A2, and A3) from the hemolymph of immunized larvae of the Udo longicorn beetle, Acalolepta luxuriosa. These peptides have the same 6 N-terminal amino acid residues and show potent antibacterial activity against some Gram-negative bacteria. The three peptides are thought to be isoforms. Reverse phase HPLC analysis of the hemolymph of immunized and naive larvae showed that acaloleptins A1, A2, and A3 were inducible and suggested that all three peptides were produced in a single insect. We determined the complete amino acid sequence of acaloleptin A1: Acaloleptin A1 consists of 71 amino acid residues and shares significant sequence similarity with coleoptericin and holotricin 2, which were isolated from other coleopteran insects. Furthermore, the 29 C-terminal residues of acaloleptin A1 had 40% identity with the 30 C-terminal residues of hymenoptaecin found in honeybees. Arch. Insect Biochem. PMID:10077828

  13. Evidence that a BCR-ABL fusion peptide does not induce lymphocyte proliferation or cytokine production in vitro.

    PubMed

    Abu-Eisha, Hazem M; Butt, Nauman M; Clark, Richard E; Christmas, Stephen E

    2007-12-01

    The BCR-ABL fusion protein is characteristic of chronic myeloid leukaemia and may be an effective tumour-specific antigen. CD8+ T cell responses to BCR-ABL fusion peptides have been reported in normal subjects and CML patients but CD4+ T cell responses have been less well characterised. Here, the 23-mer e14a2 fusion peptide VHSATGFKQSSKALQRPVASDFE has been used to stimulate T cell responses. Most normal subjects and CML patients showed no proliferative responses to this peptide, with stimulation indices not significantly greater than 1.0. Following a second stimulation with the same peptide, small proliferative responses were obtained in normal subjects but not CML patients. These responses were not improved following a third stimulation with 23-mer peptide, nor by using mature autologous dendritic cells to present the peptide. Intracellular interferon-gamma production by CD4+ T cells was also not induced by the 23-mer e14a2 peptide. Hence, this e14a2 peptide does not stimulate CD4+ T cell proliferation in vitro in most normal subjects or CML patients. The precise sequence of amino acids may be critical in defining immunogenicity for CD4+ T cell responses against BCR-ABL peptides.

  14. Antimicrobial peptide protonectin disturbs the membrane integrity and induces ROS production in yeast cells.

    PubMed

    Wang, Kairong; Dang, Wen; Xie, Junqiu; Zhu, Ranran; Sun, Mengyang; Jia, Fengjing; Zhao, Yanyan; An, Xiaoping; Qiu, Shuai; Li, Xiaoyuan; Ma, Zelin; Yan, Wenjin; Wang, Rui

    2015-10-01

    Candidiasis is often observed in immunocompromised patients and is the 4th most common cause of bloodstream infections. However, antifungals are limited, so novel antifungal agents are urgently needed. Antimicrobial peptides (AMPs) are considered as potential alternatives of conventional antibiotics. In the present study, antimicrobial peptide protonectin was chemically synthesized and its antifungal activity and mode of action were studied. Our results showed that protonectin has potent antifungal activity and fungicidal activity against the tested fungi cells. Its action mode involved the disruption of the membrane integrity and the inducing of the production of cellular ROS. Furthermore, protonectin could inhibit the formation of biofilm and kill the adherent fungi cells. In conclusion, with the increase of fungal infection, protonectin may offer a new strategy and be considered as a potential therapeutic agent against fungal disease. PMID:26209560

  15. Thioredoxin-mimetic peptides (TXM) reverse auranofin induced apoptosis and restore insulin secretion in insulinoma cells.

    PubMed

    Cohen-Kutner, Moshe; Khomsky, Lena; Trus, Michael; Aisner, Yonatan; Niv, Masha Y; Benhar, Moran; Atlas, Daphne

    2013-04-01

    The thioredoxin reductase/thioredoxin system (TrxR/Trx1) plays a major role in protecting cells from oxidative stress. Disruption of the TrxR-Trx1 system keeps Trx1 in the oxidized state leading to cell death through activation of the ASK1-Trx1 apoptotic pathway. The potential mechanism and ability of tri- and tetra-oligopeptides derived from the canonical -CxxC- motif of the Trx1-active site to mimic and enhance Trx1 cellular activity was examined. The Trx mimetics peptides (TXM) protected insulinoma INS 832/13 cells from oxidative stress induced by selectively inhibiting TrxR with auranofin (AuF). TXM reversed the AuF-effects preventing apoptosis, and increasing cell-viability. The TXM peptides were effective in inhibiting AuF-induced MAPK, JNK and p38(MAPK) phosphorylation, in correlation with preventing caspase-3 cleavage and thereby PARP-1 dissociation. The ability to form a disulfide-bridge-like conformation was estimated from molecular dynamics simulations. The TXM peptides restored insulin secretion and displayed Trx1 denitrosylase activity. Their potency was 10-100-fold higher than redox reagents like NAC, AD4, or ascorbic acid. Unable to reverse ERK1/2 phosphorylation, TXM-CB3 (NAc-Cys-Pro-Cys amide) appeared to function in part, through inhibiting ASK1-Trx dissociation. These highly effective anti-apoptotic effects of Trx1 mimetic peptides exhibited in INS 832/13 cells could become valuable in treating adverse oxidative-stress related disorders such as diabetes. PMID:23327993

  16. Amyloid beta-peptide induces cell monolayer albumin permeability, impairs glucose transport, and induces apoptosis in vascular endothelial cells.

    PubMed

    Blanc, E M; Toborek, M; Mark, R J; Hennig, B; Mattson, M P

    1997-05-01

    Amyloid beta-peptide (A beta) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that A beta25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to A beta25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by A beta25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of A beta25-35 were specific because A beta1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of A beta1-40 aggregates and because astrocytes did not undergo similar changes after exposure to A beta25-35. Damage and death of ECs induced by A beta25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that A beta induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then A beta and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation. PMID:9109512

  17. Injection of Cocaine-Amphetamine Regulated Transcript (CART) peptide into the nucleus accumbens does not inhibit caffeine-induced locomotor activity: Implications for CART peptide mechanism.

    PubMed

    Job, Martin O

    2016-09-01

    Much evidence suggests that intra-nucleus accumbens (NAc) CART peptide (CART 55-102) injection inhibits locomotor activity (LMA) when there is an increase in the release and activity of dopamine (DA) in the NAc. However, this hypothesis has not been fully tested. One way to examine this is to determine if there is a lack of effect of intra-NAc CART peptide on LMA that does not involve increases in DA release in the NAc. Several studies have suggested that caffeine-induced LMA does not involve extracellular DA release in the NAc core. Therefore, in this study, we have examined the effect of injections of CART peptide (2.5μg) into the NAc core on the locomotor effects of caffeine in male Sprague-Dawley rats. Several LMA relevant doses of caffeine were used (0, 10, 20mg/kg i.p.), and an inverted U response curve was found as expected. We determined, in the same animals, that intra-NAc CART peptide had no effect on caffeine-induced LMA whereas it blunted cocaine-mediated LMA, as shown by other reports. We also extended a previous observation in mice by showing that at a LMA activating dose of caffeine there is no alteration of CART peptide levels in the NAc of rats. Our study supports the hypothesis that the inhibitory effects of CART peptide in the NAc may be exerted only under conditions of increased extracellular DA release and activity in this region. Our results also suggest that intra-NAc CART 55-102 does not generally inhibit increases in LMA due to all drugs, but has a more specific inhibitory effect on dopaminergic neurotransmission. PMID:27168116

  18. Apolipoprotein A-I mimetic peptide 4F blocks sphingomyelinase-induced LDL aggregation.

    PubMed

    Nguyen, Su Duy; Javanainen, Matti; Rissanen, Sami; Zhao, Hongxia; Huusko, Jenni; Kivelä, Annukka M; Ylä-Herttuala, Seppo; Navab, Mohamad; Fogelman, Alan M; Vattulainen, Ilpo; Kovanen, Petri T; Öörni, Katariina

    2015-06-01

    Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.

  19. Protective effects of luteolin against cognitive impairment induced by infusion of Aβ peptide in rats.

    PubMed

    Yu, Tian-Xia; Zhang, Peng; Guan, Yan; Wang, Min; Zhen, Ming-Qing

    2015-01-01

    Luteolin can be found in many traditional Chinese medicines, it's a falconoid compound derived from Lonicera japonica Thunb. This study aims to investigate the neuroprotective effects of luteolin against cognitive impairment induced by amyloid-β (Aβ) peptide and the underlying mechanisms in rats. The animal behavioral tests showed that luteolin could ameliorate Aβ-induced learning and memory impairment. In hippocampal tissue, the activity of choline acetyl transferase (ChAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) increased after treated by luteolin. Luteolin also reversed the increased activity of acetylcholine esterase (AchE). In hippocampi homogenate, the content of acetylcholine (Ach) increased, but malondialdehyde (MDA) reduced. Moreover, luteolin can increase Bcl-2/Bax ratio. This study demonstrated that luteolin could protect Alzheimer's disease (AD) rats against Aβ-induced cognitive impairment through regulating the cholinergic system and inhibiting oxidative injuries. The results suggesting that luteolin may have potential as a therapy for AD. PMID:26261557

  20. Applicability and Limitations of Cell-Penetrating Peptides in Noncovalent Mucosal Drug or Carrier Delivery Systems.

    PubMed

    Kamei, Noriyasu; Bech Nielsen, Ebbe Juel; Nakakubo, Takayuki; Aoyama, Yukina; Rahbek, Ulrik Lytt; Pedersen, Betty Lomstein; Takeda-Morishita, Mariko

    2016-02-01

    Our recent studies show that cell-penetrating peptides (CPPs) have potential to improve the intestinal absorption of peptide and protein drugs safely and effectively when used in the noncovalent drug--CPP approach. To clarify the applicability and limitations of this strategy, the present study examined the effects of cargo size on the absorption-stimulatory effect of CPPs. Different sizes of hydrophilic macromolecular dextran (4.4, 10, and 70 kDa) and polystyrene-based nanoparticles (20, 100, and 200 nm) were chosen as the model cargos in this study. In an in situ rat intestinal absorption study, CPPs (octaarginine, Tat, penetratin, and PenetraMax) increased the intestinal absorption of dextran, and the efficiency varied according to the molecular size of dextran. Among the CPPs, D-penetratin showed an enhancing effect even when coadministered with the largest dextran (70 kDa). By contrast, an in vitro study of Caco-2 cell uptake showed that the ability of CPPs to deliver nanoparticles into epithelial cells was dependent on their particle size and that the relatively poor internalization of 200-nm nanoparticles could be facilitated by coincubation with CPPs. These findings suggest that the intrinsic uptake properties of macromolecules and particulate cargos determine the effectiveness of their intestinal mucosal delivery using the noncovalent CPP method. PMID:26869427

  1. Stress-induced analgesia and endogenous opioid peptides: the importance of stress duration.

    PubMed

    Parikh, Drupad; Hamid, Abdul; Friedman, Theodore C; Nguyen, Khanh; Tseng, Andy; Marquez, Paul; Lutfy, Kabirullah

    2011-01-15

    Stress is known to elicit pain relief, a phenomenon referred to as stress-induced analgesia. Based on stress parameters, opioid and non-opioid intrinsic pain inhibitory systems can be activated. In the present study, we assessed whether changing the duration of stress would affect the involvement of endogenous opioids in antinociception elicited by swim in warm water (32 °C), known to be opioid-mediated. Using mice lacking beta-endorphin, enkephalins or dynorphins and their respective wild-type littermates, we assessed the role of each opioid peptide in antinociception induced by a short (3 min) vs. long (15 min) swim. Mice were tested for baseline hot plate latency, exposed to swim (3 or 15 min) in warm water (32 °C) and then tested for antinociception at 5, 15 and 30 min. Our results revealed that both swim paradigms induced significant antinociception in wild-type mice. However, the short swim failed to induce antinociception in beta-endorphin-deficient mice, illustrating that beta-endorphin is important in this form of stress-induced antinociception. On the other hand, antinociception elicited by the long swim was only slightly reduced in beta-endorphin-deficient mice despite pretreatment with naloxone, a non-selective opioid receptor antagonist, significantly attenuated the antinociception elicited by the long swim. Nevertheless, a delayed hyperalgesic response developed in mice lacking beta-endorphin following exposure to either swim paradigm. On the other hand, mice lacking enkephalins or dynorphins and their respective wild-type littermates expressed a comparable antinociceptive response and did not exhibit the delayed hyperalgesic response. Together, our results suggest that the endogenous opioid peptide beta-endorphin not only mediates antinociception induced by the short swim but also prevents the delayed hyperalgesic response elicited by either swim paradigm. PMID:21044625

  2. Stress-induced analgesia and endogenous opioid peptides: the importance of stress duration

    PubMed Central

    Parikh, Drupad; Hamid, Abdul; Friedman, Theodore C.; Nguyen, Khanh; Tseng, Andy; Marquez, Paul; Lutfy, Kabirullah

    2010-01-01

    Stress is known to elicit pain relief, a phenomenon referred to as stress-induced analgesia. Based on stress parameters, opioid and non-opioid intrinsic pain inhibitory systems can be activated. In the present study, we assessed whether changing the duration of stress would affect the involvement of endogenous opioids in antinociception elicited by swim in warm water (32°C), known to be opioid-mediated. Using mice lacking beta-endorphin, enkephalins or dynorphins and their respective wild-type littermates, we assessed the role of each opioid peptide in antinociception induced by a short (3 min) vs. long (15 min) swim. Mice were tested for baseline hot plate latency, exposed to swim (3 or 15 min) in warm water (32°C) and then tested for antinociception at 5, 15 and 30 min. Our results revealed that both swim paradigms induced significant antinociception in wild-type mice. However, the short swim failed to induce antinociception in beta-endorphin-deficient mice, illustrating that beta-endorphin is important in this form of stress-induced antinociception. On the other hand, antinociception elicited by the long swim was only slightly reduced in beta-endorphin-deficient mice despite pretreatment with naloxone, a non-selective opioid receptor antagonist, significantly attenuated the antinociception elicited by the long swim. Nevertheless, a delayed hyperalgesic response developed in mice lacking beta-endorphin following exposure to either swim paradigm. On the other hand, mice lacking enkephalins or dynorphins and their respective wild-type littermates expressed a comparable antinociceptive response and did not exhibit the delayed hyperalgesic response. Together, our results suggest that the endogenous opioid peptide beta-endorphin not only mediates antinociception induced by the short swim but also prevents the delayed hyperalgesic response elicited by either swim paradigm. PMID:21044625

  3. Novel leptin OB3 peptide-induced signaling and progression in thyroid cancers: Comparison with leptin

    PubMed Central

    Hsieh, Meng-Ti; Lai, Hsuan-Yu; Ke, Chien-Chih; Crawford, Dana R.; Lee, Oscar K.; Fu, Earl; Mousa, Shaker A.; Grasso, Patricia; Liu, Leroy F.; Chang, Heng-Yu; Tang, Heng-Yuan; Lin, Hung-Yun; Davis, Paul J.

    2016-01-01

    Obesity results in increased secretion of cytokines from adipose tissue and is a risk factor for various cancers. Leptin is largely produced by adipose tissue and cancer cells. It induces cell proliferation and may serve to induce various cancers. OB3-leptin peptide (OB3) is a new class of functional leptin peptide. However, its mitogenic effect has not been determined. In the present study, because of a close link between leptin and the hypothalamic-pituitary-thyroid axis, OB3 was compared with leptin in different thyroid cancer cells for gene expression, proliferation and invasion. Neither agent stimulated cell proliferation. Leptin stimulated cell invasion, but reduced adhesion in anaplastic thyroid cancer cells. Activated ERK1/2 and STAT3 contributed to leptin-induced invasion. In contrast, OB3 did not affect expression of genes involved in proliferation and invasion. In vivo studies in the mouse showed that leptin, but not OB3, significantly increased circulating levels of thyrotropin (TSH), a growth factor for thyroid cancer. In summary, OB3 is a derivative of leptin that importantly lacks the mitogenic effects of leptin on thyroid cancer cells. PMID:27050378

  4. Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

    SciTech Connect

    Wu, Guoqiu; Wu, Hongbin; Li, Linxian; Fan, Xiaobo; Ding, Jiaxuan; Li, Xiaofang; Xi, Tao; Shen, Zilong

    2010-04-23

    Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin's antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.

  5. Production of phytotoxic cationic α-helical antimicrobial peptides in plant cells using inducible promoters.

    PubMed

    Company, Nuri; Nadal, Anna; Ruiz, Cristina; Pla, Maria

    2014-01-01

    Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, have potent and specific activities against economically important plant pathogenic bacteria. They are also recognized as valuable therapeutics and preservatives. However, highly active BP100 derivatives are often phytotoxic when expressed at high levels as recombinant peptides in plants. Here we demonstrate that production of recombinant phytotoxic peptides in transgenic plants is possible by strictly limiting transgene expression to certain tissues and conditions, and specifically that minimization of this expression during transformation and regeneration of transgenic plants is essential to obtain viable plant biofactories. On the basis of whole-genome transcriptomic data available online, we identified the Os.hsp82 promoter that fulfilled this requirement and was highly induced in response to heat shock. Using this strategy, we generated transgenic rice lines producing moderate yields of severely phytotoxic BP100 derivatives on exposure to high temperature. In addition, a threshold for gene expression in selected tissues and stages was experimentally established, below which the corresponding promoters should be suitable for driving the expression of recombinant phytotoxic proteins in genetically modified plants. In view of the growing transcriptomics data available, this approach is of interest to assist promoter selection for specific purposes.

  6. Cold denaturation induces inversion of dipole and spin transfer in chiral peptide monolayers

    PubMed Central

    Eckshtain-Levi, Meital; Capua, Eyal; Refaely-Abramson, Sivan; Sarkar, Soumyajit; Gavrilov, Yulian; Mathew, Shinto P.; Paltiel, Yossi; Levy, Yaakov; Kronik, Leeor; Naaman, Ron

    2016-01-01

    Chirality-induced spin selectivity is a recently-discovered effect, which results in spin selectivity for electrons transmitted through chiral peptide monolayers. Here, we use this spin selectivity to probe the organization of self-assembled α-helix peptide monolayers and examine the relation between structural and spin transfer phenomena. We show that the α-helix structure of oligopeptides based on alanine and aminoisobutyric acid is transformed to a more linear one upon cooling. This process is similar to the known cold denaturation in peptides, but here the self-assembled monolayer plays the role of the solvent. The structural change results in a flip in the direction of the electrical dipole moment of the adsorbed molecules. The dipole flip is accompanied by a concomitant change in the spin that is preferred in electron transfer through the molecules, observed via a new solid-state hybrid organic–inorganic device that is based on the Hall effect, but operates with no external magnetic field or magnetic material. PMID:26916536

  7. Production of Phytotoxic Cationic α-Helical Antimicrobial Peptides in Plant Cells Using Inducible Promoters

    PubMed Central

    Company, Nuri; Nadal, Anna; Ruiz, Cristina; Pla, Maria

    2014-01-01

    Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, have potent and specific activities against economically important plant pathogenic bacteria. They are also recognized as valuable therapeutics and preservatives. However, highly active BP100 derivatives are often phytotoxic when expressed at high levels as recombinant peptides in plants. Here we demonstrate that production of recombinant phytotoxic peptides in transgenic plants is possible by strictly limiting transgene expression to certain tissues and conditions, and specifically that minimization of this expression during transformation and regeneration of transgenic plants is essential to obtain viable plant biofactories. On the basis of whole-genome transcriptomic data available online, we identified the Os.hsp82 promoter that fulfilled this requirement and was highly induced in response to heat shock. Using this strategy, we generated transgenic rice lines producing moderate yields of severely phytotoxic BP100 derivatives on exposure to high temperature. In addition, a threshold for gene expression in selected tissues and stages was experimentally established, below which the corresponding promoters should be suitable for driving the expression of recombinant phytotoxic proteins in genetically modified plants. In view of the growing transcriptomics data available, this approach is of interest to assist promoter selection for specific purposes. PMID:25387106

  8. Cold denaturation induces inversion of dipole and spin transfer in chiral peptide monolayers

    NASA Astrophysics Data System (ADS)

    Eckshtain-Levi, Meital; Capua, Eyal; Refaely-Abramson, Sivan; Sarkar, Soumyajit; Gavrilov, Yulian; Mathew, Shinto P.; Paltiel, Yossi; Levy, Yaakov; Kronik, Leeor; Naaman, Ron

    2016-02-01

    Chirality-induced spin selectivity is a recently-discovered effect, which results in spin selectivity for electrons transmitted through chiral peptide monolayers. Here, we use this spin selectivity to probe the organization of self-assembled α-helix peptide monolayers and examine the relation between structural and spin transfer phenomena. We show that the α-helix structure of oligopeptides based on alanine and aminoisobutyric acid is transformed to a more linear one upon cooling. This process is similar to the known cold denaturation in peptides, but here the self-assembled monolayer plays the role of the solvent. The structural change results in a flip in the direction of the electrical dipole moment of the adsorbed molecules. The dipole flip is accompanied by a concomitant change in the spin that is preferred in electron transfer through the molecules, observed via a new solid-state hybrid organic-inorganic device that is based on the Hall effect, but operates with no external magnetic field or magnetic material.

  9. Peptides with dual mode of action: Killing bacteria and preventing endotoxin-induced sepsis.

    PubMed

    Brandenburg, Klaus; Heinbockel, Lena; Correa, Wilmar; Lohner, Karl

    2016-05-01

    Bacterial infections, with the most severe form being sepsis, can often not be treated adequately leading to high morbidity and lethality of infected patients in critical care units. In particular, the increase in resistant bacterial strains and the lack of new antibiotics are main reasons for the worsening of the current situation, As a new approach, the use of antimicrobial peptides (AMPs) seems to be promising, combining the ability of broad-spectrum bactericidal activity and low potential of induction of resistance. Peptides based on natural defense proteins or polypeptides such as lactoferrin, Limulus anti-lipopolysaccharide factor (LALF), cathelicidins, and granulysins are candidates due to their high affinity to bacteria and to their pathogenicity factors, in first line lipopolysaccharide (LPS, endotoxin) of Gram-negative origin. In this review, we discuss literature with the focus on the use of AMPs from natural sources and their variants as antibacterial as well as anti-endotoxin (anti-inflammatory) drugs. Considerable progress has been made by the design of new AMPs for acting efficiently against the LPS-induced inflammation reaction in vitro as well as in vivo (mouse) models of sepsis. Furthermore, the data indicate that efficient antibacterial compounds are not necessarily equally efficient as anti-endotoxin drugs and vice versa. The most important reason for this may be the different molecular geometry of LPS in bacteria and in free form. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. PMID:26801369

  10. Comparison of Ultraviolet Photodissociation and Collision Induced Dissociation of Adrenocorticotropic Hormone Peptides

    NASA Astrophysics Data System (ADS)

    Robotham, Scott A.; Brodbelt, Jennifer S.

    2015-09-01

    In an effort to better characterize the fragmentation pathways promoted by ultraviolet photoexcitation in comparison to collision induced dissociation (CID), six adrenocorticotropic hormone (ACTH) peptides in a range of charge states were subjected to 266 nm ultraviolet photodissociation (UVPD), 193 nm UVPD, and CID. Similar fragment ions and distributions were observed for 266 nm UVPD and 193 nm UVPD for all peptides investigated. While both UVPD and CID led to preferential cleavage of the Y-S bond for all ACTH peptides [except ACTH (1-39)], UVPD was far less dependent on charge state and location of basic sites for the production of C-terminal and N-terminal ions. For ACTH (1-16), ACTH (1-17), ACTH (1-24), and ACTH (1-39), changes in the distributions of fragment ion types ( a, b, c, x, y, z, and collectively N-terminal ions versus C-terminal ions) showed only minor changes upon UVPD for all charge states. In contrast, CID displayed significant changes in the fragment ion type distributions as a function of charge state, an outcome consistent with the dependence on the number and location of mobile protons that is not prominent for UVPD. Sequence coverages obtained by UVPD showed less dependence on charge state than those determined by CID, with the latter showing a consistent decrease in coverage as charge state increased.

  11. Catalysis of Dialanine Formation by Glycine in the Salt-Induced Peptide Formation Reaction.

    NASA Astrophysics Data System (ADS)

    Suwannachot, Yuttana; Rode, Bernd M.

    1998-02-01

    Mutual catalysis of amino acids in the salt-induced peptide formation (SIPF) reaction is demonstrated for the case of glycine/alanine. The presence of glycine enhances dialanine formation by a factor up to 50 and enables dialanine formation at much lower alanine concentrations. The actual amounts of glycine play an important role for this catalytic effect, the optimal glycine concentration is 1/8 of the alanine concentration. The mechanism appears to be based on the formation of the intermediate Gly-Ala-Ala tripeptide, connected to one coordination site of copper(II) ion, and subsequent hydrolysis to dialanine and glycine.

  12. Selective determination of arginine-containing and tyrosine-containing peptides using capillary electrophoresis and laser-induced fluorescence detection.

    PubMed

    Cobb, K A; Novotny, M V

    1992-01-01

    The use of two different amino acid-selective fluorogenic reagents for the derivatization of peptides is investigated. One such scheme utilizes a selective reaction of benzoin with the guanidine moiety to derivatize arginine residues occurring in a peptide. The second scheme involves the formylation of tyrosine, followed by reaction with 4-methoxy-1,2-phenylenediamine. The use of capillary electrophoresis and laser-induced fluorescence detection allows enhanced efficiencies and sensitivities to be obtained for the separations of either arginine- or tyrosine-containing peptides. A helium-cadmium laser (325 nm) is ideally suited for the laser-based detection system due to a close match of the excitation maxima of derivatized peptides from both reactions. A detection limit of 270 amol is achieved for model arginine-containing peptides, while the detection limit for model tyrosine-containing peptides is measured at 390 amol. Both derivatization reactions are found to be useful for high-sensitivity peptide mapping applications in which only the peptides containing the derivatized amino acids are detected.

  13. A rational approach to select immunogenic peptides that induce IFN-γ response against Toxoplasma gondii in human leukocytes.

    PubMed

    Cardona, Néstor I; Moncada, Diego M; Gómez-Marin, Jorge E

    2015-12-01

    The ideal vaccine to prevent toxoplasmosis in humans would comprise antigens that elicit a protective T cell type 1 response with high IFN-γ production. Here, we report the use of a bioinformatics pipeline to discover peptides based on biochemical characteristics that predict strong IFN-γ response by human leukocytes. We selected peptide sequences that previously were reported to induce IFN-γ to identify the biophysical characteristics that will predict HLA-A*02 high-affinity epitopes. We found that the protein motif pattern FL...L..[VL] was common in previously reported highly immunogenic sequences. We have selected new peptides with a length of 9 residues with affinities from 2 to 21 nM with peptide signal and transmembrane domains and predicted to be cleaved at the proteasome to perform ELISPOT assays with human leukocytes. Within 9 peptides with the highest scores for IFN-γ production, four peptides elicited IFN-γ levels in a range from 252 to 1763 SFC/1e6. Our pipeline uncovered Toxoplasma proteins with peptides that are processed by MHC class 1 in humans. Our results suggest that our rational strategy for the selection of immunogenic epitopes could be used to select peptides as candidates for inclusion in epitope-based vaccines. PMID:26210043

  14. Novel peptide VIP-TAT with higher affinity for PAC1 inhibited scopolamine induced amnesia.

    PubMed

    Yu, Rongjie; Yang, Yanxu; Cui, Zekai; Zheng, Lijun; Zeng, Zhixing; Zhang, Huahua

    2014-10-01

    A novel peptide VIP-TAT with a cell penetrating peptide TAT at the C-terminus of VIP was constructed and prepared using intein mediated purification with an affinity chitin-binding tag (IMPACT) system to enhance the brain uptake efficiency for the medical application in central nervous system. It was found by labeling VIP-TAT and VIP with fluorescein isothiocyanate (FITC) that the extension with TAT increased the brain uptake efficiency of VIP-TAT significantly. Then short-term and long-term treatment with scopolamine (Scop) was used to evaluate the effect of VIP-TAT or VIP on Scop induced amnesia. Both short-term and long-term administration of VIP-TAT inhibited the latent time reduction in step-through test induced by Scop significantly, but long-term administration of VIP aggravated the Scop induced amnesia. Long-term i.p. injection of VIP-TAT was shown to have positive effect by inhibiting the oxidative damage, apoptosis and the cholinergic system activity reduction that induced by Scop, while VIP exerted negative effect in brain opposite to that in periphery system. The in vitro data showed that VIP-TAT had not only protective but also proliferative effect on Neuro2a cells which was inhibited by PAC1 antagonist PACAP(6-38). Competition binding assay and cAMP assay confirmed that VIP-TAT had higher affinity and activation for PAC1 than VIP. So it was concluded that the significantly stronger protective effect of VIP-TAT against Scop induced amnesia than VIP was due to (1) the enhanced brain uptake efficiency of VIP-TAT and (2) the increased affinity and activation of VIP-TAT for receptor PAC1.

  15. Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability

    PubMed Central

    Aslam, Muhammad; Paddenberg, Renate; Quanz, Karin; Chang, Chia L.; Park, Jae-Il; Gries, Barbara; Rafiq, Amir; Faulhammer, Petra; Goldenberg, Anna; Papadakis, Tamara; Noll, Thomas; Hsu, Sheau Y. T.; Weissmann, Norbert; Kummer, Wolfgang

    2009-01-01

    Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in preventing damage of the lung by stabilizing pulmonary barrier function. Intermedin (IMD), also termed adrenomedullin-2, is the most recently identified peptide targeting this receptor. Here we investigated the effect of hypoxia on the expression of IMD in the murine lung and cultured murine pulmonary microvascular endothelial cells (PMEC) as well as the role of IMD in regulating vascular permeability. Monoclonal IMD antibodies were generated, and transcript levels were assayed by quantitative RT-PCR. The promoter region of IMD gene was analyzed, and the effect of hypoxia-inducible factor (HIF)-1α on IMD expression was investigated in HEK293T cells. Isolated murine lungs and a human lung microvascular endothelial cell monolayer model were used to study the effect of IMD on vascular permeability. IMD was identified as a pulmonary endothelial peptide by immunohistochemistry and RT-PCR. Hypoxia caused an upregulation of IMD mRNA in the murine lung and PMEC. As shown by these results, HIF-1α enhances IMD promoter activity. Our functional studies showed that IMD abolished the increase in pressure-induced endothelial permeability. Moreover, IMD decreased basal and thrombin-induced hyperpermeability of an endothelial cell monolayer in a receptor-dependent manner and activated PKA in these cells. In conclusion, IMD is a novel hypoxia-induced gene and a potential interventional agent for the improvement of endothelial barrier function in systemic inflammatory responses and hypoxia-induced vascular leakage. PMID:19684198

  16. Liquid Crystalline Nanodispersions Functionalized with Cell-Penetrating Peptides for Topical Delivery of Short-Interfering RNAs: A Proposal for Silencing a Pro-Inflammatory Cytokine in Cutaneous Diseases.

    PubMed

    Petrilli, R; Eloy, J O; Praça, F S G; Del Ciampo, J O; Fantini, M A C; Fonseca, M J V; Bentley, M V L B

    2016-05-01

    Short-interfering RNAs (siRNAs) are a potential strategy for the treatment of cutaneous diseases. In this context, liquid crystalline nanoparticles functionalized with specific proteins and peptide-transduction domains (PTDs), which act as penetration enhancers, are a promising carrier for siRNA delivery through the skin. Herein, hexagonal phase liquid crystal nanoparticles based on monoolein (MO) and/or oleic acid (OA) containing (or lacking) the cationic polymer polyethylenimine (PEI) and the cationic lipid oleylamine (OAM) were functionalized with the membrane transduction peptides transcriptional activator (TAT) or penetratin (PNT). These nanoparticles were complexed with siRNA and characterized by particle size, polydispersity, zeta potential, complexation efficiency and siRNA release. The formulations containing cationic agents presented positive zeta potentials, sizes on the nanometer scale, and complexed siRNAs at concentrations of 10 μM; these agents were successfully released in a heparin competition assay. Cell culture studies demonstrated that nanoparticles composed of MO:OA:PEI functionalized with TAT were the most efficient at transfecting L929 cells, and the uptake efficiency was enhanced by TAT peptide functionalization. Thereafter, the selected formulations were evaluated for in vivo skin irritation, penetration and in vivo efficacy using a chemically induced inflammatory animal model. These nanoparticles did not irritate the skin and provided higher siRNA penetration and delivery into the skin than control formulations. Additionally, efficacy studies in the animal model showed that the association of TAT with the nanodispersion provided higher suppression of tumor necrosis factor (TNF)-α. Thus, the development of liquid crystalline nanodispersions containing TAT may lead to improved topical siRNA delivery for the treatment of inflammatory skin diseases. PMID:27305826

  17. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    SciTech Connect

    Amand, Helene L.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from

  18. PCR-cloning of cadmium-inducible peptides in the barnacle, Megabalanus volcano.

    PubMed

    Togi, Akiko; Kamino, Kei; Shizuri, Yoshikazu

    2002-04-01

    A 340 bp DNA fragment was amplified from barnacle (Megabalanus volcano) cDNA by polymerase chain reaction using primers designed based on the amino acid sequences of barnacle cadmium-inducible peptides CdlP1 and CdlP2. The whole sequence was determined by rapid amplification of cDNA ends method. The cDNA contained an open reading frame encoding 71 amino acid residues and the sequences for CdlP1 and CdlP2 were found to be located in the center of this coding region. Although CdlP1 and CdlP2 had been detected only in the cadmium-exposed barnacles, their mRNA was present both in cadmium-exposed barnacles and in unexposed barnacles. These results suggest that posttranslational proteolytic processing may be induced in the presence of cadmium.

  19. Mitochondria related peptide MOTS-c suppresses ovariectomy-induced bone loss via AMPK activation.

    PubMed

    Ming, Wei; Lu, Gan; Xin, Sha; Huanyu, Lu; Yinghao, Jiang; Xiaoying, Lei; Chengming, Xu; Banjun, Ruan; Li, Wang; Zifan, Lu

    2016-08-01

    Therapeutic targeting bone loss has been the focus of the study in osteoporosis. The present study is intended to evaluate whether MOTS-c, a novel mitochondria related 16 aa peptide, can protect mice from ovariectomy-induced osteoporosis. After ovary removal, the mice were injected with MOTS-c at a dose of 5 mg/kg once a day for 12 weeks. Our results showed that MOTS-c treatment significantly alleviated bone loss, as determined by micro-CT examination. Mechanistically, we found that the receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclast differentiation was remarkably inhibited by MOTS-c. Moreover, MOTS-c increased phosphorylated AMPK levels, and compound C, an AMPK inhibitor, could partially abrogate the effects of the MOTS-c on osteoclastogenesis. Thus, our findings provide evidence that MOTS-c may exert as an inhibitor of osteoporosis via AMPK dependent inhibition of osteoclastogenesis. PMID:27237975

  20. NADPH oxidase mediates β-amyloid peptide-induced activation of ERK in hippocampal organotypic cultures

    PubMed Central

    Serrano, Faridis; Chang, Angela; Hernandez, Caterina; Pautler, Robia G; Sweatt, J David; Klann, Eric

    2009-01-01

    Background Previous studies have shown that beta amyloid (Aβ) peptide triggers the activation of several signal transduction cascades in the hippocampus, including the extracellular signal-regulated kinase (ERK) cascade. In this study we sought to characterize the cellular localization of phosphorylated, active ERK in organotypic hippocampal cultures after acute exposure to either Aβ (1-42) or nicotine. Results We observed that Aβ and nicotine increased the levels of active ERK in distinct cellular localizations. We also examined whether phospho-ERK was regulated by redox signaling mechanisms and found that increases in active ERK induced by Aβ and nicotine were blocked by inhibitors of NADPH oxidase. Conclusion Our findings indicate that NADPH oxidase-dependent redox signaling is required for Aβ-induced activation of ERK, and suggest a similar mechanism may occur during early stages of Alzheimer's disease. PMID:19804648

  1. Two nitrogen-containing ligands as inhibitors of metal-induced amyloid β-peptide aggregation.

    PubMed

    Chen, Tingting; Zhu, Shajun; Liu, Siyuan; Lu, Yapeng; Zhu, Li

    2014-02-01

    Abnormal interactions of Zn(2+) and Cu(2+) with the amyloid β-peptide (Aβ) are proposed to play an important role in the neuropathogenesis of Alzheimer's disease (AD). Metal chelators are potential therapeutic agents for AD because they could sequester metals ions from Aβ aggregates and reverse the aggregation. In this study, two nitrogencontaining ligands, TACN and BPA, have been investigated as possible metal chelators in the therapy of Alzheimer's disease. The interactions between the chelators and Aβ40 aggregates are studied by turbidometry, thioflavin T (ThT) fluorescence spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), BCA protein assay, circular dichroism spectroscopy (CD), and atomic force microscopy (AFM). The results demonstrates that TACN and BPA are capable of both disrupting and preventing Zn(2+) or Cu(2+)-induced Aβ40 aggregation. Moreover, they can also suppress the production of H2O2 induced by Cu-Aβ40, associated with toxic oxidative stress in AD. PMID:23844690

  2. Effect of heparin on bovine epithelial lens cell proliferation induced by heparin affin regulatory peptide.

    PubMed

    Delbé, J; Vacherot, F; Laaroubi, K; Barritault, D; Courty, J

    1995-07-01

    HARP (heparin affin regulatory peptide) is an 18 kDa heparin binding protein, also known as HB-GAM or pleiotrophin (PTN) which has been primarily isolated from brain and uterus, and displays neurite outgrowth, angiogenic and mitogenic activities. Previously, we have expressed the human cDNA encoding human HARP in NIH 3T3 cells. Purified recombinant HARP displayed mitogenic activity for endothelial cells. Its NH2-terminal sequence indicates that the HARP molecule possesses a three amino acid extension from the signal peptide more than the NH2-terminal described. For HB-GAM or PTN, these three amino acids may be essential for the stability and the mitogenic activity of this growth factor. In an attempt to further study the mode of action of this growth factor, we have investigated the mitogenic effect of HARP on various cell types. In contrast to FGF-2, HARP failed to induce stimulation of DNA synthesis on a CCL39 cell line. However, we found that in quiescent bovine epithelial lens (BEL) cells, the stimulation of DNA synthesis induced by HARP is dose-dependent (EC50: 2.5 ng/ml) and maximal stimulation is as potent as that induced by FGF-2 (EC50: 25 pg/ml). Interestingly, when BEL cells were allowed to quiesce in the presence of serum, the stimulation induced by HARP is considerably less potent. In this highly responsive cell system, heparin could potentiate the mitogenic activity of HARP at very low doses (0.1-1 microgram/ml) and inhibit this activity at concentrations of 10 micrograms/ml. In contrast to its protective effect on FGF-1 and -2, heparin was unable to preserve HARP from tryptic and chymotryptic degradations.

  3. Iron oxide nanoparticles induce Pseudomonas aeruginosa growth, induce biofilm formation, and inhibit antimicrobial peptide function.

    PubMed

    Borcherding, Jennifer; Baltrusaitis, Jonas; Chen, Haihan; Stebounova, Larissa; Wu, Chia-Ming; Rubasinghege, Gayan; Mudunkotuwa, Imali A; Caraballo, Juan Carlos; Zabner, Joseph; Grassian, Vicki H; Comellas, Alejandro P

    2014-04-01

    Given the increased use of iron-containing nanoparticles in a number of applications, it is important to understand any effects that iron-containing nanoparticles can have on the environment and human health. Since iron concentrations are extremely low in body fluids, there is potential that iron-containing nanoparticles may influence the ability of bacteria to scavenge iron for growth, affect virulence and inhibit antimicrobial peptide (AMP) function. In this study, Pseudomonas aeruginosa (PA01) and AMPs were exposed to iron oxide nanoparticles, hematite (α-Fe2O3), of different sizes ranging from 2 to 540 nm (2 ± 1, 43 ± 6, 85 ± 25 and 540 ± 90 nm) in diameter. Here we show that the greatest effect on bacterial growth, biofilm formation, and AMP function impairment is found when exposed to the smallest particles. These results are attributed in large part to enhanced dissolution observed for the smallest particles and an increase in the amount of bioavailable iron. Furthermore, AMP function can be additionally impaired by adsorption onto nanoparticle surfaces. In particular, lysozyme readily adsorbs onto the nanoparticle surface which can lead to loss of peptide activity. Thus, this current study shows that co-exposure of nanoparticles and known pathogens can impact host innate immunity. Therefore, it is important that future studies be designed to further understand these types of impacts. PMID:25221673

  4. Non-fibrillar amyloid-{beta} peptide reduces NMDA-induced neurotoxicity, but not AMPA-induced neurotoxicity

    SciTech Connect

    Niidome, Tetsuhiro; Goto, Yasuaki; Kato, Masaru; Wang, Pi-Lin; Goh, Saori; Tanaka, Naoki; Akaike, Akinori; Kihara, Takeshi; Sugimoto, Hachiro

    2009-09-04

    Amyloid-{beta} peptide (A{beta}) is thought to be linked to the pathogenesis of Alzheimer's disease. Recent studies suggest that A{beta} has important physiological roles in addition to its pathological roles. We recently demonstrated that A{beta}42 protects hippocampal neurons from glutamate-induced neurotoxicity, but the relationship between A{beta}42 assemblies and their neuroprotective effects remains largely unknown. In this study, we prepared non-fibrillar and fibrillar A{beta}42 based on the results of the thioflavin T assay, Western blot analysis, and atomic force microscopy, and examined the effects of non-fibrillar and fibrillar A{beta}42 on glutamate-induced neurotoxicity. Non-fibrillar A{beta}42, but not fibrillar A{beta}42, protected hippocampal neurons from glutamate-induced neurotoxicity. Furthermore, non-fibrillar A{beta}42 decreased both neurotoxicity and increases in the intracellular Ca{sup 2+} concentration induced by N-methyl-D-aspartate (NMDA), but not by {alpha}-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA). Our results suggest that non-fibrillar A{beta}42 protects hippocampal neurons from glutamate-induced neurotoxicity through regulation of the NMDA receptor.

  5. Identification of a Possible Role for Atrial Natiuretic Peptide in MDMA-induced hyperthermia

    PubMed Central

    Hrometz, Sandra L; Thatcher, Karen E; Ebert, Jeremy A; Mills, Edward M; Sprague, Jon E

    2011-01-01

    MDMA (3,4-methylenedioxymethamphetamine) induces thermogenesis in a mitochondrial uncoupling protein 3-dependent manner. There is evidence that this hyperthermia is mediated in part by the lipolytic release of free fatty acids, that subsequently activate uncoupling protein 3 in skeletal muscle mitochondria. We hypothesize that atrial natriuretic peptide (ANP), a strong lipolytic mediator, may contribute to the induction and maintenance of MDMA-induced thermogenesis. The specific aims of this study were to 1) determine if ANP is released following MDMA administration, and 2) use the ANP receptor antagonist, Anantin, to ascertain the role of ANP in MDMA-induced hyperthermia. ANP levels were measured in plasma at baseline, 10, 20, 30 and 60 min following MDMA (40 mg/kg, sc) administration in 16 male Sprague-Dawley rats. A robust increase in ANP was seen within ten min of MDMA administration. ANP levels returned to baseline at 20 min and then gradually rose over the 60 min monitoring period. The administration of Anantin (40 mg, ip), 15 min before and after MDMA, significantly attenuated the MDMA-induced hyperthermia. We conclude that ANP signaling contributes to the hyperthermia induced by MDMA. PMID:21827841

  6. Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons.

    PubMed

    Kelly, J F; Furukawa, K; Barger, S W; Rengen, M R; Mark, R J; Blanc, E M; Roth, G S; Mattson, M P

    1996-06-25

    Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C, resulting in the liberation of inositol trisphosphate and Ca2+ release from intracellular stores. We now report that amyloid beta-peptide (Abeta), which forms plaques in the brain in AD, impairs muscarinic receptor activation of G proteins in cultured rat cortical neurons. Exposure of rodent fetal cortical neurons to Abeta25-35 and Abeta1-40 resulted in a concentration and time-dependent attenuation of carbachol-induced GTPase activity without affecting muscarinic receptor ligand binding parameters. Downstream events in the signal transduction cascade were similarly attenuated by Abeta. Carbachol-induced accumulation of inositol phosphates (IP, IP2, IP3, and IP4) was decreased and calcium imaging studies revealed that carbachol-induced release of calcium was severely impaired in neurons pretreated with Abeta. Muscarinic cholinergic signal transduction was disrupted with subtoxic levels of exposure to AP. The effects of Abeta on carbachol-induced GTPase activity and calcium release were attenuated by antioxidants, implicating free radicals in the mechanism whereby Abeta induced uncoupling of muscarinic receptors. These data demonstrate that Abeta disrupts muscarinic receptor coupling to G proteins that mediate induction of phosphoinositide accumulation and calcium release, findings that implicate Abeta in the impairment of cholinergic transmission that occurs in AD. PMID:8692890

  7. DCP-LA neutralizes mutant amyloid beta peptide-induced impairment of long-term potentiation and spatial learning.

    PubMed

    Nagata, Tetsu; Tomiyama, Takami; Tominaga, Takemi; Mori, Hiroshi; Yaguchi, Takahiro; Nishizaki, Tomoyuki

    2010-01-01

    Long-term potentiation (LTP) was monitored from the CA1 region of the intact rat hippocampus by delivering high frequency stimulation (HFS) to the Schaffer collateral commissural pathway. Intraventricular injection with mutant amyloid beta(1-42) peptide lacking glutamate-22 (Abeta(1-42)E22Delta), favoring oligomerization, 10 min prior to HFS, inhibited expression of LTP, with the potency more than wild-type amyloid beta(1-42) peptide. Intraperitoneal injection with the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) 70 min prior to HFS neutralized mutant Abeta(1-42)E22Delta peptide-induced LTP inhibition. In the water maze test, continuous intraventricular injection with mutant Abeta(1-42)E22Delta peptide for 14 days prolonged the acquisition latency as compared with that for control, with the potency similar to wild-type Abeta(1-42) peptide, and intraperitoneal injection with DCP-LA shortened the prolonged latency to control levels. The results of the present study indicate that DCP-LA neutralizes mutant Abeta(1-42)E22Delta peptide-induced impairment of LTP and spatial learning.

  8. Osmolyte Induced Changes in Peptide Conformational Ensemble Correlate with Slower Amyloid Aggregation: A Coarse-Grained Simulation Study.

    PubMed

    Sukenik, Shahar; Sapir, Liel; Harries, Daniel

    2015-12-01

    Stabilizing osmolytes are known to impact the process of amyloid aggregation, often altering aggregation kinetics. Recent evidence further suggests that osmolytes modify the peptide conformational dynamics, as well as change the physical characteristics of assembling amyloid fibrils. To resolve how these variations emerge on the molecular level, we simulated the initial aggregation steps of an amyloid-forming peptide in the presence and absence of the osmolyte sorbitol, a naturally occurring polyol. To this end, a coarse-grained force field was extended and implemented to access larger aggregate sizes and longer time scales. The force field optimization procedure placed emphasis on calibrating the solution thermodynamics of sorbitol, the aggregating peptide in its monomeric form, and the interaction of both of these components with each other and with water. Our simulations show a difference in aggregation kinetics and structural parameters in the presence of sorbitol compared to water, which qualitatively agree well with our experimentally resolved aggregation kinetics of the same peptide. The kinetic changes induced by sorbitol can be traced in our simulations to changes in monomer conformations resulting from osmolyte presence. These translate into changes in peptide conformations within the aggregated clusters and into differences in rates of monomer nucleation and of association to formed fibrils. We find that, compared to pure water as solvent, the presence of sorbitol induces formation of more aggregates each containing fewer peptides, with an increased tendency toward parallel interpeptide contacts. PMID:26587669

  9. Phosphorylation-induced conformational changes in short peptides probed by fluorescence resonance energy transfer in the 10A domain.

    PubMed

    Sahoo, Harekrushna; Nau, Werner M

    2007-03-26

    Phosphorylation-induced conformational changes in short polypeptides were probed by a fluorescence resonance energy transfer (FRET) method by employing a short-distance FRET pair (R(0) approximately 10 A) based on tryptophan as natural donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as synthetic acceptor. Two substrates for kinases, LeuArgArgTrpSerLeuGly-Dbo (peptide I) and TrpLysArgThrLeuArgArg-Dbo (peptide II), were investigated, with serine and threonine, respectively, as phosphorylation sites. Steady-state and time-resolved fluorescence experiments in H(2)O revealed a decrease in FRET efficiency for peptide I and an increase for peptide II; this suggested that the effective distances between donor and acceptor increased and decreased, respectively. The same trends and similar absolute variations in effective donor-acceptor distances were observed in propylene glycol, a less polar and highly viscous solvent; this suggested that the variations are due to intrinsic structural preferences. Fitting of the time-resolved decay traces according to a distribution function model (Gaussian distribution) provided the mean donor-acceptor distances, which showed an increase upon phosphorylation for peptide I (from 9.7 to 10.5 A) and a decrease for peptide II (from 10.9 to 9.3 A) in H(2)O. The broadness (half-width) of the distributions, which provides a measure of the rigidity of the peptides, remained similar upon phosphorylation of peptide I (3.0 versus 3.1 A), but decreased for peptide II (from 3.1 to 0.73 A in H(2)O); this suggests a more compact, structured conformation upon phosphorylation of the latter peptide. The elongation of the peptide backbone (by ca. 0.7 A) for peptide I is attributed to an increase in steric demand upon phosphorylation, which favors an extended conformation. The contraction (by ca. 1.4 A) and structural rigidification of peptide II is attributed to attractive Coulombic interactions and hydrogen bonding between the

  10. Nutrient-induced glucagon like peptide-1 release is modulated by serotonin.

    PubMed

    Ripken, Dina; van der Wielen, Nikkie; Wortelboer, Heleen M; Meijerink, Jocelijn; Witkamp, Renger F; Hendriks, Henk F J

    2016-06-01

    Glucagon like peptide-1 (GLP-1) and serotonin are both involved in food intake regulation. GLP-1 release is stimulated upon nutrient interaction with G-protein coupled receptors by enteroendocrine cells (EEC), whereas serotonin is released from enterochromaffin cells (ECC). The central hypothesis for the current study was that nutrient-induced GLP-1 release from EECs is modulated by serotonin through a process involving serotonin receptor interaction. This was studied by assessing the effects of serotonin reuptake inhibition by fluoxetine on nutrient-induced GLP-1, PYY and CCK release from isolated pig intestinal segments. Next, serotonin-induced GLP-1 release was studied in enteroendocrine STC-1 cells, where effects of serotonin receptor inhibition were studied using specific and non-specific antagonists. Casein (1% w/v), safflower oil (3.35% w/v), sucrose (50mM) and rebaudioside A (12.5mM) stimulated GLP-1 release from intestinal segments, whereas casein only stimulated PYY and CCK release. Combining nutrients with fluoxetine further increased nutrient-induced GLP-1, PYY and CCK release. Serotonin release from intestinal tissue segments was stimulated by casein and safflower oil while sucrose and rebaudioside A had no effect. The combination with fluoxetine (0.155μM) further enhanced casein and safflower oil induced-serotonin release. Exposure of ileal tissue segments to serotonin (30μM) stimulated GLP-1 release whereas it did not induce PYY and CCK release. Serotonin (30 and 100μM) also stimulated GLP-1 release from STC-1 cells, which was inhibited by the non-specific 5HT receptor antagonist asenapine (1 and 10μM). These data suggest that nutrient-induced GLP-1 release is modulated by serotonin through a receptor mediated process. PMID:27142747

  11. Neuroprotective Peptide humanin inhibits inflammatory response in astrocytes induced by lipopolysaccharide.

    PubMed

    Zhao, Shen-Ting; Zhao, Li; Li, Jian-Hua

    2013-03-01

    Humanin (HN) has been proved to be an extensive neuroprotective peptide against AD-related and unrelated insults, but little is know about the effect of HN in inflammation response. Current studies indicated the receptors of HN have a close relationship with immune system, which led us to hypothesize HN might have a role in inflammatory response. In this study, we used lipopolysaccharide (LPS) to induce astrocyte inflammation response. This model in vitro allowed us to study the effect of HN on the pure response of astrocyte without the exogenous influence between cells in vivo. Our results showed that 1.0 μg/ml LPS induced a significant activation of astrocyte, shown as the marked increase in the glial fibrillary acidic protein (GFAP) expression, the cell viability and the number of 5-bromo-2'-deoxyuridine (BrdU)-positive living cells. Pretreatment with HN (5, 10, 20 μM) led to a significant inhibition in astrocyte overactivation in a concentration dependent manner. We also found pretreatment with HN decreased the level of proinflammatory cytokines, interleukin (IL)-6, IL-1β and tumor necrosis factor α (TNFα) induced by LPS. Furthermore, we noticed HN couldn't completely reverse the above inflammatory injury. Our findings imply that HN partly antagonizes inflammation injury induced by LPS and the protective effect of HN on astrocyte is concentration-dependent.

  12. Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization.

    PubMed Central

    Rodrigues, C. M.; Solá, S.; Silva, R.; Brites, D.

    2000-01-01

    BACKGROUND: The pathogenesis of bilirubin encephalopathy and Alzheimer's disease appears to result from accumulation of unconjugated bilirubin (UCB) and amyloid-beta (Abeta) peptide, respectively, which may cause apoptosis. Permeabilization of the mitochondrial membrane, with release of intermembrane proteins, has been strongly implicated in cell death. Inhibition of the mitochondrial permeability is one pathway by which ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) protect against apoptosis in hepatic and nonhepatic cells. In this study, we further characterize UCB- and Abeta-induced cytotoxicty in isolated neural cells, and investigate membrane perturbation during incubation of isolated mitochondria with both agents. In addition, we evaluate whether the anti-apoptotic drugs UDC and TUDC prevent any changes from occurring. MATERIALS AND METHODS: Primary rat neuron and astrocyte cultures were incubated with UCB or Abeta peptide, either alone or in the presence of UDC. Apoptosis was assessed by DNA fragmentation and nuclear morphological changes. Isolated mitochondria were treated with each toxic, either alone or in combination with UDC, TUDC, or cyclosporine A. Mitochondrial swelling was measured spectrophotometrically and cytochrome c protein levels determined by Western blot. RESULTS: Incubation of neural cells with both UCB and Abeta induced apoptosis (p < 0.01). Coincubation with UDC reduced apoptosis by > 50% (p < 0.05). Both toxins caused membrane permeabilization in isolated mitochondria (p < 0.001); whereas, pretreatment with UDC was protective (p < 0.05). TUDC was even more effective at preventing matrix swelling mediated by Abeta (p < 0.01). UDC and TUDC markedly reduced cytochrome c release associated with mitochondrial permeabilization induced by UCB and Abeta, respectively (p < 0.05). Moreover, cyclosporine A significantly inhibited mitochondrial swelling and cytochrome c efflux mediated by UCB (p < 0.05). CONCLUSION: UCB and Abeta peptide

  13. Atrial natriuretic peptide attenuates agonist-induced pulmonary edema in mice with targeted disruption of the gene for natriuretic peptide receptor-A

    PubMed Central

    Tsai, Shu-Whei; Green, Sabrina; Grinnell, Katie L.; Machan, Jason T.; Harrington, Elizabeth O.

    2013-01-01

    Atrial natriuretic peptide (ANP) inhibits agonist-induced pulmonary edema formation, but the signaling pathway responsible is not well defined. To investigate the role of the particulate guanylate cyclase-linked receptor, natriuretic peptide receptor-A (NPR-A), we measured acute lung injury responses in intact mice and pulmonary microvascular endothelial cells (PMVEC) with normal and disrupted expression of NPR-A. NPR-A wild-type (NPR-A+/+), heterozygous (NPR-A+/−), and knockout (NPR-A−/−) mice were anesthetized and treated with thrombin receptor agonist peptide (TRAP) or lipopolysaccharide (LPS). Lung injury was assessed by lung wet-to-dry (W/D) weight and by protein and cell concentration of bronchoalveolar lavage (BAL) fluid. No difference in pulmonary edema formation was seen between NPR-A genotypes under baseline conditions. TRAP and LPS increased lung W/D weight and BAL fluid cell counts more in NPR-A−/− mice than in NPR-A+/− or NPR-A+/+ mice, but no genotype-related differences were seen in TRAP-induced increases in bloodless lung W/D weight or LPS-induced increases in BAL protein concentration. Pretreatment with ANP infusion completely blocked TRAP-induced increases in lung W/D weight and blunted LPS-induced increases in BAL cell counts and protein concentration in both NPR-A−/− and NPR-A+/+ mice. Thrombin decreased transmembrane electrical resistance in monolayers of PMVECs in vitro, and this effect was attenuated by ANP in PMVECs isolated from both genotypes. Administration of the NPR-C-specific ligand, cANF, also blocked TRAP-induced increases in lung W/D weight and LPS-induced increases in BAL cell count and protein concentration in NPR-A+/+ and NPR-A−/− mice. We conclude that ANP is capable of attenuating agonist-induced lung edema in the absence of NPR-A. The protective effect of ANP on agonist-induced lung injury and pulmonary barrier function may be mediated by NPR-C. PMID:23195629

  14. Peptidyl transferase inhibition by the nascent leader peptide of an inducible cat gene.

    PubMed Central

    Gu, Z; Rogers, E J; Lovett, P S

    1993-01-01

    The site of ribosome stalling in the leader of cat transcripts is critical to induction of downstream translation. Site-specific stalling requires translation of the first five leader codons and the presence of chloramphenicol, a sequence-independent inhibitor of ribosome elongation. We demonstrate in this report that a synthetic peptide (the 5-mer) corresponding to the N-terminal five codons of the cat-86 leader inhibits peptidyl transferase in vitro. The N-terminal 2-, 3-, and 4-mers and the reverse 5-mer (reverse amino acid sequence of the 5-mer) are virtually without effect on peptidyl transferase. A missense mutation in the cat-86 leader that abolishes induction in vivo corresponds to an amino acid replacement in the 5-mer that completely relieves peptidyl transferase inhibition. In contrast, a missense mutation that does not interfere with in vivo induction corresponds to an amino acid replacement in the 5-mer that does not significantly alter peptidyl transferase inhibition. Our results suggest that peptidyl transferase inhibition by the nascent cat-86 5-mer peptide may be the primary determinant of the site of ribosome stalling in the leader. A model based on this concept can explain the site specificity of ribosome stalling as well as the response of induction to very low levels of the antibiotic inducer. Images PMID:7690023

  15. Corn Peptides Protect Against Thioacetamide-Induced Hepatic Fibrosis in Rats

    PubMed Central

    Lv, Jie; Nie, Zhi-Kui; Zhang, Jiu-Liang; Liu, Feng-Yan; Wang, Zhen-Zhen; Ma, Zhi-Li

    2013-01-01

    Abstract Certain bioactive peptides are reported to be able to alleviate hepatic fibrosis. Our previous work has confirmed the hepatoprotective effect of corn peptides (CPs) that are prepared from a high protein by-product, corn gluten meal, on acute liver injury in an animal model. However, the antifibrotic activity of CPs remained to be elucidated. In this study, the hepatoprotective effect of CPs on thioacetamide (TAA)-induced liver fibrosis was tested. Results showed that CPs (100 mg/kg body weight) significantly decreased the levels of alanine transaminase/aspartate transaminase, laminin, type IV collagen, and type III collagen in serum and increased the serum albumin levels and total antioxidant capacity. Additionally, with CP treatment (100 mg/kg body weight), a significant decrease was observed in the levels of malondialdehyde, nitric oxide, hydroxyproline, transforming growth factor β1, and lactate dehydrogenase activity as well as the liver index, while the activity of superoxidedismutase was significantly increased in livers. The histological and morphological analysis showed that the hepatocyte structure in CP-treated rats was superior to that of TAA-injured rats, and inflammation and fibrosis were also ameliorated. Therefore, CPs can be used as an option for prevention and adjuvant therapy of liver fibrosis. PMID:24063407

  16. Mechanisms of Ultrasonically Induced Fibrillation of Amyloid β1-40 Peptides

    NASA Astrophysics Data System (ADS)

    Uesugi, Kentaro; Ogi, Hirotsugu; Fukushima, Masahiko; So, Masatomo; Yagi, Hisashi; Goto, Yuji; Hirao, Masahiko

    2013-07-01

    We systematically study the relationship between the ultrasonically induced aggregation behavior of amyloid β1-40 peptide and acoustic pressures to clarify the dominant mechanism of the aggregation. With ultrasonic irradiation, the thioflavin-T (ThT) level of the Aβ solution rises after a lag time, takes a maximum at ˜5 h, and remains unchanged or decreases. Thus, we monitor the ThT level at 5 h to evaluate the progress of the β-sheet structure and investigate its correlation with the acoustic pressures of fundamental and harmonics waves. The second-harmonics-wave amplitude shows the highest correlation with the ThT level, indicating the dominant contribution of cavitation bubbles to the fibrillation phenomenon. The influence of solution pH and Ar gas are investigated to identify the aggregation mechanism. As a result, local condensation of the peptide due to the high affinity of hydrophobic residues to the bubble-solution interface causes a highly supersaturated solution, leading to precipitation of β-sheet-rich nuclei.

  17. Unique properties of cd-binding peptides induced in fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    Hayashi, Y.; Nakagawa, C.W.; Murasugi, A.

    1986-03-01

    Metallothioneins, a class of low molecular weight cysteine-rich proteins that bind heavy metal ions, have been found in various eucaryotic organisms. When fission yeasts are grown in the presence of high concentration of CdCl/sub 2/, large amounts of Cd-binding peptides (Cd-BP1 and Cd-BP2) are synthesized. While Cd-BP2 shows similarities to mammalian Cd-thioneins in UV and CD spectra, Cd-BP1has a characteristic shoulder at 265 nm in the UV absorption spectrum and shows two marked Cotton bands at 257 nm (negative) and 275 nm (positive). These characteristics of Cd-BP1 are not found in the other Cd-thioneins. The UV and CD spectra differences between reconstituted and native Cd-BP1 suggest the requirement for some additional molecular architecture including another peptide-Cd/sup 2 +/ interaction. Induction of cadystin synthesis is almost exclusive for Cd, but an exception is a small amount of cadystin also induced by the higher concentration of CuCl/sub 2/ (2.5 mM). The UV spectrum of the natural Cu-cadystin complex was similar to that of Cd-BP1. On the basis of these findings the models for Cd-BP1 and Cd-BP2 are proposed.

  18. Improved Ligand Discrimination by Force-Induced Unbinding of the T Cell Receptor from Peptide-MHC

    PubMed Central

    Klotzsch, Enrico; Schütz, Gerhard J.

    2013-01-01

    T cell activation is mediated via the recognition of peptides by the T cell receptor (TCR). This receptor ligand interaction is highly specific, and the TCR has to discriminate between a huge number of peptides presented by the products of the major histocompatibility complexes (MHCs). Recent studies indicate that cells probe the TCR-pMHC interaction by imposing force on the interaction. Here we investigated in a theoretical analysis the consequences of such force-induced unbinding for T cell recognition. Our findings are as follows. First, the bond rupture under force is much faster, improving the time resolution of the discrimination process. Second, cells can access additional parameters characterizing the shape of the binding energy surface. Third, load-induced unbinding yields a reduced coefficient of variation of the bond lifetimes, which improves the discriminative power even between peptide/MHCs (pMHCs) with similar off-rates. PMID:23601314

  19. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced Pluripotent Stem Cells

    PubMed Central

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-01-01

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies. PMID:26561805

  20. Peptide-decorated polyvinyl alcohol/hyaluronan nanofibers for human induced pluripotent stem cell culture.

    PubMed

    Deng, Yi; Zhang, Xiaohong; Zhao, Yinghui; Liang, Shangshang; Xu, Anxiu; Gao, Xiang; Deng, Feng; Fang, Jing; Wei, Shicheng

    2014-01-30

    Realization of the full potential of human induced pluripotent stem cells (hiPSCs) in clinical applications requires development of well-defined conditions for their growth and differentiation. A novel fully defined polyvinyl alcohol/hyaluronan (PVA/HA) polysaccharide nanofiber was developed for hiPSCs culture in commercially available xeno-free, chemically defined medium. Vitronectin peptide (VP) was immobilized to PVA/HA nanofibers through NHS/EDC chemistry. The hiPSCs successfully grew and proliferated on the VP-decorated PVA/HA nanofibers, similar to those on Matrigel™. Such well-defined, xeno-free and safe nanofiber substrate that supports culture of hiPSCs will not only help to accelerate the translational perspectives of hiPSCs, but also provide a platform to investigate the cell-nanofiber interaction mechanisms that regulate stem cell proliferation and differentiation.

  1. Plant natriuretic peptides induce proteins diagnostic for an adaptive response to stress

    PubMed Central

    Turek, Ilona; Marondedze, Claudius; Wheeler, Janet I.; Gehring, Chris; Irving, Helen R.

    2014-01-01

    In plants, structural and physiological evidence has suggested the presence of biologically active natriuretic peptides (PNPs). PNPs are secreted into the apoplast, are systemically mobile and elicit a range of responses signaling via cGMP. The PNP-dependent responses include tissue specific modifications of cation transport and changes in stomatal conductance and the photosynthetic rate. PNP also has a critical role in host defense responses. Surprisingly, PNP-homologs are produced by several plant pathogens during host colonization suppressing host defense responses. Here we show that a synthetic peptide representing the biologically active fragment of the Arabidopsis thaliana PNP (AtPNP-A) induces the production of reactive oxygen species in suspension-cultured A. thaliana (Col-0) cells. To identify proteins whose expression changes in an AtPNP-A dependent manner, we undertook a quantitative proteomic approach, employing tandem mass tag (TMT) labeling, to reveal temporal responses of suspension-cultured cells to 1 nM and 10 pM PNP at two different time-points post-treatment. Both concentrations yield a distinct differential proteome signature. Since only the higher (1 nM) concentration induces a ROS response, we conclude that the proteome response at the lower concentration reflects a ROS independent response. Furthermore, treatment with 1 nM PNP results in an over-representation of the gene ontology (GO) terms “oxidation-reduction process,” “translation” and “response to salt stress” and this is consistent with a role of AtPNP-A in the adaptation to environmental stress conditions. PMID:25505478

  2. Ultrasensitive detection of closely related angiotensin I peptides using capillary electrophoresis with near-infrared laser-induced fluorescence detection.

    PubMed

    Baars, M J; Patonay, G

    1999-02-01

    A novel near-infrared (NIR) fluorescent dye (NN382, LICOR, Inc.) was evaluated as an ultrasensitive peptide-labeling reagent for use with capillary electrophoresis (CE). Six angiotensin I (Ang-I) variants were selected as model peptides for the derivatization and separation studies. The closely related decapeptides were labeled with the NIR dye, separated using CE, and detected by NIR laser-induced fluorescence. Derivatization of the peptides was achieved under aqueous conditions using 2.5-500 pmol of Ang-I in a 50-microL sample (5 x 10(-8)-1 x 10(-5)M), and between 1.3 and 254 amol of the labeled peptides were injected on column. The fluorescence response was linear over a 200-fold range (correlation r > or = 0.9986). The limit of detection (SNR = 3, signal/RMS noise) ranged from 100 to 300 zmol, for the six Ang-I variants. Four of six peptides were resolved from each other and excess dye using capillary zone electrophoresis with a simple 50 mM phosphate run buffer, pH 7.2. Two pairs of coeluting peptides were successfully resolved using micellar electrokinetic chromatography with a nonionic surfactant, Triton X-100. The NIR amine-labeling reagent NN382 is a viable alternative to using visible fluorophores for CE methods requiring high sensitivity. PMID:9989384

  3. Ethanol alters vasoactive intestinal peptide-induced steroid release from immature rat ovaries in vitro

    SciTech Connect

    Dees, W.L.; Hiney, J.K.; Fuentes, F.; Forrest, D.W. )

    1990-01-01

    The present study was conducted to examine the acute effects of ethanol (ETOH) on basal and VIP-induced release of testosterone (T) and estradiol (E{sub 2}) from immature ovaries in vitro. Ovaries were collected from anestrus (A) and both naturally occurring and pregnant mare's serum gonadotropin (PMSG)-induced early proestrus (EP) animals. The ovaries were incubated in wither media alone, media plus 1 {mu}M VIP, media plus ETOH in doses ranging from 25 to 100 mM, or media plus each dose of ETOH containing VIP. The present results demonstrate that ETOH did not affect either basal or VIP-induced steroid release from ovaries collected from A animals. Likewise, the ETOH did not alter basal steroid secretion from EP animals; however, the drug significantly reduced the VIP-stimulated release of both T and E, from EP ovaries. Thus, these data demonstrate for the first time that ETOH is capable of altering prepubertal ovarian responsiveness to VIP, a peptide known to be involved in the developmental regulation of ovarian function.

  4. Isoflurane and desflurane at clinically relevant concentrations induce amyloid {beta}-peptide oligomerization: An NMR study

    SciTech Connect

    Mandal, Pravat K Fodale, Vincenzo

    2009-02-13

    Current understanding on Alzheimer's disease (AD) reveals that soluble amyloid {beta}-peptide (A{beta}) oligomeric formation plays an important role in AD pathophysiology. A potential role for several inhaled anesthetics in promoting A{beta} oligomer formation has been suggested. Using a nuclear magnetic resonance (NMR) study, we previously demonstrated that at a high concentration (higher than clinically relevant concentrations), the inhaled anesthetics halothane and isoflurane, interact with specific amino acid residues (G29, A30, and I31) and induce A{beta} oligomerization. The present study confirms this is true at a clinically relevant concentration. Isoflurane and desflurane induce A{beta} oligomerization by inducing chemical shift changes of the critical amino acid residues (G29, A30, and I31), reinforcing the evidence that perturbation of these three crucial residues indeed plays an important role in oligomerization. These findings support the emerging hypothesis that several commonly used inhaled anesthetics could be involved in neurodegeneration, as well as risk factor for accelerating the onset of AD.

  5. Induced expression of neuronal membrane attack complex and cell death by Alzheimer's beta-amyloid peptide.

    PubMed

    Shen, Y; Sullivan, T; Lee, C M; Meri, S; Shiosaki, K; Lin, C W

    1998-06-15

    beta-amyloid peptide (A beta) and complement-derived membrane attack complex (MAC) are co-localized in senile plaques of brains from Alzheimer's disease (AD) patients. But the relationship between A beta and complement activation is unclear. We have used human neurotypic cells, differentiated SH-SY5Y, as a model system to examine regulation of neuronal MAC expression and cell death by A beta. We demonstrated that mRNAs (C1q, C2, C3, C4, C5, C6, C7, C8 and C9) and proteins (C1q, C3 and C9) for the major components of the classical complement cascade are present in the SH-SY5Y neurotypic cells, indicating that neuronal cells can synthesize the necessary proteins required for MAC formation. Furthermore, immunocytochemical studies showed the A beta-induced neuronal MAC expression on the SH-SY5Y cells after CD59 was removed by PIPLC or blocked by anti-CD59 antibody. Meanwhile, increased A beta-induced neuronal cell death was observed following treatment with anti-CD59. Taken together, these results suggest that A beta activates neuronal complement cascade to induce MAC, and a deficiency of endogenous complement regulatory proteins, e.g., CD59, may increase the vulnerability of neurons to complement-mediated cytotoxicity. PMID:9689469

  6. Transfected human neuropeptide Y cDNA expression in mouse pituitary cells. Inducible high expression, peptide characterization, and secretion.

    PubMed

    Dickerson, I M; Dixon, J E; Mains, R E

    1987-10-01

    An expression vector was constructed that placed the cDNA for human neuropeptide Y (NPY) under the control of the mouse metallothionein promoter and was used to transfect the AtT-20 mouse anterior pituitary corticotrope cell line. AtT-20 cells normally process the pro-ACTH/endorphin precursor but do not produce detectable levels of NPY. The resulting AtT-20/NPY cell line (Mt.NPY1a) was used to study the ability of the corticotrope cells to synthesize, process, and secrete the foreign proNPY-related peptide products. The stable cell line created contains approximately 40 copies of proNPY cDNA per cell. NPY mRNA levels and proNPY synthesis were increased at least 35-fold when maximally induced with cadmium; proNPY synthesis was also induced by glucocorticoids. Upon induction the NPY secretion rate was equimolar to that of the endogenous peptides. ProNPY, NPY, and the COOH-terminal peptide produced by this cell line had molecular weight and amino acid-labeling pattern predicted from cDNA sequence data and from previous isolation of NPY-related molecules from NPY-producing cells. The structures of secreted proNPY, NPY, and COOH-terminal peptide, as well as determination of the site of proteolytic cleavage between NPY and the COOH-terminal peptide, were determined by tryptic mapping and Edman degradation of secreted biosynthetically labeled peptide products. The proNPY molecule appears to be processed in the same pathway responsible for cleavage of the endogenous pro-ACTH/endorphin precursor. Secretion of proNPY-derived peptides paralleled secretion of endogenous pro-ACTH/endorphin-derived products, under both basal and stimulated conditions. With induction proNPY expression there is a dose-dependent inhibition of both proNPY and pro-ACTH/endorphin proteolytic processing.

  7. In vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models

    PubMed Central

    Almeida, Joao Paulo Mattos; Lin, Adam Yuh; Figueroa, Elizabeth Raquel

    2015-01-01

    Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, we hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16-OVA tumor model. AuNP delivery of OVA (AuNP-OVA) and of CpG (AuNP-CpG) enhanced the efficacy of both agents and induced strong antigen-specific responses. In addition, we found that AuNP-OVA delivery alone, without CpG, was sufficient to promote significant antigen-specific responses, leading to subsequent anti-tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy was likely due to the adjuvant effect of peptide coated AuNPs, as they induced inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, we demonstrate that AuNP mediated OVA peptide delivery can produce significant therapeutic benefit without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination. PMID:25354691

  8. Synthetic Amphipathic Helical Peptides Targeting CD36 Attenuate Lipopolysaccharide-Induced Inflammation and Acute Lung Injury.

    PubMed

    Bocharov, Alexander V; Wu, Tinghuai; Baranova, Irina N; Birukova, Anna A; Sviridov, Denis; Vishnyakova, Tatyana G; Remaley, Alan T; Eggerman, Thomas L; Patterson, Amy P; Birukov, Konstantin G

    2016-07-15

    Synthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema. PMID:27316682

  9. Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium

    PubMed Central

    Geldart, Kathryn; Borrero, Juan

    2015-01-01

    Antibiotic-resistant enterococcal infections are a major concern in hospitals where patients with compromised immunity are readily infected. Enterococcus faecium bacteria are of particular interest as these pathogens account for over 80% of vancomycin-resistant enterococcal infections. Antimicrobial peptides (AMPs) produced at the site of infection by engineered bacteria may offer a potential alternative to traditional antibiotics for the treatment of resistant bacteria such as E. faecium. For this mode of delivery to be effective, it is essential to identify a suitable protein expression system that can be used in the desired delivery bacterium. In this study, we describe a promising chloride-inducible promoter and its application in the bacterial delivery of AMPs from Lactococcus lactis to reduce counts of E. faecium bacteria in vitro. Reporter gene studies show that at chloride concentrations found within the human intestines, the chloride-inducible promoter exhibits high levels of protein expression compared to those of the commonly used nisin-inducible promoter. These results indicate that this system is powerful and would not require the exogenous administration of an inducer molecule. In its application for AMP production against E. faecium in vitro, L. lactis producing AMPs under the chloride promoter rapidly decreased E. faecium counts by nearly 10,000-fold. As an extension of this application, we also demonstrate the potential in using this type of delivery system in combination with traditional antibiotics to slow the development of resistance. Collectively, this study shows the promise of using a chloride-inducible promoter for the bacterial delivery of AMPs in the body for the treatment of vancomycin-resistant enterococci (VRE) and other antibiotic-resistant bacteria. PMID:25841002

  10. Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium.

    PubMed

    Geldart, Kathryn; Borrero, Juan; Kaznessis, Yiannis N

    2015-06-01

    Antibiotic-resistant enterococcal infections are a major concern in hospitals where patients with compromised immunity are readily infected. Enterococcus faecium bacteria are of particular interest as these pathogens account for over 80% of vancomycin-resistant enterococcal infections. Antimicrobial peptides (AMPs) produced at the site of infection by engineered bacteria may offer a potential alternative to traditional antibiotics for the treatment of resistant bacteria such as E. faecium. For this mode of delivery to be effective, it is essential to identify a suitable protein expression system that can be used in the desired delivery bacterium. In this study, we describe a promising chloride-inducible promoter and its application in the bacterial delivery of AMPs from Lactococcus lactis to reduce counts of E. faecium bacteria in vitro. Reporter gene studies show that at chloride concentrations found within the human intestines, the chloride-inducible promoter exhibits high levels of protein expression compared to those of the commonly used nisin-inducible promoter. These results indicate that this system is powerful and would not require the exogenous administration of an inducer molecule. In its application for AMP production against E. faecium in vitro, L. lactis producing AMPs under the chloride promoter rapidly decreased E. faecium counts by nearly 10,000-fold. As an extension of this application, we also demonstrate the potential in using this type of delivery system in combination with traditional antibiotics to slow the development of resistance. Collectively, this study shows the promise of using a chloride-inducible promoter for the bacterial delivery of AMPs in the body for the treatment of vancomycin-resistant enterococci (VRE) and other antibiotic-resistant bacteria. PMID:25841002

  11. Exploiting anthracene photodimerization within peptides: light induced sequence-selective DNA binding.

    PubMed

    Bullen, Gemma A; Tucker, James H R; Peacock, Anna F A

    2015-05-11

    The unprecedented use of anthracene photodimerization within a protein or peptide system is explored through its incorporation into a DNA-binding peptide, derived from the GCN4 transcription factor. This study demonstrates an effective and dynamic interplay between a photoreaction and a peptide-DNA assembly, with each process able to exert control over the other.

  12. HER-3 peptide vaccines/mimics: Combined therapy with IGF-1R, HER-2, and HER-1 peptides induces synergistic antitumor effects against breast and pancreatic cancer cells

    PubMed Central

    Miller, Megan Jo; Foy, Kevin C; Overholser, Jay P; Nahta, Rita; Kaumaya, Pravin TP

    2014-01-01

    The human epidermal growth factor receptor 3 (HER-3/ErbB3) is a unique member of the human epidermal growth factor family of receptors, because it lacks intrinsic kinase activity and ability to heterodimerize with other members. HER-3 is frequently upregulated in cancers with epidermal growth factor receptor (EGFR/HER-1/ErbB1) or human epidermal growth factor receptor 2 (HER-2/ErBB2) overexpression, and targeting HER-3 may provide a route for overcoming resistance to agents that target EGFR or HER-2. We have previously developed vaccines and peptide mimics for HER-1, HER-2 and vascular endothelial growth factor (VEGF). In this study, we extend our studies by identifying and evaluating novel HER-3 peptide epitopes encompassing residues 99–122, 140–162, 237–269 and 461–479 of the HER-3 extracellular domain as putative B-cell epitopes for active immunotherapy against HER-3 positive cancers. We show that the HER-3 vaccine antibodies and HER-3 peptide mimics induced antitumor responses: inhibition of cancer cell proliferation, inhibition of receptor phosphorylation, induction of apoptosis and antibody dependent cellular cytotoxicity (ADCC). Two of the HER-3 epitopes 237–269 (domain II) and 461–479 (domain III) significantly inhibited growth of xenografts originating from both pancreatic (BxPC3) and breast (JIMT-1) cancers. Combined therapy of HER-3 (461–471) epitope with HER-2 (266–296), HER-2 (597–626), HER-1 (418–435) and insulin-like growth factor receptor type I (IGF-1R) (56–81) vaccine antibodies and peptide mimics show enhanced antitumor effects in breast and pancreatic cancer cells. This study establishes the hypothesis that combination immunotherapy targeting different signal transduction pathways can provide effective antitumor immunity and long-term control of HER-1 and HER-2 overexpressing cancers. PMID:25941588

  13. A novel immunogenic CS1-specific peptide inducing antigen-specific cytotoxic T lymphocytes targeting multiple myeloma

    PubMed Central

    Bae, Jooeun; Song, Weihua; Smith, Robert; Daley, John; Tai, Yu-Tzu; Anderson, Kenneth C.; Munshi, Nikhil C.

    2013-01-01

    The CS1 antigen provides a unique target for the development of an immunotherapeutic strategy to treat patients with multiple myeloma (MM). This study aimed to identify HLA-A2+ immunogenic peptides from the CS1 antigen, which induce peptide-specific cytotoxic T lymphocytes (CTL) against HLA-A2+ MM cells. We identified a novel immunogenic HLA-A2-specific CS1239–247 (SLFVLGLFL) peptide, which induced CS1-specific CTL (CS1-CTL) to MM cells. The CS1-CTL showed a distinct phenotype, with an increased percentage of effector memory and activated CTL and a decreased percentage of naive CTL. CS1239–247 peptide-specific CD8+ T cells were detected by DimerX analyses and demonstrated functional activities specific to the peptide. The CTL displayed HLA-A2-restricted and antigen-specific cytotoxicity, proliferation, degranulation and γ-interferon (IFN-γ)production against both primary MM cells and MM cell lines. In addition, the effector memory cells subset (CD45RO−CCR7−/CD3+CD8+) within CS1-CTL showed a higher level of CD107a degranulation and IFN-γproduction as compared to effector cells (CD45RO−CCR7−/CD3+CD8+) against HLA-A2+ primary MM cells or MM cell lines. In conclusion, this study introduced a novel immunogenic HLA-A2-specific CS1239–247 peptide capable of inducing antigen-specific CTL against MM cells that will provide a framework for its application as a novel MM immunotherapy. PMID:22533610

  14. A novel immunogenic CS1-specific peptide inducing antigen-specific cytotoxic T lymphocytes targeting multiple myeloma.

    PubMed

    Bae, Jooeun; Song, Weihua; Smith, Robert; Daley, John; Tai, Yu-Tzu; Anderson, Kenneth C; Munshi, Nikhil C

    2012-06-01

    The CS1 antigen provides a unique target for the development of an immunotherapeutic strategy to treat patients with multiple myeloma (MM). This study aimed to identify HLA-A2(+) immunogenic peptides from the CS1 antigen, which induce peptide-specific cytotoxic T lymphocytes (CTL) against HLA-A2(+) MM cells. We identified a novel immunogenic HLA-A2-specific CS1(239-247) (SLFVLGLFL) peptide, which induced CS1-specific CTL (CS1-CTL) to MM cells. The CS1-CTL showed a distinct phenotype, with an increased percentage of effector memory and activated CTL and a decreased percentage of naïve CTL. CS1(239-247) peptide-specific CD8(+) T cells were detected by DimerX analyses and demonstrated functional activities specific to the peptide. The CTL displayed HLA-A2-restricted and antigen-specific cytotoxicity, proliferation, degranulation and γ-interferon (IFN-γ) production against both primary MM cells and MM cell lines. In addition, the effector memory cells subset (CD45RO(+) CCR7(-) /CD3(+) CD8(+) ) within CS1-CTL showed a higher level of CD107a degranulation and IFN-γ production as compared to effector cells (CD45RO(-) CCR7(-) /CD3(+) CD8(+) ) against HLA-A2(+) primary MM cells or MM cell lines. In conclusion, this study introduced a novel immunogenic HLA-A2-specific CS1(239-247) peptide capable of inducing antigen-specific CTL against MM cells that will provide a framework for its application as a novel MM immunotherapy. PMID:22533610

  15. Antimicrobial Peptide Lactoferricin B-Induced Rapid Leakage of Internal Contents from Single Giant Unilamellar Vesicles.

    PubMed

    Moniruzzaman, Md; Alam, Jahangir Md; Dohra, Hideo; Yamazaki, Masahito

    2015-09-29

    Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to

  16. Antimicrobial Peptide Lactoferricin B-Induced Rapid Leakage of Internal Contents from Single Giant Unilamellar Vesicles.

    PubMed

    Moniruzzaman, Md; Alam, Jahangir Md; Dohra, Hideo; Yamazaki, Masahito

    2015-09-29

    Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to

  17. The Peptide Vaccine Combined with Prior Immunization of a Conventional Diphtheria-Tetanus Toxoid Vaccine Induced Amyloid β Binding Antibodies on Cynomolgus Monkeys and Guinea Pigs

    PubMed Central

    Yano, Akira; Ito, Kaori; Miwa, Yoshikatsu; Kanazawa, Yoshito; Chiba, Akiko; Iigo, Yutaka; Kashimoto, Yoshinori; Kanda, Akira; Murata, Shinji; Makino, Mitsuhiro

    2015-01-01

    The reduction of brain amyloid beta (Aβ) peptides by anti-Aβ antibodies is one of the possible therapies for Alzheimer's disease. We previously reported that the Aβ peptide vaccine including the T-cell epitope of diphtheria-tetanus combined toxoid (DT) induced anti-Aβ antibodies, and the prior immunization with conventional DT vaccine enhanced the immunogenicity of the peptide. Cynomolgus monkeys were given the peptide vaccine subcutaneously in combination with the prior DT vaccination. Vaccination with a similar regimen was also performed on guinea pigs. The peptide vaccine induced anti-Aβ antibodies in cynomolgus monkeys and guinea pigs without chemical adjuvants, and excessive immune responses were not observed. Those antibodies could preferentially recognize Aβ40, and Aβ42 compared to Aβ fibrils. The levels of serum anti-Aβ antibodies and plasma Aβ peptides increased in both animals and decreased the brain Aβ40 level of guinea pigs. The peptide vaccine could induce a similar binding profile of anti-Aβ antibodies in cynomolgus monkeys and guinea pigs. The peptide vaccination could be expected to reduce the brain Aβ peptides and their toxic effects via clearance of Aβ peptides by generated antibodies. PMID:26539559

  18. Quantitative Peptidomics Study Reveals That a Wound-Induced Peptide from PR-1 Regulates Immune Signaling in Tomato[W][OPEN

    PubMed Central

    Chen, Ying-Lan; Lee, Chi-Ying; Cheng, Kai-Tan; Chang, Wei-Hung; Huang, Rong-Nan; Nam, Hong Gil

    2014-01-01

    Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (Solanum lycopersicum) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in Arabidopsis thaliana, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species. PMID:25361956

  19. Calcitonin gene-related peptide-induced suppression of luteinizing hormone pulses in the rat: the role of endogenous opioid peptides

    PubMed Central

    Bowe, JE; Li, XF; Kinsey-Jones, JS; Paterson, S; Brain, SD; Lightman, SL; O'Byrne, KT

    2005-01-01

    Calcitonin gene-related peptide (CGRP) is involved in a variety of stress responses in the rat. Central administration of CGRP activates the hypothalamo–pituitary–adrenal axis resulting in increased corticosterone secretion. We have previously shown that central CGRP suppresses the gonadotrophin-releasing hormone (GnRH) pulse generator, specifically LH pulses. Endogenous opioid peptides (EOPs) have been shown to play an important role in stress-induced suppression of the reproductive axis. The aim of the present study was to test the hypothesis that EOPs mediate CGRP-induced suppression of pulsatile LH secretion. Ovariectomized rats were implanted with intracerebroventricular (i.c.v.) and i.v. cannulae. Intravenous administration of the opioid antagonist naloxone (250 μg) completely blocked the suppression of LH pulses induced by 1.5 μg i.c.v. CGRP and significantly attenuated the suppression of pulsatile LH secretion induced by 5 μg i.c.v. CGRP. Furthermore, intravenous administration of naloxone was found to immediately restore normal LH pulse frequency in animals treated 90 min earlier with 1.5 μg i.c.v. CGRP. Co-administration (i.c.v.) of CGRP (1.5 μg) with the μ and κ opioid receptor-specific antagonists naloxone (10 μg) and norbinaltorphimine (5 μg), respectively, blocked the CGRP-induced suppression of LH pulses, whilst i.c.v. co-administration of CGRP (1.5 μg) with the δ opioid receptor-specific antagonist naltrindole (5 μg) did not. These data provide evidence that EOPs play a pivotal role in mediating the inhibitory effects of CGRP on pulsatile LH secretion in the rat. They also suggest that the μ and κ, but not the δ, opioid receptors may be responsible for mediating the effects of CGRP on LH pulses. PMID:15905218

  20. Peptides as modifiers of Na+-induced pinocytosis in starved Amoeba proteus.

    PubMed

    Josefsson, J O; Johansson, P

    1985-01-01

    Low concentrations of six peptide hormones; glucagon, vasoactive intestinal peptide, substance P, angiotensin II, lysine-vasopressin, arginine-vasopressin, and the chemotactic peptide fMet-Leu-Phe, activated the capacity for pinocytosis in starved Amoeba proteus. Competitive inhibitors of the chemotactic peptide in leucocytes inhibited activation by fMet-Leu-Phe, suggesting that its action in the amoeba is mediated by specific receptors. The opioid peptides, beta-endorphin, dynorphin (1-13) and leu-enkephalin abolished through a naloxone-sensitive mechanism activation by hormones and several other activating agents. Also, low concentrations of beef and pork insulin inhibited activation by peptide hormones. An insulin analogue of low potency in mammalian cells was inactive in the amoeba. These results support the hypothesis that besides opioid receptors, there may be insulin receptors and possibly receptors for several other peptide hormones in Amoeba proteus.

  1. Smad-induced alterations of matrix metabolism by a myristoyl tetra peptide.

    PubMed

    Kwon, Haeyoung; Lee, Yun Sub; Kim, Myung Ok; Chang, Min Youl; Won, Bo Mi; Jin, Byung Suk; Park, Seyeon

    2014-12-01

    Regulation of extracellular matrix (ECM) components is essential for tissue homeostasis and function. We screened a small peptide that induces ECM protein synthesis for its usefulness in protecting keratinocytes. In this report, we demonstrate that myristoyl tetrapeptide Ala-Ala-Pro-Val (mAAPV) stimulates the expression of ECM proteins and inhibits the expression of metalloproteinases (MMPs) that degrade ECM proteins in Hs68 human fibroblast cells. In order to elucidate the underlying molecular mechanisms for the effects of mAAVP, we investigated the changes in gene expression in the presence of mAAPV using a cDNA microarray. Treatment with mAAPV resulted in decreased expression of MMP-related genes such as MMP1, MMP3, TIMP1 and TIMP3 and increased expression of collagen genes, including COL1A1, COL1A2, COL3A1, COL5A1 and COL6A3. The pattern of gene expression regulated by mAAPV was very similar to that of gene expression induced by transforming growth factor (TGF)-β, indicating that the TGF-β signaling pathway is crucial for simultaneous activation of several ECM-related genes by mAAPV. We examined whether the activation of SMAD, a downstream protein of TGF-β receptor, is involved in the signal transduction pathway induced by mAAPV. The results demonstrate that mAAVP directly activates SMAD2 and induces SMAD3 to bind to DNA. In conclusion, our results demonstrate that mAAPV both enhances the expression of collagen and inhibits its degradation via production of protease inhibitors that prevent enzymatic breakdown of the ECM. The results suggest that mAAPV would be a useful ECM-protecting agent. PMID:25289880

  2. Neuropeptide FF and related peptides attenuates warm-, but not cold-water swim stress-induced analgesia in mice.

    PubMed

    Li, Ning; Han, Zheng-lan; Fang, Quan; Wang, Zi-long; Tang, Hong-zhu; Ren, Hui; Wang, Rui

    2012-08-01

    Neuropeptide FF (NPFF) belongs to a neuropeptide family including two receptors (NPFF(1) and NPFF(2)). NPFF system has been reported to play important roles in pain transmission. The aim of the present study was to investigate the roles of NPFF related peptides and their receptors in swim stress-induced analgesia (SIA). Nociceptive test was performed in mice stressed by forced swimming in water at 15 °C (cold water swimming) or 32 °C (warm water swimming). Warm water swimming produced a naloxone-mediated antinociceptive effect. This warm water swim SIA was dose-dependently antagonized by i.c.v. injection of NPFF and two related peptides (3-30 nmol), NPVF and dNPA, which exhibited the highest selectivities for NPFF(1) and NPFF(2) receptors, respectively. Moreover, the selective NPFF receptor antagonist RF9 (30 nmol) was inactive by itself, but prevented the effects of NPFF and related peptides. Cold-water swimming produced a wilder analgesic effect that was blocked by MK-801, but not naloxone. However, NPFF system failed to modify the cold water swim stress-induced analgesia. These findings demonstrated that NPFF and related peptides attenuated opioid-mediated form of SIA via NPFF receptors in the brain, but not non-opioid swim stress-induced analgesia. These data further support an anti-opioid character of NPFF system.

  3. Asymmetrical myocardial expression of natriuretic peptides in pacing-induced heart failure.

    PubMed

    Del Ry, Silvia; Cabiati, Manuela; Lionetti, Vincenzo; Simioniuc, Anca; Caselli, Chiara; Prescimone, Tommaso; Emdin, Michele; Giannessi, Daniela

    2009-09-01

    High-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive heart failure (HF) with pronounced differences in regional contractility. Aim of this study was to evaluate possible changes in brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) as compared to the infero-septal region (opposite site, OS) and to explore possible association between the contractiling pattern and biomarker expression. Cardiac tissue was collected from minipigs with pacing-induced HF (n=8) and without (control, n=6). The samples were selectively harvested from the anterior left ventricular (LV) wall, PS, and from an area remote to the pacing-site, OS. BNP and CNP mRNA expression was evaluated by semi-quantitative polymerase chain reaction (PCR). A significant difference in BNP expression was found in the PS between HF animals and controls (BNP/GAPDH: 0.65+/-0.11 vs. 0.35+/-0.04, p=0.02), but not in the OS (BNP/GAPDH: 0.36+/-0.05, ns vs. controls). CNP expression was not different compared to controls, although higher levels were observed in the PS and in the OS with respect to the controls (CNP/GAPDH: controls 0.089+/-0.036, PS 0.289+/-0.23, OS 0.54+/-0.16). This finding was in tune with an increase of CNP tissue concentration (controls: 0.69+/-0.13; PS=1.56+/-0.19; OS=1.70+/-0.42 pg/mg protein; p=0.039 controls vs. OS). Higher BNP mRNA expression in the PS is consistent with a reduction in contractile function in this region, while higher CNP mRNA expression in the OS suggests the presence of concomitant endothelial dysfunction in the remote region.

  4. LXXLL Peptide Converts Transportan 10 to a Potent Inducer of Apoptosis in Breast Cancer Cells

    PubMed Central

    Tints, Kairit; Prink, Madis; Neuman, Toomas; Palm, Kaia

    2014-01-01

    Degenerate expression of transcription coregulator proteins is observed in most human cancers. Therefore, in targeted anti-cancer therapy development, intervention at the level of cancer-specific transcription is of high interest. The steroid receptor coactivator-1 (SRC-1) is highly expressed in breast, endometrial, and prostate cancer. It is present in various transcription complexes, including those containing nuclear hormone receptors. We examined the effects of a peptide that contains the LXXLL-motif of the human SRC-1 nuclear receptor box 1 linked to the cell-penetrating transportan 10 (TP10), hereafter referred to as TP10-SRC1LXXLL, on proliferation and estrogen-mediated transcription of breast cancer cells in vitro. Our data show that TP10-SRC1LXXLL induced dose-dependent cell death of breast cancer cells, and that this effect was not affected by estrogen receptor (ER) status. Surprisingly TP10-SRC1LXXLL severely reduced the viability and proliferation of hormone-unresponsive breast cancer MDA-MB-231 cells. In addition, the regulation of the endogenous ERα direct target gene pS2 was not affected by TP10-SRC1LXXLL in estrogen-stimulated MCF-7 cells. Dermal fibroblasts were similarly affected by treatment with higher concentrations of TP10-SRC1LXXLL and this effect was significantly delayed. These results suggest that the TP10-SRC1LXXLL peptide may be an effective drug candidate in the treatment of cancers with minimal therapeutic options, for example ER-negative tumors. PMID:24705462

  5. Proteinase-activated receptors induce nonoxidative, antimicrobial peptides and increased antimicrobial activity in human mononuclear phagocytes.

    PubMed

    Lippuner, Nadine; Morell, Bernhard; Schaffner, Andreas; Schaer, Dominik J

    2007-02-01

    As thrombin and SFLLRNPNDKYEPF (SFLLRN-14), a synthetic ligand, mainly of the proteinase-activated receptor-1 (PAR-1), induce in monocytes the synthesis and secretion of chemokines, the PAR pathway can be viewed as a mononuclear phagocyte-activating principle. Classically, antimicrobial activity of mononuclear phagocytes is the measure for activation. Here, we investigated whether thrombin or SFLLRN-14 increases the antimicrobial activity of human monocytes and compared these effects to those of IFN-gamma. Furthermore, we measured the effects of these agents on the secretion of reactive oxygen intermediates and the antimicrobial activity of acid peptide extracts from monocytes. Human monocytes were exposed to maximally active concentrations of thrombin, SFLLRN-14, and IFN-gamma. Human monocytes treated with thrombin or SFLLRN-14 and then challenged with Salmonella enterica serovar typhimurium, including its attenuated mutant phoP, or Listeria monocytogenes killed, within 3 h, significantly more bacteria than control cells, an effect comparable with or surpassing the effect of IFN-gamma. This finding establishes the proteinase-PAR pathway as a potent, alternate activation pathway of mononuclear phagocytes. Thrombin and SFLLRN-14 had no significant effects on the amount of H(2)O(2) secreted by monocytes. This was in contrast to IFN-gamma, which as expected, increased the secretion of H(2)O(2) by approximately fourfold. Thrombin and SFLLRN-14, but not IFN-gamma, however, significantly increased the antimicrobial activity of acid peptide extracts of monocytes in a radial diffusion assay. Taken together, these findings suggest that IFN-gamma and thrombin differentially regulate oxidative and nonoxidative killing systems of human monocytes. PMID:17095611

  6. Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion.

    PubMed Central

    Kahya, N; Pécheur, E I; de Boeij, W P; Wiersma, D A; Hoekstra, D

    2001-01-01

    In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state. PMID:11509360

  7. Polysaccharide peptide induces a tumor necrosis factor-α-dependent drop of body temperature in rats.

    PubMed

    Jedrzejewski, Tomasz; Piotrowski, Jakub; Wrotek, Sylwia; Kozak, Wieslaw

    2014-08-01

    Polysaccharide peptide (PSP) extracted from the Coriolus versicolor mushroom is frequently suggested as an adjunct to the chemo- or radiotherapy in cancer patients. It improves quality of the patients' life by decreasing pain, fatigue, loss of appetite, nausea, and vomiting. However, the effect of PSP on body temperature has not thus far been studied, although it is well known that treatment with other polysaccharide adjuvants, such as lipopolysaccharides, may induce fever. The aim of the present study, therefore, was to investigate the influence of PSP on temperature regulation in rats. We report that intraperitoneal injection of PSP provoked a dose-dependent decrease of temperature in male Wistar rats equipped with biotelemetry devices to monitor deep body temperature (Tb). The response was rapid (i.e., with latency of 15-20min), transient (lasting up to 5h post-injection), and accompanied by a significant elevation of the blood tumor necrosis factor-α (TNF-α) level. Pretreatment of the rats with anti-TNF-α antibody prevented the PSP-induced drop in Tb. Based on these data, we conclude that rats may develop an anapyrexia-like response to the injection of peptidopolysaccharide rather than fever, and the response was TNF-α-dependent.

  8. Involvement of atrial natriuretic peptide in blood pressure reduction induced by estradiol in spontaneously hypertensive rats.

    PubMed

    Belo, Najara O; Silva-Barra, Juliana; Carnio, Evelin C; Antunes-Rodrigues, Jose; Gutkowska, Jolanta; Dos Reis, Adelina M

    2004-01-15

    The aim of the present study was to determine the involvement of atrial natriuretic peptide (ANP) in blood pressure (BP) alterations induced by estradiol treatment. Spontaneously hypertensive rats (SHR) and Wistar rats (WR) were ovariectomized and, after 3 weeks, were injected daily for 4 days with estradiol benzoate (E2; 5 microg/100 g/day) or a vehicle. One day after the last injection, the animals were decapitated, blood was collected, and both right and left atrial appendages were quickly removed for determination of ANP by radioimmunoassay (RIA), or used for ANP mRNA determination. Estradiol treatment induced a significant reduction of blood pressure in SHR, but not in WR. This reduction was correlated with the increase of plasma ANP levels that were significantly increased in E2-treated, compared with vehicle-treated, SHR. E2-treated SHR showed significant increases in ANP concentration in the right and left atria compared to the vehicle-treated animals. These observations were confirmed by ANP mRNA. In summary, the present study shows that short-term estradiol treatment reduces the blood pressure of ovariectomized SHR, but not of WR. This reduction was highly correlated with increased plasma estradiol and ANP levels. These results suggest that ANP is involved in mediating the effect of estradiol on blood pressure reduction.

  9. SARS Coronavirus Fusion Peptide-Derived Sequence Suppresses Collagen-Induced Arthritis in DBA/1J Mice

    PubMed Central

    Shen, Zu T.; Sigalov, Alexander B.

    2016-01-01

    During the co-evolution of viruses and their hosts, the viruses have evolved numerous strategies to counter and evade host antiviral immune responses in order to establish a successful infection, replicate and persist in the host. Recently, based on our model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model, we suggested specific molecular mechanisms used by different viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (MIRRs). This family includes T cell receptor (TCR) that is critically involved in immune diseases such as autoimmune arthritis. In the present study, we provide compelling experimental in vivo evidence in support of our hypothesis. Using the SCHOOL approach and the SARS-CoV fusion peptide sequence, we rationally designed a novel immunomodulatory peptide that targets TCR. We showed that this peptide ameliorates collagen-induced arthritis in DBA/1J mice and protects against bone and cartilage damage. Incorporation of the peptide into self-assembling lipopeptide nanoparticles that mimic native human high density lipoproteins significantly increases peptide dosage efficacy. Together, our data further confirm that viral immune evasion strategies that target MIRRs can be transferred to therapeutic strategies that require similar functionalities. PMID:27349522

  10. SARS Coronavirus Fusion Peptide-Derived Sequence Suppresses Collagen-Induced Arthritis in DBA/1J Mice.

    PubMed

    Shen, Zu T; Sigalov, Alexander B

    2016-01-01

    During the co-evolution of viruses and their hosts, the viruses have evolved numerous strategies to counter and evade host antiviral immune responses in order to establish a successful infection, replicate and persist in the host. Recently, based on our model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model, we suggested specific molecular mechanisms used by different viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (MIRRs). This family includes T cell receptor (TCR) that is critically involved in immune diseases such as autoimmune arthritis. In the present study, we provide compelling experimental in vivo evidence in support of our hypothesis. Using the SCHOOL approach and the SARS-CoV fusion peptide sequence, we rationally designed a novel immunomodulatory peptide that targets TCR. We showed that this peptide ameliorates collagen-induced arthritis in DBA/1J mice and protects against bone and cartilage damage. Incorporation of the peptide into self-assembling lipopeptide nanoparticles that mimic native human high density lipoproteins significantly increases peptide dosage efficacy. Together, our data further confirm that viral immune evasion strategies that target MIRRs can be transferred to therapeutic strategies that require similar functionalities. PMID:27349522

  11. Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides

    NASA Astrophysics Data System (ADS)

    Samgina, Tatiana Y.; Vorontsov, Egor A.; Gorshkov, Vladimir A.; Artemenko, Konstantin A.; Zubarev, Roman A.; Ytterberg, Jimmy A.; Lebedev, Albert T.

    2013-07-01

    Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S-S bonds is often used in "bottom up" sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the "hidden" C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S-S bond rupture does not occur in this case.

  12. A mucin-like peptide from Fasciola hepatica induces parasite-specific Th1-type cell immunity.

    PubMed

    Noya, Verónica; Brossard, Natalie; Berasaín, Patricia; Rodríguez, Ernesto; Chiale, Carolina; Mazal, Daniel; Carmona, Carlos; Freire, Teresa

    2016-03-01

    Fasciolosis, caused by the liver fluke Fasciola hepatica, is a major parasitic disease of livestock that causes significant economic losses worldwide. Although drugs are effective against liver flukes, they do not prevent reinfection, and continuous treatment is costly. Moreover, resistant fluke strains are emerging. In this context, vaccination is a good alternative since it provides a cost-effective long-term prevention strategy to control fasciolosis. In this paper, we evaluate the Fhmuc peptide as a potential vaccine against fasciolosis. This peptide derives from a mucin-like protein highly expressed in the infective stage of Fasciola hepatica. Mucin-like molecules expressed by parasites can contribute to several infection processes by protecting the parasite from host proteases and recognition by the immune system. We show that the Fhmuc peptide induces Th1-like immune responses specific for F. hepatica excretion-secretion products (FhESP) with a high production of IFNγ. We also investigated whether this peptide could protect animals from infection, and present preliminary data indicating that animals treated with Fhmuc exhibited reduced liver damage compared to non-immunised animals and that this protection was associated with a recruitment of B and T lymphocytes in the peritoneum, as well as eosinophils and mature dendritic cells. These results suggest that the mucin-like peptide Fhmuc could constitute a potential vaccine candidate against fasciolosis and pave the way towards the development of vaccines against parasites.

  13. Inhibition of interleukin-1 beta-induced pyresis in the rabbit by peptide 204-212 of lipocortin 5.

    PubMed

    Palmi, M; Frosini, M; Sgaragli, G P; Becherucci, C; Perretti, M; Parente, L

    1995-07-25

    The intracerebroventricular administration of interleukin-1 beta (12.5 ng/kg) in rabbits caused a prompt rise of prostaglandin E2 concentration (+ 632.6 +/- 243.9%) in the cerebrospinal fluid followed by hyperthermia (+ 1.61 +/- 0.14 delta degrees C). The intracerebroventricular administration of an anti-inflammatory nonapeptide (amino acids 204-212, SHLRKVFDK) derived from lipocortin 5, thereafter referred to as lipocortin 5-(204-212)-peptide, inhibited in a significant manner both the increase in cerebrospinal fluid [prostaglandin E2] and the febrile response induced by the cytokine. This inhibitory effect is probably due to interference by the peptide with phospholipase A2 activity. A control peptide (FKRVHDLKS) formed by the same amino acids in a randomly shuffled sequence had no effect. These results show that, in addition to the anti-inflammatory effect previously reported, the peptide 204-212 of lipocortin 5 possesses, like glucocorticoids, anti-pyretic activity. The research on lipocortin-derived peptides may lead to the development of novel anti-inflammatory and anti-pyretic compounds. PMID:8566125

  14. Amyloid-β peptide induces temporal membrane biphasic changes in astrocytes through cytosolic phospholipase A2

    PubMed Central

    Hicks, Jacob B.; Lai, Yinzhi; Sheng, Wenwen; Yang, Xiaoguang; Zhu, Donghui; Sun, Grace Y.; Lee, James C-M

    2008-01-01

    Oligomeric amyloid-β peptide (Aβ) is known to induce cytotoxic effects and to damage cell functions in Alzheimer’s disease. However, mechanisms underlying the effects of Aβ on cell membranes have yet to be fully elucidated. In this study, Aβ 1–42 (Aβ42) was shown to cause a temporal biphasic change in membranes of astrocytic DITNC cells using fluorescence microscopy of Laurdan. Aβ42 made astrocyte cell membranes became more molecularly-disordered within the first 30 minutes to 1 hour, but gradually changed to more molecularly-ordered after 3 hours. However, Aβ42 caused artificial membranes of vesicles made of rat whole brain lipid extract to become more disordered only. The trend for more molecularly-ordered membranes in astrocytes induced by Aβ42 was abrogated by either an NADPH oxidase inhibitor, apocynin, or an inhibitor of cytosolic phospholipase A2 (cPLA2), but not by an inhibitor of calcium-independent PLA2 (iPLA2). Apocynin also suppressed the increased production of superoxide anions (O2.−) and phosphorylation of cPLA2 induced by Aβ42. In addition, hydrolyzed products of cPLA2, arachidonic acid (AA), but not lysophosphatidylcholine (LPC) caused astrocyte membranes to become more molecularly-ordered. These results suggest (1) a direct interaction of Aβ42 with cell membranes making them more molecularly-disordered, and (2) Aβ42 also indirectly makes membranes become more molecularly-ordered by triggering the signaling pathway involving NADPH oxidase and cPLA2 in astrocytes. PMID:18725190

  15. Antihepatotoxic effect of corn peptides against Bacillus Calmette-Guerin/lipopolysaccharide-induced liver injury in mice.

    PubMed

    Guo, Hui; Sun, Jie; He, Hui; Yu, Guo-Cai; Du, Jing

    2009-10-01

    Hepatitis is a severe disease with a high incidence rate around the world [Hwang, J.M., Tseng, T.H., Tsai, Y.Y., Lee, H.J., Chou, F.P., Wang, C.J., Chu, C.Y., 2005. Protective effects of baicalein on tert-butyl hydroperoxide-induced hepatic toxicity in rat hepatocytes. J. Biomed. Sci. 12, 389-397]. Corn gluten meal is a byproduct of starch industry with abundant protein. However, the application of corn protein is limited because of its low solubility and short of essential amino acids such as lysine and tryptophan. The hepatoprotective activity of corn peptides (CP) from corn gluten meal hydrolysate was evaluated against Bacillus Calmette-Guerin (BCG)/lipopolysaccharide (LPS) induced immunological liver injury (ILI) in mice. Results showed that ILI was manifested by a significant increase in levels of serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT) and liver malondialdehyde (MDA)/nitric oxide (NO) levels (p<0.01), and by a significant decrease in levels of superoxide dismutase (SOD)/glutathione peroxidase (GPX) and glutathione (GSH) in liver (p<0.01). Pretreatment of mice with CP reversed these altered parameters to normal values. The effect of CP was further demonstrated by histopathological examination of liver sections. The best hepatoprotective effect of CP treatment was observed at the dose of 600 mg/kg bw, which was evidenced from biochemical parameters and liver histopathological characters. Results of this study revealed that CP could afford a significant protection against BCG/LPS-induced hepatocellular injury. It will broaden the application and increase the value of corn gluten meal, byproduct from starch industry.

  16. Natriuretic Peptide-Induced Catecholamine Release from Cardiac Sympathetic Neurons: Inhibition by Histamine H3 and H4 Receptor Activation

    PubMed Central

    Chan, Noel Yan-Ki; Robador, Pablo A.

    2012-01-01

    We reported previously that natriuretic peptides, including brain natriuretic peptide (BNP), promote norepinephrine release from cardiac sympathetic nerves and dopamine release from differentiated pheochromocytoma PC12 cells. These proexocytotic effects are mediated by an increase in intracellular calcium secondary to cAMP/protein kinase A (PKA) activation caused by a protein kinase G (PKG)-mediated inhibition of phosphodiesterase type 3 (PDE3). The purpose of the present study was to search for novel means to prevent the proadrenergic effects of natriuretic peptides. For this, we focused our attention on neuronal inhibitory Gαi/o-coupled histamine H3 and H4 receptors. Our findings show that activation of neuronal H3 and H4 receptors inhibits the release of catecholamines elicited by BNP in cardiac synaptosomes and differentiated PC12 cells. This effect results from a decrease in intracellular Ca2+ due to reduced intracellular cAMP/PKA activity, caused by H3 and H4 receptor-mediated PKG inhibition and consequent PDE3-induced increase in cAMP metabolism. Indeed, selective H3 and H4 receptor agonists each synergized with a PKG inhibitor and a PDE3 activator in attenuating BNP-induced norepinephrine release from cardiac sympathetic nerve endings. This indicates that PKG inhibition and PDE3 stimulation are pivotal for the H3 and H4 receptor-mediated attenuation of BNP-induced catecholamine release. Cardiac sympathetic overstimulation is characteristic of advanced heart failure, which was recently found not to be improved by the administration of recombinant BNP (nesiritide), despite the predicated beneficial effects of natriuretic peptides. Because excessive catecholamine release is likely to offset the desirable effects of natriuretic peptides, our findings suggest novel means to alleviate their adverse effects and improve their therapeutic potential. PMID:22923736

  17. Peptide sequencing using a patchwork approach and surface-induced dissociation in sector-TOF and dual quadrupole mass spectrometers.

    PubMed

    Fernández, Facundo M; Smith, Lori L; Kuppannan, Krishnamoorthy; Yang, Xi; Wysocki, Vicki H

    2003-12-01

    Surface-induced ion activation in combination with a database search strategy based on the Patchwork concept is applied to the determination of peptide sequences. Surface-induced dissociation (SID) is performed in a tandem quadrupole mass spectrometer and in a hybrid sector/time-of-flight mass spectrometer in order to evaluate the importance of accurate mass analysis of the SID fragment ions for peptide identification. The modified Patchwork approach is based on piecing together the peptide blocks in a bidirectional way, simultaneously using low-mass fragments originating from the C-terminus and N-terminus of the molecule, and relying on the measurement of the peptide's molecular weight with moderate mass accuracy. The results from this analysis are used as search filters in MASCOT's (http://www.matrixscience.com) Sequence Query search engine, with the simultaneous addition of the full MS/MS peak list. SID is performed with collision targets coated with pure and mixed composition self-assembled monolayers produced by fluorocarbon and hydrocarbon alkanethiolate solutions of varying chemical composition. The resulting MS/MS spectra produced on pure and mixed hydrocarbon SAMs are submitted to the modified version of Patchwork sequencing. It is found that hydrocarbon surfaces improve the relative abundance of larger fragments. Under the moderate mass accuracy conditions (+/-0.3 u) offered by our linear-TOF-SID instrument, it is found that increasing the abundance of larger fragments dramatically improves the sequencing scores.

  18. A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera

    PubMed Central

    Mita, Masatoshi; Yoshikuni, Michiyasu; Ohno, Kaoru; Shibata, Yasushi; Paul-Prasanth, Bindhu; Pitchayawasin, Suthasinee; Isobe, Minoru; Nagahama, Yoshitaka

    2009-01-01

    Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera, from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturation-inducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin. PMID:19470645

  19. Amyloidogenic amyloid-β-peptide variants induce microbial agglutination and exert antimicrobial activity.

    PubMed

    Spitzer, Philipp; Condic, Mateja; Herrmann, Martin; Oberstein, Timo Jan; Scharin-Mehlmann, Marina; Gilbert, Daniel F; Friedrich, Oliver; Grömer, Teja; Kornhuber, Johannes; Lang, Roland; Maler, Juan Manuel

    2016-01-01

    Amyloid-β (Aβ) peptides are the main components of the plaques found in the brains of patients with Alzheimer's disease. However, Aβ peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aβ peptides. Recently, anti-infective properties of Aβ peptides have been reported. Here, we investigated the interaction of Aβ peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aβ1-42, Aβ2-42, and Aβ3p-42 but not the non-amyloidogenic peptides Aβ1-40 and Aβ2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aβ peptide variants ending at position 42 (Aβx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aβx-40. Furthermore, Aβx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aβ1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aβ1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aβx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system. PMID:27624303

  20. Amyloidogenic amyloid-β-peptide variants induce microbial agglutination and exert antimicrobial activity

    PubMed Central

    Spitzer, Philipp; Condic, Mateja; Herrmann, Martin; Oberstein, Timo Jan; Scharin-Mehlmann, Marina; Gilbert, Daniel F.; Friedrich, Oliver; Grömer, Teja; Kornhuber, Johannes; Lang, Roland; Maler, Juan Manuel

    2016-01-01

    Amyloid-β (Aβ) peptides are the main components of the plaques found in the brains of patients with Alzheimer’s disease. However, Aβ peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aβ peptides. Recently, anti-infective properties of Aβ peptides have been reported. Here, we investigated the interaction of Aβ peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aβ1-42, Aβ2-42, and Aβ3p-42 but not the non-amyloidogenic peptides Aβ1-40 and Aβ2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aβ peptide variants ending at position 42 (Aβx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aβx-40. Furthermore, Aβx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aβ1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aβ1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aβx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system. PMID:27624303

  1. Interactions of peptide triazole thiols with Env gp120 induce irreversible breakdown and inactivation of HIV-1 virions

    PubMed Central

    2013-01-01

    Background We examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity. Results KR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions. Conclusions The antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion. PMID:24330857

  2. Melittin peptide kills Trypanosoma cruzi parasites by inducing different cell death pathways.

    PubMed

    Adade, Camila M; Oliveira, Isabelle R S; Pais, Joana A R; Souto-Padrón, Thaïs

    2013-07-01

    Antimicrobial peptides (AMPs) are components of the innate immune response that represent desirable alternatives to conventional pharmaceuticals, as they have a fast mode of action, a low likelihood of resistance development and can act in conjunction with existing drug regimens. AMPs exhibit strong inhibitory activity against both Gram-positive and Gram-negative bacteria, fungi, viruses, metazoans and other parasites, such as the protozoan Leishmania. Melittin is a naturally occurring AMP, which comprises 40-50% of the dry weight of Apis mellifera venom. Our group has recently shown that crude A. mellifera venom is lethal to Trypanosoma cruzi, the Chagas disease etiologic agent, and generates a variety of cell death phenotypes among treated parasites. Here, we demonstrate that the melittin affected all of T. cruzi developmental forms, including the intracellular amastigotes. The ultrastructural changes induced by melittin suggested the occurrence of different programmed cell death pathways, as was observed in A. mellifera-treated parasites. Autophagic cell death appeared to be the main death mechanism in epimastigotes. In contrast, melittin-treated trypomastigotes appeared to be dying via an apoptotic mechanism. Our findings confirm the great potential of AMPs, including melittin, as a potential source of new drugs for the treatment of neglected diseases, such as Chagas disease. PMID:23562368

  3. The role of atrial natriuretic peptide (ANP) in cold-induced diuresis (CID)

    SciTech Connect

    Agnew, J.W.; Freund, B.J.; DuBose, D.A.; McKay, J.M.; Hashiro, G.M. Tripler Army Medical Center, Honolulu, HI )

    1991-03-11

    The hormonal control of cold-induced diuresis (CID) remains unresolved. This study investigated the role of ANP, plasma vasopressin (AVP), and aldosterone (ALDO) on CID. Four semi-nude men participated in a 210 min exposure to 15C and 29C air, on separate days. These subjects drank 300 mL of water and had an intravenous saline drip throughout both exposures to replace blood and insensible fluid losses. CID was observed in 15C but not in the 29C experiment, as indicated by a greater urine output. In 15C, atrial natriuretic peptide (ANP) increased after 90 min by 41% and remained elevated for 2 h relative to 29C. No differences were observed in AVP between 15C and 29C. In the 15C versus the 29C experiment, ALDO was approximately 37% lower at the pre, 15 and 90 min time periods. Mean arterial blood pressure was generally greater but only significant at 60 min during the 15C versus the 29C experiment. Urinary NA{sup +} excretion was elevated in 15C relative to 29C while no difference in K{sup +} excretion was observed. Although pressure effects may contribute, the observed natriuresis in the absence of a kaliuresis in the cold suggests a physiological role of ANP in CID.

  4. Glucagon-like peptide-2-induced memory improvement and anxiolytic effects in mice.

    PubMed

    Iwai, Takashi; Jin, Kazushi; Ohnuki, Tomoko; Sasaki-Hamada, Sachie; Nakamura, Minami; Saitoh, Akiyoshi; Sugiyama, Azusa; Ikeda, Masaatsu; Tanabe, Mitsuo; Oka, Jun-Ichiro

    2015-02-01

    We investigated the effectiveness of glucagon-like peptide-2 (GLP-2) on memory impairment in lipopolysaccharide (LPS)-treated mice, and anxiety-like behavior in adrenocorticotropic hormone (ACTH)-treated mice. In the Y-maze test, LPS (10 µg/mouse, i.c.v.) significantly decreased spontaneous alternation, which was prevented by pretreatment with GLP-2 (0.01-0.3 µg/mouse, i.c.v.). The GLP-2 treatment just before the Y-maze test also improved LPS-induced memory impairment. Continuous treatment with GLP-2 (3 µg/mouse, i.c.v.) had no effect on the open-field test in saline-treated or ACTH-treated mice. Chronic ACTH treatment did not cause anxiogenic effects in the elevated plus-maze test. GLP-2 showed weak anxiolytic-like effects in the elevated plus-maze test in ACTH-treated, but not saline-treated mice. Moreover, GLP-2 increased 5-HT, but not 5-HIAA and tryptophan hydroxylase 2 levels in the amygdala of ACTH-treated mice. Pharmacological depletion of 5-HT prevented the anxiolytic effects of GLP-2. These results suggest that GLP-2 protected and improved memory function in LPS-treated mice, and also had anxiolytic effects due to changes in the 5-HT system.

  5. Internal energy distribution of peptides in electrospray ionization : ESI and collision-induced dissociation spectra calculation.

    PubMed

    Pak, Alireza; Lesage, Denis; Gimbert, Yves; Vékey, Károly; Tabet, Jean-Claude

    2008-04-01

    The internal energy of ions and the timescale play fundamental roles in mass spectrometry. The main objective of this study is to estimate and compare the internal energy distributions of different ions (different nature, degree of freedom 'DOF' and fragmentations) produced in an electrospray source (ESI) of a triple-quadrupole instrument (Quattro I Micromass). These measurements were performed using both the Survival Yield method (as proposed by De Pauw) and the MassKinetics software (kinetic model introduced by Vékey). The internal energy calibration is the preliminary step for ESI and collision-induced dissociation (CID) spectra calculation. meta-Methyl-benzylpyridinium ion and four protonated peptides (YGGFL, LDIFSDF, LDIFSDFR and RLDIFSDF) were produced using an electrospray source. These ions were used as thermometer probe compounds. Cone voltages (V(c)) were linearly correlated with the mean internal energy values () carried by desolvated ions. These mean internal energy values seem to be slightly dependent on the size of the studied ion. ESI mass spectra and CID spectra were then simulated using the MassKinetics software to propose an empirical equation for the mean internal energy () versus cone voltage (V(c)) for different source temperatures (T): < E(int) > = [405 x 10(-6) - 480 x 10(-9) (DOF)] V(c)T + E(therm)(T). In this equation, the E(therm)(T) parameter is the mean internal energy due to the source temperature at 0 V(c).

  6. Complement-related proteins control the flavivirus infection of Aedes aegypti by inducing antimicrobial peptides.

    PubMed

    Xiao, Xiaoping; Liu, Yang; Zhang, Xiaoyan; Wang, Jing; Li, Zuofeng; Pang, Xiaojing; Wang, Penghua; Cheng, Gong

    2014-04-01

    The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry in vertebrates and invertebrates as an immune defense mechanism. Thioester (TE)-containing proteins (TEPs), which show high similarity to mammalian complement C3, are thought to play a key role in innate immunity in arthropods. Herein, we report that a viral recognition cascade composed of two complement-related proteins limits the flaviviral infection of Aedes aegypti. An A. aegypti macroglobulin complement-related factor (AaMCR), belonging to the insect TEP family, is a crucial effector in opposing the flaviviral infection of A. aegypti. However, AaMCR does not directly interact with DENV, and its antiviral effect requires an A. aegypti homologue of scavenger receptor-C (AaSR-C), which interacts with DENV and AaMCR simultaneously in vitro and in vivo. Furthermore, recognition of DENV by the AaSR-C/AaMCR axis regulates the expression of antimicrobial peptides (AMPs), which exerts potent anti-DENV activity. Our results both demonstrate the existence of a viral recognition pathway that controls the flaviviral infection by inducing AMPs and offer insights into a previously unappreciated antiviral function of the complement-like system in arthropods.

  7. BMAP-28, an Antibiotic Peptide of Innate Immunity, Induces Cell Death through Opening of the Mitochondrial Permeability Transition Pore

    PubMed Central

    Risso, Angela; Braidot, Enrico; Sordano, Maria Concetta; Vianello, Angelo; Macrì, Francesco; Skerlavaj, Barbara; Zanetti, Margherita; Gennaro, Renato; Bernardi, Paolo

    2002-01-01

    BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca2+ and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability. PMID:11865069

  8. Amyloid-β peptide fibrils induce nitro-oxidative stress in neuronal cells.

    PubMed

    Ill-Raga, Gerard; Ramos-Fernández, Eva; Guix, Francesc X; Tajes, Marta; Bosch-Morató, Mónica; Palomer, Ernest; Godoy, Juan; Belmar, Sebastián; Cerpa, Waldo; Simpkins, James W; Inestrosa And, Nibaldo C; Muñoz, Francisco J

    2010-01-01

    Different mechanisms including oxidative stress are proposed for amyloid-β peptide (Aβ) neurotoxicity, and here we contribute to demonstrate that nitro-oxidative stress is playing a key role. Yeasts are a well-known model for H2O2 toxicity. Interestingly, yeast cell wall prevents interaction of Aβ fibrils with membrane receptors or calcium channels and we found a significant viability reduction in yeasts when challenged with Aβ fibrils. Furthermore, iron and copper chelators, as well as the antioxidants glutathione and trolox, were neuroprotective on neuroblastoma cells and mouse hippocampal neurons challenged with Aβ fibrils. Glutathione prevents the oxidation, glycation and nitrotyrosination of cell proteins induced by Aβ. Trolox protected neurons in cell viability studies, maintaining the vesicular transport integrity and preventing the trigger of apoptotic mechanisms. Interestingly, we have also found that brain derived neuronal factor (BDNF) and neurotrophin-3 (NT-3) were able to protect mouse hippocampal and cortical neurons against H2O2 and Aβ fibrils. Considering that superoxide anion, produced by Aβ cell damage, and nitric oxide, whose production is altered in AD, react to form the highly reactive peroxynitrite anion, we studied the role of trolox to ameliorate the peroxynitrite cell damage. Finally, one of the major proteins to be nitrotyrosinated in AD, the triose phosphate isomerase (TPI) was assayed searching for a denitrase activity that could reverse intracellular nitrotyrosination. We have found that human neuroblastoma SH-SY5Y cells express a constitutive denitrase activity that partially denitrated nitro-TPI. Altogether, our results support a key role of nitro-oxidative stress in the neuronal damage induced by Aβ fibrils. PMID:20858976

  9. Parabrachial Nucleus Contributions to Glucagon-Like Peptide-1 Receptor Agonist-Induced Hypophagia

    PubMed Central

    Swick, Jennifer C; Alhadeff, Amber L; Grill, Harvey J; Urrea, Paula; Lee, Stephanie M; Roh, Hyunsun; Baird, John-Paul

    2015-01-01

    Exendin-4 (Ex4), a glucagon-like peptide-1 receptor (GLP-1R) agonist approved to treat type 2 diabetes mellitus, is well known to induce hypophagia in human and animal models. We evaluated the contributions of the hindbrain parabrachial nucleus (PBN) to systemic Ex4-induced hypophagia, as the PBN receives gustatory and visceral afferent relays and descending input from several brain nuclei associated with feeding. Rats with ibotenic-acid lesions targeted to the lateral PBN (PBNx) and sham controls received Ex4 (1 μg/kg) before 24 h home cage chow or 90 min 0.3 M sucrose access tests, and licking microstructure was analyzed to identify components of feeding behavior affected by Ex4. PBN lesion efficacy was confirmed using conditioned taste aversion (CTA) tests. As expected, sham control but not PBNx rats developed a CTA. In sham-lesioned rats, Ex4 reduced chow intake within 4 h of injection and sucrose intake within 90 min. PBNx rats did not show reduced chow or sucrose intake after Ex4 treatment, indicating that the PBN is necessary for Ex4 effects under the conditions tested. In sham-treated rats, Ex4 affected licking microstructure measures associated with hedonic taste evaluation, appetitive behavior, oromotor coordination, and inhibitory postingestive feedback. Licking microstructure responses in PBNx rats after Ex4 treatment were similar to sham-treated rats with the exception of inhibitory postingestive feedback measures. Together, the results suggest that the PBN critically contributes to the hypophagic effects of systemically delivered GLP-1R agonists by enhancing visceral feedback. PMID:25703200

  10. Septal Glucagon-Like Peptide 1 Receptor Expression Determines Suppression of Cocaine-Induced Behavior

    PubMed Central

    Harasta, Anne E; Power, John M; von Jonquieres, Georg; Karl, Tim; Drucker, Daniel J; Housley, Gary D; Schneider, Miriam; Klugmann, Matthias

    2015-01-01

    Glucagon-like peptide 1 (GLP-1) and its receptor GLP-1R are a key component of the satiety signaling system, and long-acting GLP-1 analogs have been approved for the treatment of type-2 diabetes mellitus. Previous reports demonstrate that GLP-1 regulates glucose homeostasis alongside the rewarding effects of food. Both palatable food and illicit drugs activate brain reward circuitries, and pharmacological studies suggest that central nervous system GLP-1 signaling holds potential for the treatment of addiction. However, the role of endogenous GLP-1 in the attenuation of reward-oriented behavior, and the essential domains of the mesolimbic system mediating these beneficial effects, are largely unknown. We hypothesized that the central regions of highest Glp-1r gene activity are essential in mediating responses to drugs of abuse. Here, we show that Glp-1r-deficient (Glp-1r−/−) mice have greatly augmented cocaine-induced locomotor responses and enhanced conditional place preference compared with wild-type (Glp-1r+/+) controls. Employing mRNA in situ hybridization we located peak Glp-1r mRNA expression in GABAergic neurons of the dorsal lateral septum, an anatomical site with a crucial function in reward perception. Whole-cell patch-clamp recordings of dorsal lateral septum neurons revealed that genetic Glp-1r ablation leads to increased excitability of these cells. Viral vector-mediated Glp-1r gene delivery to the dorsal lateral septum of Glp-1r−/− animals reduced cocaine-induced locomotion and conditional place preference to wild-type levels. This site-specific genetic complementation did not affect the anxiogenic phenotype observed in Glp-1r−/− controls. These data reveal a novel role of GLP-1R in dorsal lateral septum function driving behavioral responses to cocaine. PMID:25669605

  11. Protective Role of Glucagon-Like Peptide-1 Against High-Glucose-Induced Endothelial Oxidative Damage

    PubMed Central

    Guo, Lixin; Qiao, Yue; Zhang, Lina; Pan, Qi

    2015-01-01

    Abstract To investigate the protective effect of glucagon-like peptide-1 (GLP-1) against cell damage induced by high glucose. Human umbilical vein endothelial cells (HUVECs) were divided into control group (5.5 mmol/L) and high glucose groups (19, 33, or 47 mmol/L), which were cultured with different concentrations of glucose for 48 hours, respectively. Cell viability was measured with MTT assay. Levels of intracellular reactive oxygen species (ROS) were monitored by flow cytometry and apoptotic cell death was measured by staining with Annexin V-FITC and propidium iodide. Cultured cells were detected with intercellular adhesion molecule 1 (ICAM-1), VCAM-1, and JNK on protein. Compared with the control group, cell viability was decreased by 20% and 37%, respectively, when cultured under 33 and 47 mM, while increased in different GLP-1-treated groups (0.01 L, 0.1, 1, and 10 nmol/L). The GLP-1 treatment significantly reduced the ROS level of high glucose treatment group but not impact on the control group. Meanwhile, the level of apoptosis was elevated in the high glucose treatment group. Early apoptosis was significantly reversed in the GLP-1-treated group (0.1, 1, and 10 nmol/L). Late apoptosis was uniquely decreased in the GLP-1 concentrations of 10 nmol/L. Furthermore, GLP-1 could also reduce the protein levels of ICAM-1, VCAM-1, and phospho JNK in the endothelial cells with high glucose treatment. GLP-1 could inhibit cell apoptosis and reduce ROS generation and JNK-Bax signaling pathway activation, which were induced by high glucose treatment. PMID:26632709

  12. Alcohol-Induced Changes in Opioid Peptide Levels in Adolescent Rats Are Dependent on Housing Conditions

    PubMed Central

    Palm, Sara; Nylander, Ingrid

    2014-01-01

    Background Endogenous opioids are implicated in the mechanism of action of alcohol and alcohol affects opioids in a number of brain areas, although little is known about alcohol's effects on opioids in the adolescent brain. One concern, in particular when studying young animals, is that alcohol intake models often are based on single housing that may result in alcohol effects confounded by the lack of social interactions. The aim of this study was to investigate short- and long-term alcohol effects on opioids and the influence of housing conditions on these effects. Methods In the first part, opioid peptide levels were measured after one 24-hour session of single housing and 2-hour voluntary alcohol intake in adolescent and adult rats. In the second part, a model with a cage divider inserted during 2-hour drinking sessions was tested and the effects on opioids were examined after 6 weeks of adolescent voluntary intake in single-and pair-housed rats, respectively. Results The effects of single housing were age specific and affected Met-enkephalin-Arg6Phe7 (MEAP) in particular. In adolescent rats, it was difficult to distinguish between effects induced by alcohol and single housing, whereas alcohol-specific effects were seen in dynorphin B (DYNB), beta-endorphin (BEND), and MEAP levels in adults. Voluntary drinking affected several brain areas and the majority of alcohol-induced effects were not dependent on housing. However, alcohol effects on DYNB and BEND in the amygdala were dependent on housing. Housing alone affected MEAP in the cingulate cortex. Conclusions Age-specific housing- and alcohol-induced effects on opioids were found. In addition, prolonged voluntary alcohol intake under different housing conditions produced several alcohol-induced effects independent of housing. However, housing-dependent effects were found in areas implicated in stress, emotionality, and alcohol use disorder. Housing condition and age may therefore affect the reasons and

  13. Hypoglycemic, hypolipidemic and antioxidant effects of peptides from red deer antlers in streptozotocin-induced diabetic mice.

    PubMed

    Jiang, Ning; Zhang, Shuangjian; Zhu, Jing; Shang, Jing; Gao, Xiangdong

    2015-01-01

    Diabetes mellitus is a serious chronic metabolic disorder. To develop novel anti-diabetic drugs from nature sources has always been the focus of research. Red deer (Cervus elaphu Linnaeus) antler is one of the most famous Chinese traditional medicines. We found that the peptides of 5-10 kDa from red deer antlers (PRDA) promoted the growth of cultured rat islet cells. The purpose of this study was to investigate the anti-diabetic actions of PRDA in vivo and purify a pure active peptide. We therefore investigated the hypoglycemic, hypolipidemic and antioxidant effects of PRDA in streptozotocin-induced diabetic mice and isolated a pure anti-diabetic peptide. PRDA, given intraperitoneally (75, 150, or 300 μg/kg), significantly decreased the blood glucose levels, significantly increased the insulin concentrations, and remarkably improved the lipid metabolism in the diabetic mice. PRDA significantly increased the superoxide dismutase activity, catalase activity and the total antioxidant capacity in the serum and liver, and simultaneously decreased the malondialdehyde levels. The activities of hexokinase and pyruvate kinase, two important enzymes involved in glucose utilization, were also significantly increased in the liver of the PRDA-treated diabetic mice. Moreover, a novel anti-diabetic peptide isolated from PRDA significantly promoted the viability of cultured rat insulinoma cells. The molecular mass of the purified peptide was 7064.8 Da under mass spectrometry, and its N-terminal amino acid sequence was identified as LSPFTTKTYFPHFDLSHGSA. Thus, PRDA may be useful in managing the hyperglycemia, hyperlipidemia, and oxidative stress in diabetes, and the anti-diabetic peptide is a promising drug for the treatment of diabetes. PMID:25985857

  14. Exercise-induced changes in atrial peptides in relation to neuroendocrine responses and fluid balance in the horse.

    PubMed

    Kokkonen, U M; Pösö, A R; Hyyppä, S; Huttunen, P; Leppäluoto, J

    2002-04-01

    Previous data show that, in horses, plasma atrial natriuretic peptides (ANP and NT-ANP) remain elevated for a long time after exercise. To study whether exercise-induced changes in hormonal and fluid balance explain this, we measured plasma concentrations of COOH- and NH2-terminal atrial natriuretic peptides (ANP(99-129) and NT-ANP(1-98) together with arginine vasopressin (AVP), adrenocorticotrophin (ACTH), beta-endorphin, cortisol, catecholamines, and indicators of fluid balance in six Finnhorses after a graded submaximal exercise test on a treadmill. After exercise, AVP and catecholamines diminished rapidly; atrial peptides, ACTH, beta-endorphin, and cortisol remained elevated longer. ANP reached its peak value at 5 min and NT-ANP at 30 min post-exercise. At 60 min, ANP was still significantly increased and NT-ANP even above its level at the end of exercise. The different temporal patterns of ANP and NT-ANP are most probably explained by differences in their plasma half-lives. The post-exercise increase in NT-ANP indicates that the release of atrial peptides is stimulated during recovery after exercise. The rapid decrease in AVP and catecholamines suggests that these hormones do not explain the long-lasting increase in atrial peptides. Cortisol remained elevated longer and it may have contributed to some extent. After exercise, the packed cell volume (PCV) decreased more slowly than plasma total protein and electrolytes, which refers to a slow post-exercise return in blood volume. Taken together, the present results show that the long-lasting post-exercise increase in plasma atrial peptides in horses is most probably explained by elevated central blood volume and that the role of vasoactive hormones is small. PMID:12019955

  15. Release of Periplasmic Nucleotidase Induced by Human Antimicrobial Peptide in E. coli Causes Accumulation of the Immunomodulator Adenosine.

    PubMed

    Estrela, Andreia Bergamo; Türck, Patrick; Stutz, Elaine; Abraham, Wolf-Rainer

    2015-01-01

    Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado) in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5'-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado).

  16. Release of Periplasmic Nucleotidase Induced by Human Antimicrobial Peptide in E. coli Causes Accumulation of the Immunomodulator Adenosine.

    PubMed

    Estrela, Andreia Bergamo; Türck, Patrick; Stutz, Elaine; Abraham, Wolf-Rainer

    2015-01-01

    Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado) in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5'-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado). PMID:26371472

  17. Release of Periplasmic Nucleotidase Induced by Human Antimicrobial Peptide in E. coli Causes Accumulation of the Immunomodulator Adenosine

    PubMed Central

    Estrela, Andreia Bergamo; Türck, Patrick; Stutz, Elaine; Abraham, Wolf-Rainer

    2015-01-01

    Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado) in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5’-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado). PMID:26371472

  18. Pri sORF peptides induce selective proteasome-mediated protein processing.

    PubMed

    Zanet, J; Benrabah, E; Li, T; Pélissier-Monier, A; Chanut-Delalande, H; Ronsin, B; Bellen, H J; Payre, F; Plaza, S

    2015-09-18

    A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

  19. STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-{alpha}2b peptide

    SciTech Connect

    Blank, Viviana C.; Pena, Clara; Roguin, Leonor P.

    2010-02-15

    In the search of mimetic peptides of the interferon-{alpha}2b molecule (IFN-{alpha}2b), we have previously designed and synthesized a chimeric cyclic peptide of the IFN-{alpha}2b that inhibits WISH cell proliferation by inducing an apoptotic response. Here, we first studied the ability of this peptide to activate intracellular signaling pathways and then evaluated the participation of some signals in the induction of apoptosis. Stimulation of WISH cells with the cyclic peptide showed tyrosine phosphorylation of Jak1 and Tyk2 kinases, tyrosine and serine phosphorylation of STAT1 and STAT3 transcription factors and activation of p38 MAPK pathway, although phosphorylation levels or kinetics were in some conditions different to those obtained under IFN-{alpha}2b stimulus. JNK and p44/42 pathways were not activated by the peptide in WISH cells. We also showed that STAT1 and STAT3 downregulation by RNA interference decreased the antiproliferative activity and the amount of apoptotic cells induced by the peptide. Pharmacological inhibition of p38 MAPK also reduced the peptide growth inhibitory activity and the apoptotic effect. Thus, we demonstrated that the cyclic peptide regulates WISH cell proliferation through the activation of Jak/STAT signaling pathway. In addition, our results indicate that p38 MAPK may also be involved in cell growth regulation. This study suggests that STAT1, STAT3 and p38 MAPK would be mediating the antitumor and apoptotic response triggered by the cyclic peptide in WISH cells.

  20. An ALS-mutant TDP-43 neurotoxic peptide adopts an anti-parallel β-structure and induces TDP-43 redistribution.

    PubMed

    Zhu, Li; Xu, Meng; Yang, Mengxue; Yang, Yanlian; Li, Yang; Deng, Jianwen; Ruan, Linhao; Liu, Jianghong; Du, Sidan; Liu, Xuehui; Feng, Wei; Fushimi, Kazuo; Bigio, Eileen H; Mesulam, Marsel; Wang, Chen; Wu, Jane Y

    2014-12-20

    TDP-43 proteinopathies are clinically and genetically heterogeneous diseases that had been considered distinct from classical amyloid diseases. Here, we provide evidence for the structural similarity between TDP-43 peptides and other amyloid proteins. Atomic force microscopy and electron microscopy examination of peptides spanning a previously defined amyloidogenic fragment revealed a minimal core region that forms amyloid fibrils similar to the TDP-43 fibrils detected in FTLD-TDP brain tissues. An ALS-mutant A315E amyloidogenic TDP-43 peptide is capable of cross-seeding other TDP-43 peptides and an amyloid-β peptide. Sequential Nuclear Overhauser Effects and double-quantum-filtered correlation spectroscopy in nuclear magnetic resonance (NMR) analyses of the A315E-mutant TDP-43 peptide indicate that it adopts an anti-parallel β conformation. When added to cell cultures, the amyloidogenic TDP-43 peptides induce TDP-43 redistribution from the nucleus to the cytoplasm. Neuronal cultures in compartmentalized microfluidic-chambers demonstrate that the TDP-43 peptides can be taken up by axons and induce axonotoxicity and neuronal death, thus recapitulating key neuropathological features of TDP-43 proteinopathies. Importantly, a single amino acid change in the amyloidogenic TDP-43 peptide that disrupts fibril formation also eliminates neurotoxicity, supporting that amyloidogenesis is critical for TDP-43 neurotoxicity.

  1. An Amphiphilic Peptide Induces Apoptosis Through the miR29b-p53 Pathway in Cancer Cells

    PubMed Central

    Kim, Soyoung; Lee, Jung Hyun; Kang, Igojo; Hyun, Soonsil; Yu, Jaehoon; Shin, Chanseok

    2016-01-01

    Peptides have been in the limelight, as therapeutic agents for cancer treatment through various applications due to their high target selectivity and exceptional ability to penetrate the cell membrane. Recent studies have revealed that synthesized peptides bind to hairpin structures of RNA that affect their activities such as changing the efficacy of microRNA maturation. MicroRNA-mediated p53 activation by the microRNA-29 (miR29) family is one of the most important regulatory pathways in cancer therapeutics. By targeting the suppressors of p53, a tumor suppressor protein, miR29 induces apoptosis of cancer cells through p53 stabilization. Here, we identify a novel synthesized amphiphilic peptide, LK-L1C/K6W/L8C, which enhances expression of miR29b and promotes p53 activity. In the presence of LK-L1C/K6W/L8C, pre-miR29b preferentially forms a complex with the Dicer protein through interaction of LK-L1C/K6W/L8C with the terminal loop region of pre-miR29b, leading to an increase in Dicer processing. Furthermore, LK-L1C/K6W/L8C stimulates apoptosis by improving p53 stability in miR29-inducible HeLa and MCF7 cells. Collectively, our study shows that a peptide can directly influence the miR29b-mediated p53 activation pathway in cancer cells. Therefore, our findings provide the basis for a new, potentially promising peptide-based drug for cancer therapy. PMID:27377134

  2. A synthetic peptide derived from the D1 domain of flagellin induced the expression of proinflammatory cytokines in fish macrophages.

    PubMed

    González-Stegmaier, Roxana; Guzmán, Fanny; Albericio, Fernando; Villarroel-Espíndola, Franz; Romero, Alex; Mulero, Victoriano; Mercado, Luis

    2015-11-01

    Flagellin is the main protein component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in fish and mammals. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines, and other immunological functions. We have previously reported that recombinant flagellin from Vibrio anguillarum and its ND1 domain are able to upregulate the expression of genes encoding major the proinflammatory mediators in gilthead seabream and rainbow trout macrophages. Considering the key role of D1 domain of flagellin for binding to TLR5M and its immunostimulatory activity, we designed and chemically synthesized a peptide derived of this region. The effects of the synthetic peptide were evaluated in vitro using head kidney macrophages from gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species the expression of genes encoding the proinflammatory cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), and the chemokine IL-8, was induced upon stimulation of macrophages with the D1 domain synthetic peptide. IL-1β and IL-8 were the most upregulated genes and to a lesser extent TNF-α. Interestingly, however, the induction activity of the synthetic peptide was higher in gilthead seabream than in rainbow trout macrophages. The results were confirmed at the protein levels for IL-8. Collectively, these results suggest that synthetic peptide derived from flagelling could be a promising approach for the immunostimulation and vaccination of farmed fish. PMID:26363237

  3. An Amphiphilic Peptide Induces Apoptosis Through the miR29b-p53 Pathway in Cancer Cells.

    PubMed

    Kim, Soyoung; Lee, Jung Hyun; Kang, Igojo; Hyun, Soonsil; Yu, Jaehoon; Shin, Chanseok

    2016-01-01

    Peptides have been in the limelight, as therapeutic agents for cancer treatment through various applications due to their high target selectivity and exceptional ability to penetrate the cell membrane. Recent studies have revealed that synthesized peptides bind to hairpin structures of RNA that affect their activities such as changing the efficacy of microRNA maturation. MicroRNA-mediated p53 activation by the microRNA-29 (miR29) family is one of the most important regulatory pathways in cancer therapeutics. By targeting the suppressors of p53, a tumor suppressor protein, miR29 induces apoptosis of cancer cells through p53 stabilization. Here, we identify a novel synthesized amphiphilic peptide, LK-L1C/K6W/L8C, which enhances expression of miR29b and promotes p53 activity. In the presence of LK-L1C/K6W/L8C, pre-miR29b preferentially forms a complex with the Dicer protein through interaction of LK-L1C/K6W/L8C with the terminal loop region of pre-miR29b, leading to an increase in Dicer processing. Furthermore, LK-L1C/K6W/L8C stimulates apoptosis by improving p53 stability in miR29-inducible HeLa and MCF7 cells. Collectively, our study shows that a peptide can directly influence the miR29b-mediated p53 activation pathway in cancer cells. Therefore, our findings provide the basis for a new, potentially promising peptide-based drug for cancer therapy. PMID:27377134

  4. 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.

  5. TAT-mediated intracellular delivery of NPM-derived peptide induces apoptosis in leukemic cells and suppresses leukemogenesis in mice.

    PubMed

    Zhou, Yun; Du, Wei; Koretsky, Tara; Bagby, Grover C; Pang, Qishen

    2008-09-15

    Nucleophosmin (NPM) is frequently overexpressed in leukemias and other tumors. NPM has been reported to suppress oncogene-induced senescence and apoptosis and may represent a therapeutic target for cancer. We fused a NPM-derived peptide to the HIV-TAT (TAT-NPMDeltaC) and found that the fusion peptide inhibited proliferation and induced apoptotic death of primary fibroblasts and preleukemic stem cells. TAT-NPMDeltaC down-regulated several NF-kappaB-controlled survival and inflammatory proteins and suppressed NF-kappaB-driven reporter gene activities. Using an inflammation-associated leukemia model, we demonstrate that TAT-NPMDeltaC induced proliferative suppression and apoptosis of preleukemic stem cells and significantly delayed leukemic development in mice. Mechanistically, TAT-NPMDeltaC associated with wild-type NPM proteins and formed complexes with endogenous NPM and p65 at promoters of several antiapoptotic and inflammatory genes and abrogated their transactivation by NF-kappaB in leu-kemic cells. Thus, TAT-delivered NPM peptide may provide a novel therapy for inflammation-associated tumors that require NF-kappaB signaling for survival.

  6. Chemical dynamics simulations of energy transfer, surface-induced dissociation, soft-landing, and reactive-landing in collisions of protonated peptide ions with organic surfaces.

    PubMed

    Pratihar, Subha; Barnes, George L; Hase, William L

    2016-07-01

    There are two components to the review presented here regarding simulations of collisions of protonated peptide ions peptide-H(+) with organic surfaces. One is a detailed description of the classical trajectory chemical dynamics simulation methodology. Different simulation approaches are used, and identified as MM, QM + MM, and QM/MM dependent on the potential energy surface used to represent the peptide-H(+) + surface collision. The second are representative examples of the information that may be obtained from the simulations regarding energy transfer and peptide-H(+) surface-induced dissociation, soft-landing, and reactive-landing for the peptide-H(+) + surface collisions. Good agreement with experiment is obtained for each of these four collision properties. The simulations provide atomistic interpretations of the peptide-H(+) + surface collision dynamics. PMID:26563571

  7. Systemic inflammatory response syndrome following burns is mediated by brain natriuretic peptide/natriuretic peptide A receptor-induced shock factor 1 signaling pathway.

    PubMed

    Xu, Yang-Cheng; Luo, Cheng-Qun; Li, Xiong

    2016-10-01

    The aim of this study was to determine whether systemic inflammatory response syndrome (SIRS) in burn patients is mediated by the brain natriuretic peptide (BNP)/natriuretic peptide A receptor (NPRA)-induced heat shock factor 1 (HSF-1) signalling pathway. Mononuclear cells (MNCs) that were isolated from patients with burn injuries and SIRS mouse models and a RAW264.7 cell line were treated with normal serum or serum obtained from animals with burn injuries. In parallel, small hairpin RNAs (shRNAs) against BNP or NPRA were transfected in both cell types. Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) were used to detect protein expression and inflammatory factor levels, respectively. We found that interleukin (IL)-12, tumour necrosis factor (TNF)-α, C-reactive protein (CRP), and BNP levels were increased and IL-10 levels were decreased in the plasma and MNCs in vivo in the animal model of SIRS. Additionally, NPRA was upregulated, whereas HSF-1 was downregulated in monocytes in vivo. Treatment of RAW264.7 cells with burn serum or BNP induced IL-12, TNF-α, and CRP secretion as well as HSF-1 expression. Finally, silencing BNP with shRNA interrupted the effect of burn serum on RAW264.7 cells, and silencing NPRA blocked burn serum- and BNP-mediated changes in RAW264.7 cells. These results suggest that the interaction of NPRA with BNP secreted from circulatory MNCs as well as mononuclear macrophages leads to inflammation via HSF-1 during SIRS development following serious burn injury.

  8. Satiation and Stress-Induced Hypophagia: Examining the Role of Hindbrain Neurons Expressing Prolactin-Releasing Peptide or Glucagon-Like Peptide 1

    PubMed Central

    Maniscalco, James W.; Kreisler, Alison D.; Rinaman, Linda

    2013-01-01

    Neural circuits distributed within the brainstem, hypothalamus, and limbic forebrain interact to control food intake and energy balance under normal day-to-day conditions, and in response to stressful conditions under which homeostasis is threatened. Experimental studies using rats and mice have generated a voluminous literature regarding the functional organization of circuits that inhibit food intake in response to satiety signals, and in response to stress. Although the central neural bases of satiation and stress-induced hypophagia often are studied and discussed as if they were distinct, we propose that both behavioral states are generated, at least in part, by recruitment of two separate but intermingled groups of caudal hindbrain neurons. One group comprises a subpopulation of noradrenergic (NA) neurons within the caudal nucleus of the solitary tract (cNST; A2 cell group) that is immunopositive for prolactin-releasing peptide (PrRP). The second group comprises non-adrenergic neurons within the cNST and nearby reticular formation that synthesize glucagon-like peptide 1 (GLP-1). Axonal projections from PrRP and GLP-1 neurons target distributed brainstem and forebrain regions that shape behavioral, autonomic, and endocrine responses to actual or anticipated homeostatic challenge, including the challenge of food intake. Evidence reviewed in this article supports the view that hindbrain PrRP and GLP-1 neurons contribute importantly to satiation and stress-induced hypophagia by modulating the activity of caudal brainstem circuits that control food intake. Hindbrain PrRP and GLP-1 neurons also engage hypothalamic and limbic forebrain networks that drive parallel behavioral and endocrine functions related to food intake and homeostatic challenge, and modulate conditioned and motivational aspects of food intake. PMID:23346044

  9. Diabetes-induced loss of gastric ICC accompanied by up-regulation of natriuretic peptide signaling pathways in STZ-induced diabetic mice.

    PubMed

    Wu, Yi-Song; Lu, Hong-Li; Huang, Xu; Liu, Dong-Hai; Meng, Xiang-Min; Guo, Xin; Kim, Young-chul; Xu, Wen-Xie

    2013-02-01

    Our previous study demonstrated that natriuretic peptides (NPs) play an inhibitory role in regulation of gastric smooth muscle motility. However, it is not clear whether NPs are involved in diabetics-induced loss of gastric interstitial cell of Cajal (ICC). The present study was designed to investigate the relationship between diabetics-induced loss of gastric ICC and natriuretic peptide signaling pathway in streptozotocin (STZ)-induced diabetic mice. The results showed that the protein expression levels of c-Kit and membrane-bound stem cell factor (mSCF) in gastric smooth muscle layers were decreased in STZ-induced diabetic mice. However, both mRNA and protein expression levels of natriuretic peptide receptor (NPR)-A, B and C were increased in the same place of the diabetic mice. The amplitude of spontaneous contraction in gastric antral smooth muscles was inhibited by C-type natriuretic peptide (CNP) dose-dependently and the inhibitory effect was potentiated in diabetic mice. Pretreatment of the cultured gastric smooth muscle cells (GSMCs) with different concentration of CNP can significantly decrease the mSCF expression level. 8-Bromoguanosine-3',5'-cyclomo-nophosphate (8-Br-cGMP), a membrane permeable cGMP analog, mimicked the effect of CNP but not cANF (a specific NPR-C agonist). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay showed that high concentration of cANF (10(-6) mol/L) inhibited cell proliferation in cultured GSMCs. These findings suggest that up-regulation of NPs/NPR-A, B/cGMP and NPs/NPR-C signaling pathways may be involved in diabetes-induced loss of gastric ICC.

  10. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.

    PubMed

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G

    2012-11-23

    The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

  11. Glucagon like peptide-2 induces intestinal restitution through VEGF release from subepithelial myofibroblasts.

    PubMed

    Bulut, Kerem; Pennartz, Christian; Felderbauer, Peter; Meier, Juris J; Banasch, Matthias; Bulut, Daniel; Schmitz, Frank; Schmidt, Wolfgang E; Hoffmann, Peter

    2008-01-14

    Glucagon like peptide-2 (GLP-2) exerts intestinotrophic actions, but the underlying mechanisms are still a matter of debate. Recent studies demonstrated the expression of the GLP-2 receptor on fibroblasts located in the subepithelial tissue, where it might induce the release of growth factors such as keratinocyte growth factor (KGF) or vascular endothelial growth factor (VEGF). Therefore, in the present studies we sought to elucidate the downstream mechanisms involved in improved intestinal adaptation by GLP-2. Human colonic fibroblasts (CCD-18Co), human colonic cancer cells (Caco-2 cells) and rat ileum IEC-18 cells were used. GLP-2 receptor mRNA expression was determined using real time RT-PCR. Conditioned media from CCD-18Co cells were obtained following incubation with GLP-2 (50-250 nM) for 24 h. Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay, and wound healing was determined with an established migration-assay. Transforming Growth Factor beta (TGF-beta), VEGF and KGF mRNA levels were determined by RT-PCR. Protein levels of VEGF and TGF-beta in CCD-18Co cells following GLP-2 stimulation were determined using ELISA. Neutralizing TGF-beta and VEGF-A antibodies were utilized to assess the role of TGF-beta and VEGF-A in the process of wound healing. GLP-2 receptor expression was detected in CCD-18Co cells. Conditioned media from CCD-18Co cells dose-dependently induced proliferation in Caco-2 cells, but not in IEC-18 cells. Conditioned media also enhanced cell migration in IEC-18 cells (P<0.01), while migration was even inhibited in Caco-2 cells (P<0.0012). GLP-2 significantly stimulated mRNA expression of VEGF and TGF-beta, but not of KGF in CCD-18Co. The migratory effects of GLP-2 were completely abolished in the presence of TGF-beta and VEGF-A antibodies. GLP-2 exerts differential effects on the epithelium of the small intestine and the colon. Thus, in small intestinal cells GLP-2 stimulates wound

  12. 1–42 β-Amyloid peptide requires PDK1/nPKC/Rac 1 pathway to induce neuronal death

    PubMed Central

    Manterola, L; Hernando-Rodríguez, M; Ruiz, A; Apraiz, A; Arrizabalaga, O; Vellón, L; Alberdi, E; Cavaliere, F; Lacerda, H M; Jimenez, S; Parada, L A; Matute, C; Zugaza, J L

    2013-01-01

    1–42 β-Amyloid (Aβ1–42) peptide is a key molecule involved in the development of Alzheimer's disease. Some of its effects are manifested at the neuronal morphological level. These morphological changes involve loss of neurites due to cytoskeleton alterations. However, the mechanism of Aβ1–42 peptide activation of the neurodegenerative program is still poorly understood. Here, Aβ1–42 peptide-induced transduction of cellular death signals through the phosphatidylinositol 3-kinase (PI3K)/phosphoinositol-dependent kinase (PDK)/novel protein kinase C (nPKC)/Rac 1 axis is described. Furthermore, pharmacological inhibition of PDK1 and nPKC activities blocks Rac 1 activation and neuronal cell death. Our results provide insights into an unsuspected connection between PDK1, nPKCs and Rac 1 in the same signal-transduction pathway and points out nPKCs and Rac 1 as potential therapeutic targets to block the toxic effects of Aβ1–42 peptide in neurons. PMID:23340502

  13. 1-42 β-amyloid peptide requires PDK1/nPKC/Rac 1 pathway to induce neuronal death.

    PubMed

    Manterola, L; Hernando-Rodríguez, M; Ruiz, A; Apraiz, A; Arrizabalaga, O; Vellón, L; Alberdi, E; Cavaliere, F; Lacerda, H M; Jimenez, S; Parada, L A; Matute, C; Zugaza, J L

    2013-01-22

    1-42 β-Amyloid (Aβ(1-42)) peptide is a key molecule involved in the development of Alzheimer's disease. Some of its effects are manifested at the neuronal morphological level. These morphological changes involve loss of neurites due to cytoskeleton alterations. However, the mechanism of Aβ(1-42) peptide activation of the neurodegenerative program is still poorly understood. Here, Aβ(1-42) peptide-induced transduction of cellular death signals through the phosphatidylinositol 3-kinase (PI3K)/phosphoinositol-dependent kinase (PDK)/novel protein kinase C (nPKC)/Rac 1 axis is described. Furthermore, pharmacological inhibition of PDK1 and nPKC activities blocks Rac 1 activation and neuronal cell death. Our results provide insights into an unsuspected connection between PDK1, nPKCs and Rac 1 in the same signal-transduction pathway and points out nPKCs and Rac 1 as potential therapeutic targets to block the toxic effects of Aβ(1-42) peptide in neurons.

  14. Thiosemicarbazone modification of 3-acetyl coumarin inhibits Aβ peptide aggregation and protect against Aβ-induced cytotoxicity.

    PubMed

    Ranade, Dnyanesh S; Bapat, Archika M; Ramteke, Shefali N; Joshi, Bimba N; Roussel, Pascal; Tomas, Alain; Deschamps, Patrick; Kulkarni, Prasad P

    2016-10-01

    Aggregation of amyloid β peptide (Aβ) is an important event in the progression of Alzheimer's disease. Therefore, among the available therapeutic approaches to fight with disease, inhibition of Aβ aggregation is widely studied and one of the promising approach for the development of treatments for Alzheimer's disease. Thiosemicarbazone compounds are known for their variety of biological activities. However, the potential of thiosemicarbazone compounds towards inhibition of Aβ peptide aggregation and the subsequent toxicity is little explored. Herein, we report synthesis and x-ray crystal structure of novel compound 3-acetyl coumarin thiosemicarbazone and its efficacy toward inhibition of Aβ(1-42) peptide aggregation. Our results indicate that 3-acetyl coumarin thiosemicarbazone inhibits Aβ(1-42) peptide aggregation up to 80% compared to the parent 3-acetyl coumarin which inhibits 52%. Further, 3-acetyl coumarin thiosemicarbazone provides neuroprotection against Aβ-induced cytotoxicity in SH-SY5Y cell line. These findings indicate that thiosemicarbazone modification renders 3-acetyl coumarin neuroprotective properties.

  15. Acyclovir or Aβ42 peptides attenuate HSV-1-induced miRNA-146a levels in human primary brain cells.

    PubMed

    Lukiw, Walter J; Cui, Jian Guo; Yuan, Li Yuan; Bhattacharjee, Partha S; Corkern, Madelyn; Clement, Christian; Kammerman, Eli M; Ball, M J; Zhao, Yuhai; Sullivan, Patrick M; Hill, James M

    2010-10-01

    Human brains harbor herpes simplex virus type-1 (HSV-1) DNA, which normally remains quiescent throughout many decades of life. HSV-1 is associated with viral encephalopathy and with the amyloid beta 42 (Abeta42) peptide-enriched lesions that characterize Alzheimer's disease neuropathology. Here we report that infection of human neuronal-glial cells in primary co-culture with HSV-1 induces an irregular hypertrophy of human neuronal-glial cell bodies, an induction of HSV-1 DNA polymerase, and an up-regulation of micro-RNA-146a associated with altered innate-immune responses. Presence of the antiviral acyclovir or soluble Abeta42 peptide significantly attenuated these neuropathological responses. The inhibitory effects of Abeta42 peptide were also observed in an HSV-1-infected CV-1 cell-based viral plaque assay. The results suggest that soluble Abeta42 peptide can invoke non-pathological and anti-viral effects through inactivation of an HSV-1 challenge to human brain cells by simple viral sequestration, viral destruction, or by complex neurogenetic mechanisms.

  16. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    SciTech Connect

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  17. Thiosemicarbazone modification of 3-acetyl coumarin inhibits Aβ peptide aggregation and protect against Aβ-induced cytotoxicity.

    PubMed

    Ranade, Dnyanesh S; Bapat, Archika M; Ramteke, Shefali N; Joshi, Bimba N; Roussel, Pascal; Tomas, Alain; Deschamps, Patrick; Kulkarni, Prasad P

    2016-10-01

    Aggregation of amyloid β peptide (Aβ) is an important event in the progression of Alzheimer's disease. Therefore, among the available therapeutic approaches to fight with disease, inhibition of Aβ aggregation is widely studied and one of the promising approach for the development of treatments for Alzheimer's disease. Thiosemicarbazone compounds are known for their variety of biological activities. However, the potential of thiosemicarbazone compounds towards inhibition of Aβ peptide aggregation and the subsequent toxicity is little explored. Herein, we report synthesis and x-ray crystal structure of novel compound 3-acetyl coumarin thiosemicarbazone and its efficacy toward inhibition of Aβ(1-42) peptide aggregation. Our results indicate that 3-acetyl coumarin thiosemicarbazone inhibits Aβ(1-42) peptide aggregation up to 80% compared to the parent 3-acetyl coumarin which inhibits 52%. Further, 3-acetyl coumarin thiosemicarbazone provides neuroprotection against Aβ-induced cytotoxicity in SH-SY5Y cell line. These findings indicate that thiosemicarbazone modification renders 3-acetyl coumarin neuroprotective properties. PMID:26232353

  18. Glucagon-like peptide-2 induces rapid digestive adaptation following intestinal resection in preterm neonates

    PubMed Central

    Vegge, Andreas; Thymann, Thomas; Lund, Pernille; Stoll, Barbara; Bering, Stine B.; Hartmann, Bolette; Jelsing, Jacob; Qvist, Niels; Burrin, Douglas G.; Jeppesen, Palle B.; Holst, Jens J.

    2013-01-01

    Short bowel syndrome (SBS) is a frequent complication after intestinal resection in infants suffering from intestinal disease. We tested whether treatment with the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) increases intestinal volume and function in the period immediately following intestinal resection in preterm pigs. Preterm pigs were fed enterally for 48 h before undergoing resection of 50% of the small intestine and establishment of a jejunostomy. Following resection, pigs were maintained on total parenteral nutrition (TPN) without (SBS, n = 8) or with GLP-2 treatment (3.5 μg/kg body wt per h, SBS+GLP-2, n = 7) and compared with a group of unresected preterm pigs (control, n = 5). After 5 days of TPN, all piglets were fed enterally for 24 h, and a nutrient balance study was performed. Intestinal resection was associated with markedly reduced endogenous GLP-2 levels. GLP-2 increased the relative absorption of wet weight (46 vs. 22%), energy (79 vs. 64%), and all macronutrients (all parameters P < 0.05). These findings were supported by a 200% increase in sucrase and maltase activities, a 50% increase in small intestinal epithelial volume (P < 0.05), as well as increased DNA and protein contents and increased total protein synthesis rate in SBS+GLP-2 vs. SBS pigs (+100%, P < 0.05). Following intestinal resection in preterm pigs, GLP-2 induced structural and functional adaptation, resulting in a higher relative absorption of fluid and macronutrients. GLP-2 treatment may be a promising therapy to enhance intestinal adaptation and improve digestive function in preterm infants with jejunostomy following intestinal resection. PMID:23764891

  19. Structural transformation of peptide amphiphile self-assembly induced by headgroup charge and size regulation

    NASA Astrophysics Data System (ADS)

    Gao, Changrui; Bedzyk, Michael; Olvera, Monica; Kewalramani, Sumit; Palmer, Liam

    The ability to control the nano and the meso-scale architecture of molecular assemblies is one of the major challenges in nanoscience. Significantly, structural transformations of amphiphilic aggregates induced by variations in environmental conditions have attracted attention due to their biotechnological relevance. Here, we study the assembly in aqueous solution for a modular series of peptide amphiphiles with 3, 2 or 1 lysine groups conjugated to a C16 carbon tail (C16K3, C16K2 and C16K1) . This system design allow us to probe how the equilibrium structure of the self-assembly can be tuned by controlling the coupling between steric (via choice of headgroup: K3, K2, or K1) and electrostatic (via solution pH) interactions. Solution small- and wide-angle X-ray scattering (SAXS/WAXS) and transmission electron microscopy (TEM) studies reveal that depending on pH and number of lysines in the lipid headgroup, amphiphiles can assemble into a range of structures: spherical micelles, bilayer ribbons and vesicles. We also perform detailed phase space mapping of pH-and headgroup size dependency of the structures of assembly over 0.1-100 nm length scales via SAXS/WAXS. The experimental results in conjunction with molecular dynamics (MD) simulations deduce quantitative relations between pH-dependent molecular charges, steric constraints and self-assembly morphologies, which is significant for developing experimental routes to obtain assembly structures with specific nano- and meso-scale features through controlled external stimuli.

  20. The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells

    PubMed Central

    Eike, Liv-Marie; Yang, Nannan; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2015-01-01

    Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models. PMID:26472184

  1. Histone peptide AKRHRK enhances H{sub 2}O{sub 2}-induced DNA damage and alters its site specificity

    SciTech Connect

    Midorikawa, Kaoru; Murata, Mariko; Kawanishi, Shosuke . E-mail: kawanisi@doc.medic.mie-u.ac.jp

    2005-08-12

    Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H{sub 2}O{sub 2}, leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH{sub 3}CO-AKRHRK-CONH{sub 2}, which has a metal-binding site. This histone peptide enhanced DNA damage induced by H{sub 2}O{sub 2} and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H{sub 2}O{sub 2} and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H{sub 2}O{sub 2}, may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition.

  2. The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

    PubMed

    Vizán, J L; Hernández-Chico, C; del Castillo, I; Moreno, F

    1991-02-01

    Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.

  3. Synthetic peptides from a conserved region of gp120 induce broadly reactive anti-HIV responses.

    PubMed Central

    Morrow, W J; Williams, W M; Whalley, A S; Ryskamp, T; Newman, R; Kang, C Y; Chamat, S; Köhler, H; Kieber-Emmons, T

    1992-01-01

    In our efforts to identify products that might be used for active immunotherapy in human immunodeficiency virus (HIV) infection, we have studied synthetic peptides derived from the CD4 attachment site of gp120. Two peptides have emerged with particularly interesting properties. The first (B138) is linear and spans the envelope residues 421-438; the second (1005/45) encompasses amino acids 418-445 and is cyclized by way of a disulphide bond joining its terminal cysteines. Both species have been shown to inhibit syncytial formation in a conventional bioassay, B138 being the most efficient. Both peptides elicit high titres of anti-peptide antibodies in immunized mice, rabbits and goats, with titres exceeding 1:10(5) in many cases. A substantial portion of this response is directed against gp120 as determined by enzyme-linked immunosorbent assay (ELISA). Analysis by flow cytometry has demonstrated that the antisera are broadly reactive with multiple diverse strains of HIV. The anti-gp120 activity of the anti-peptide antiserum was further confirmed by radioimmuno-precipitation (RIP) assays. Furthermore, RIP analysis and inhibition experiments in a GD4-gp120 binding assay have revealed that anti-peptide sera contain antibodies directed against the CD4 attachment site on gp120 and interfere with this receptor-ligand interaction. Images Figure 4 PMID:1592430

  4. Rational Optimization of Conformational Effects Induced By Hydrocarbon Staples in Peptides and their Binding Interfaces

    NASA Astrophysics Data System (ADS)

    Lama, Dilraj; Quah, Soo T.; Verma, Chandra S.; Lakshminarayanan, Rajamani; Beuerman, Roger W.; Lane, David P.; Brown, Christopher J.

    2013-12-01

    eIF4E is frequently over-expressed in different cancers and causes increased translation of oncogenic proteins via deregulated cap-dependent translation. Inhibitors of the eIF4E:eIF4G interactions represent an approach that would normalize cap-dependent translation. Stapled peptides represent an emerging class of therapeutics that can target protein: protein interactions. We present here molecular dynamics simulations for a set of rationally designed stapled peptides in solution and in complex with eIF4E, supported with biophysical and crystallographic data. Clustering of the simulated structures revealed the favoured conformational states of the stapled peptides in their bound or free forms in solution. Identifying these populations has allowed us to design peptides with improved affinities by introducing mutations into the peptide sequence to alter their conformational distributions. These studies emphasise the effects that engineered mutations have on the conformations of free and bound peptides, and illustrate that both states must be considered in efforts to attain high affinity binding.

  5. The antimicrobial peptide sublancin ameliorates necrotic enteritis induced by Clostridium perfringens in broilers.

    PubMed

    Wang, S; Zeng, X F; Wang, Q W; Zhu, J L; Peng, Q; Hou, C L; Thacker, P; Qiao, S Y

    2015-10-01

    Sublancin is an antimicrobial peptide produced by 168 containing 37 amino acids. The objective of this study was to investigate its inhibitory efficacy against both in vitro and in vivo. In the in vitro study, we determined that sublancin had a minimum inhibitory concentration of 8 μM against , which was much higher than the antibiotic lincomycin (0.281 μM). Scanning electron microscopy showed that sublancin damaged the morphology of . The in vivo study was conducted on broilers for a 28-d period using a completely randomized design. A total of 252 chickens at 1 d of age were randomly assigned to 1 of 6 treatments including an uninfected control; an infected control; 3 infected groups supplemented with sublancin at 2.88, 5.76, or 11.52 mg activity/L of water; and an infected group supplemented with lincomycin at 75 mg activity/L of water (positive control). Necrotic enteritis was induced in the broilers by oral inoculation of on d 15 through 21. Thereafter, the sublancin or lincomycin were administered fresh daily for a period of 7 days. The challenge resulted in a significant decrease in ADG ( < 0.05) and a remarkable deterioration in G:F ( < 0.05) during d 15 to 21 of the experiment. There was a sharp increase of numbers in the cecum ( < 0.05). The addition of sublancin or lincomycin reduced caecal counts ( < 0.05). The counts had a tendency to decrease in the lincomycin treatment ( = 0.051) but were the highest in the sublancin treatment (5.76 mg activity/L of water). A higher villus height to crypt depth ratio in the duodenum and jejunum as well as a higher villus height in the duodenum were observed in broilers treated with sublancin or lincomycin ( < 0.05) compared with infected control broilers. It was observed that sublancin and lincomycin decreased IL-1β, IL-6, and tumor necrosis factor-α levels ( < 0.05) in the ileum compared with the infected control. In conclusion, although sublancin's minimum inhibitory concentration is much higher than lincomycin

  6. Exendin-4, a glucagon-like peptide 1 receptor agonist, protects against amyloid-β peptide-induced impairment of spatial learning and memory in rats.

    PubMed

    Jia, Xiao-Tao; Ye-Tian; Yuan-Li; Zhang, Ge-Juan; Liu, Zhi-Qin; Di, Zheng-Li; Ying, Xiao-Ping; Fang, Yan; Song, Er-Fei; Qi, Jin-Shun; Pan, Yan-Fang

    2016-05-15

    Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) share specific molecular mechanisms, and agents with proven efficacy in one may be useful against the other. The glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 has similar properties to GLP-1 and is currently in clinical use for T2DM treatment. Thus, this study was designed to characterize the effects of exendin-4 on the impairment of learning and memory induced by amyloid protein (Aβ) and its probable molecular underlying mechanisms. The results showed that (1) intracerebroventricular (i.c.v.) injection of Aβ1-42 resulted in a significant decline of spatial learning and memory of rats in water maze tests; (2) pretreatment with exendin-4 effectively and dose-dependently protected against the Aβ1-42-induced impairment of spatial learning and memory; (3) exendin-4 treatment significantly decreased the expression of Bax and cleaved caspase-3 and increased the expression of Bcl2 in Aβ1-42-induced Alzheimer's rats. The vision and swimming speed of the rats among all groups in the visible platform tests did not show any difference. These findings indicate that systemic pretreatment with exendin-4 can effectively prevent the behavioral impairment induced by neurotoxic Aβ1-42, and the underlying protective mechanism of exendin-4 may be involved in the Bcl2, Bax and caspase-3 pathways. Thus, the application of exendin-4 or the activation of its signaling pathways may be a promising strategy to ameliorate the degenerative processes observed in AD. PMID:26992957

  7. Liposome-entrapped T-cell peptide provides help for a co-entrapped B-cell peptide to overcome genetic restriction in mice and induce immunological memory.

    PubMed Central

    Gregoriadis, G; Wang, Z; Barenholz, Y; Francis, M J

    1993-01-01

    We have investigated the possibility of a T-cell epitope peptide providing help for a B-cell epitope peptide when both peptides are co-entrapped in the same liposomes. Epitope models used were a 28 amino acid peptide from the S region of the hepatitis B surface antigen (HBsAg) (subtype adw) containing an H-2s Th-cell epitope, and a 33 amino acid peptide from the pre-S1 region of the HBsAg (subtype adw) designed to exclude an adjacent H-2s T-cell epitope, the latter (pre-S1) peptide being recognized by SJL (H-2s) mice as a B-cell epitope. SJL(H-2s) mice were immunized twice intramuscularly with S or pre-S1 peptide alone, co-entrapped in the same liposomes or entrapped in separate liposomes which were mixed before injection. Analysis of sera for anti-peptide IgG1 antibodies revealed that the Th-cell peptide provided help for the pre-S1 peptide only when the two peptides were co-entrapped in the same vesicles. This helper effect was found to correlate with the ability of S peptide (co-entrapped with the pre-S1) to stimulate T-cell proliferation in vitro. There was no IgG1 response against pre-S1 peptide in mice immunized with a mixture of the free peptides or a mixture of separately entrapped peptides. A helper effect, albeit much weaker, was also observed in mice immunized with the two peptides emulsified in incomplete Freund's adjuvant. Antisera from mice immunized with both peptides co-entrapped in liposomes were found to bind to full length (pre-S1 containing) recombinant HBsAg. Moreover, binding values were much higher than those seen with antisera from animals immunized with the liposomal S peptide above, presumably because of full access of anti-pre-S1 antibodies to the pre-S1 region of the rHBsAg. It is concluded that liposomes could serve not only as an immunological adjuvant for peptides but also as a carrier for Th- and B-cell epitopes thus eliminating the need for covalent linkage to a carrier protein. PMID:7508417

  8. The interplay of T1- and T2-relaxation on T1-weighted MRI of hMSCs induced by Gd-DOTA-peptides.

    PubMed

    Cao, Limin; Li, Binbin; Yi, Peiwei; Zhang, Hailu; Dai, Jianwu; Tan, Bo; Deng, Zongwu

    2014-04-01

    Three Gd-DOTA-peptide complexes with different peptide sequence are synthesized and used as T1 contrast agent to label human mesenchymal stem cells (hMSCs) for magnetic resonance imaging study. The peptides include a universal cell penetrating peptide TAT, a linear MSC-specific peptide EM7, and a cyclic MSC-specific peptide CC9. A significant difference in labeling efficacy is observed between the Gd-DOTA-peptides as well as a control Dotarem. All Gd-DOTA-peptides as well as Dotarem induce significant increase in T1 relaxation rate which is in favor of T1-weighted MR imaging. Gd-DOTA-CC9 yields the maximum labeling efficacy but poor T1 contrast enhancement. Gd-DOTA-EM7 yields the minimum labeling efficacy but better T1 contrast enhancement. Gd-DOTA-TAT yields a similar labeling efficacy as Gd-DOTA-CC9 and similar T1 contrast enhancement as Gd-DOTA-EM7. The underlying mechanism that governs T1 contrast enhancement effect is discussed. Our results suggest that T1 contrast enhancement induced by Gd-DOTA-peptides depends not only on the introduced cellular Gd content, but more importantly on the effect that Gd-DOTA-peptides exert on the T1-relaxation and T2-relaxation processes/rates. Both T1 and particularly T2 relaxation rate have to be taken into account to interpret T1 contrast enhancement. In addition, the interpretation has to be based on cellular instead of aqueous longitudinal and transverse relaxivities of Gd-DOTA-peptides.

  9. Evaluation of β-Amyloid Peptides Fibrillation Induced by Nanomaterials Based on Molecular Dynamics and Surface Plasmon Resonance.

    PubMed

    Hou, Yafei; Li, Pengfei; Zhou, Hongjian; Zhu, Xiaoli; Chen, Haifeng; Lee, Jaebeom; Koh, Kwangnak; Shen, Zhongming; Chen, Hongxia

    2015-02-01

    This report investigated the effect of carbon nanomaterials, single-wall carbon nanotube (SWCNT) and graphene oxide, on fibrillation of β-amyloid 40 (Aβ40) based on surface plasmon resonance (SPR) and molecular dynamics (MD). MD simulations are carried out in order to reveal the molecular mechanisms of the interaction between nanomaterials and Aβ40. The strong interaction between Aβ40 and nanomaterials is related to Van der Waals forces and the Coulomb force, inducing delicate manipulation of the main bonding energy for fibrillation of Aβ40. The interaction energy between the Aβ peptide and graphene is higher than that of SWCNT. Experimental results show both carbon nanomaterials enhance the appearance of a critical nucleus for nucleation of peptide fibrils. Graphene is more beneficial to assist the nucleation process than SWCNT. Combination of SPR and molecular dynamics could be a high-throughput method to screen protein fibrillation.

  10. Effect of Leu-enkephalin and delta sleep inducing peptide (DSIP) on endogenous noradrenaline release by rat brain synaptosomes

    SciTech Connect

    Lozhanets, V.V.; Anosov, A.K.

    1986-01-01

    The nonapeptide delta-sleep inducing peptide (DSIP) causes specific changes in the encephalogram of recipient animals: It prolongs the phase of long-wave or delta sleep. The cellular mechanism of action of DSIP has not yet been explained. To test the hyporhesis that this peptide or its degradation product may be presynaptic regulators of catecholamine release, the action of Leu-enkephaline, DSIP, and amino acids composing DSIP on release of endogenous noradrenalin (NA) from synaptosomes during depolarization was compared. Subcellular fractions from cerebral hemisphere of noninbred male albino rats were isolated. Lactate dehydrogenase activity was determined in the suspension of synaptosomes before and after addition of 0.5% Triton X-100. The results were subjected to statistical analysis, using the Wilcoxon-Mann-Whitney nonparametric test.

  11. Tuning the autophagy-inducing activity of lanthanide-based nanocrystals through specific surface-coating peptides

    NASA Astrophysics Data System (ADS)

    Zhang, Yunjiao; Zheng, Fang; Yang, Tianlong; Zhou, Wei; Liu, Yun; Man, Na; Zhang, Li; Jin, Nan; Dou, Qingqing; Zhang, Yong; Li, Zhengquan; Wen, Long-Ping

    2012-09-01

    The induction of autophagy on exposure of cells to a variety of nanoparticles represents both a safety concern and an application niche for engineered nanomaterials. Here, we show that a short synthetic peptide, RE-1, identified by means of phage display, binds to lanthanide (LN) oxide and upconversion nanocrystals (UCN), forms a stable coating layer on the nanoparticles’ surface, and effectively abrogates their autophagy-inducing activity. Furthermore, RE-1 peptide variants exhibit a differentially reduced binding capability, and correspondingly, a varied ability to reduce the autophagic response. We also show that the addition of an arginine-glycine-aspartic acid (RGD) motif to RE-1 enhances autophagy for LN UCN through the interaction with integrins. RE-1 and its variants provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy, and may prove useful for the various diagnostic and therapeutic applications of LN-based nanomaterials and nanodevices.

  12. Neonatal androgenization increases vasoactive intestinal peptide levels in rat anterior pituitary: possible involvement of vasoactive intestinal peptide in the neonatal androgenization-induced hyperprolactinemia.

    PubMed

    Watanobe, H; Sasaki, S; Takebe, K

    1991-11-01

    Accumulating evidence suggests that vasoactive intestinal peptide (VIP) may be a physiological PRL-releasing factor. In the present study, we examined a possible involvement of VIP in the neonatal androgenization (NA)-induced hyperprolactinemia. Twenty-four hours after birth, newborn female rats were injected sc with 1,000 micrograms of testosterone (NA) or with oil vehicle only (control). Both groups were sacrificed at 8 weeks of age. Compared to controls, NA rats showed significantly higher plasma PRL levels (7.3 fold), anterior pituitary (AP) PRL content (2.1 fold) and plasma estradiol levels (2.1 fold). AP VIP content was extremely higher (61 fold) in NA rats than in controls. However, NA did not affect VIP content in the suprachiasmatic nucleus, paraventricular nucleus or median eminence. These results suggest that the NA-induced hyperprolactinemia may be mediated, at least in part, by paracrine and/or autocrine effects of the increased AP VIP on PRL secretion. However, since the potentiation by NA of the AP VIP content was extremely marked compared to those of the other parameters, the possibility was also raised that the increased AP VIP may be involved in other endocrine and/or nonendocrine events occurring in the AP. PMID:1802925

  13. Predicted Peptides from Non-Structural Proteins of Porcine Reproductive and Respiratory Syndrome Virus Are Able to Induce IFN-γ and IL-10

    PubMed Central

    Burgara-Estrella, Alexel; Díaz, Ivan; Rodríguez-Gómez, Irene M.; Essler, Sabine E.; Hernández, Jesús; Mateu, Enric

    2013-01-01

    This work describes peptides from non-structural proteins (nsp) of porcine reproductive and respiratory syndrome virus (PRRSV) predicted as potential T cell epitopes by bioinfornatics and tested for their ability to induce IFN-γ and IL-10 responses. Pigs immunized with either genotype 1 or genotype 2 PRRSV attenuated vaccines (n=5/group) and unvaccinated pigs (n = 4) were used to test the peptides. Swine leukocyte antigen haplotype of each pig was also determined. Pigs were initially screened for IFN-γ responses (ELISPOT) and three peptides were identified; two of them in non-conserved segments of nsp2 and nsp5 and the other in a conserved region of nsp5 peptide. Then, peptides were screened for IL-10 inducing properties. Six peptides were found to induce IL-10 release in PBMC and some of them were also able to inhibit IFN-γ responses on PHA-stimulated cells. Interestingly, the IFN-γ low responder pigs against PRRSV were mostly homozygous for their SLA haplotypes. In conclusion, these results indicate that nsp of PRRSV contain T-cell epitopes inducing IFN-γ responses as well as IL-10 inducing segments with inhibitory capabilities. PMID:23435238

  14. Wheat peptides reduce oxidative stress and inhibit NO production through modulating μ-opioid receptor in a rat NSAID-induced stomach damage model.

    PubMed

    Yin, Hong; Cai, Hui-Zhen; Wang, Shao-Kang; Yang, Li-Gang; Sun, Gui-Ju

    2015-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) induce tissue damage and oxidative stress in animal models of stomach damage. In the present study, the protective effects of wheat peptides were evaluated in a NSAID-induced stomach damage model in rats. Different doses of wheat peptides or distilled water were administered daily by gavage for 30 days before the rat stomach damage model was established by administration of NSAIDs (aspirin and indomethacin) into the digestive tract twice. The treatment of wheat peptides decreased the NSAID-induced gastric epithelial cell degeneration and oxidative stress and NO levels in the rats. Wheat peptides significantly increased the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and decreased iNOS activity in stomach. The mRNA expression level of μ-opioid receptor was significantly decreased in wheat peptides-treated rats than that in in the control rats. The results suggest that NSAID drugs induced stomach damage in rats, wchih can be prevented by wheat peptides. The mechanisms for the protective effects were most likely through reducing NSAID-induced oxidative stress.

  15. Silibinin attenuates amyloid beta(25-35) peptide-induced memory impairments: implication of inducible nitric-oxide synthase and tumor necrosis factor-alpha in mice.

    PubMed

    Lu, P; Mamiya, T; Lu, L L; Mouri, A; Niwa, M; Hiramatsu, M; Zou, L B; Nagai, T; Ikejima, T; Nabeshima, T

    2009-10-01

    In Alzheimer's disease (AD), the deposition of amyloid peptides is invariably associated with oxidative stress and inflammatory responses. Silibinin (silybin), a flavonoid derived from the herb milk thistle, has potent anti-inflammatory and antioxidant activities. However, it remains unclear whether silibinin improves amyloid beta (Abeta) peptide-induced neurotoxicity. In this study, we examined the effect of silibinin on the fear-conditioning memory deficits, inflammatory response, and oxidative stress induced by the intracerebroventricular injection of Abeta peptide(25-35) (Abeta(25-35)) in mice. Mice were treated with silibinin (2, 20, and 200 mg/kg p.o., once a day for 8 days) from the day of the Abeta(25-35) injection (day 0). Memory function was evaluated in cued and contextual fear-conditioning tests (day 6). Nitrotyrosine levels in the hippocampus and amygdala were examined (day 8). The mRNA expression of inducible nitric-oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) in the hippocampus and amygdala was measured 2 h after the Abeta(25-35) injection. We found that silibinin significantly attenuated memory deficits caused by Abeta(25-35) in the cued and contextual fear-conditioning test. Silibinin significantly inhibited the increase in nitrotyrosine levels in the hippocampus and amygdala induced by Abeta(25-35). Nitrotyrosine levels in these regions were negatively correlated with memory performance. Moreover, real-time RT-PCR revealed that silibinin inhibited the overexpression of iNOS and TNF-alpha mRNA in the hippocampus and amygdala induced by Abeta(25-35). These findings suggest that silibinin (i) attenuates memory impairment through amelioration of oxidative stress and inflammatory response induced by Abeta(25-35) and (ii) may be a potential candidate for an AD medication. PMID:19638571

  16. The vaccine-site microenvironment induced by injection of incomplete Freund's adjuvant, with or without melanoma peptides

    PubMed Central

    Harris, Rebecca C.; Chianese-Bullock, Kimberly A.; Petroni, Gina R.; Schaefer, Jochen T.; Brill, Louis B.; Molhoek, Kerrington R.; Deacon, Donna H.; Patterson, James W.; Slingluff, Craig L.

    2011-01-01

    Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in two clinical trials of melanoma patients. In one study (Mel38), patients received one injection with an adjuvant mixture alone, comprised of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature dendritic cells (DC) and vessels resembling high endothelial venules (HEV). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DC, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4+ and CD8+ T lymphocytes, as well as FoxP3+CD4+ lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination. PMID:22130163

  17. The vaccine-site microenvironment induced by injection of incomplete Freund's adjuvant, with or without melanoma peptides.

    PubMed

    Harris, Rebecca C; Chianese-Bullock, Kimberly A; Petroni, Gina R; Schaefer, Jochen T; Brill, Louis B; Molhoek, Kerrington R; Deacon, Donna H; Patterson, James W; Slingluff, Craig L

    2012-01-01

    Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell (DC) activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in 2 clinical trials of melanoma patients. In 1 study (Mel38), patients received 1 injection with an adjuvant mixture alone, composed of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature DCs, and vessels resembling high endothelial venules (HEVs). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DCs, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4and CD8 T lymphocytes, as well as FoxP3CD4 lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination. PMID:22130163

  18. Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

    PubMed

    Cardozo, Alessandra K; Buchillier, Valérie; Mathieu, Marc; Chen, Jianhua; Ortis, Fernanda; Ladrière, Laurence; Allaman-Pillet, Nathalie; Poirot, Olivier; Kellenberger, Stephan; Beckmann, Jacques S; Eizirik, Decio L; Bonny, Christophe; Maurer, Fabienne

    2007-09-01

    We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

  19. Modulation of carrier-induced epitopic suppression by Bordetella pertussis components and muramyl peptide.

    PubMed

    Vogel, F R; Leclerc, C; Schutze, M P; Jolivet, M; Audibert, F; Klein, T W; Chedid, L

    1987-06-01

    Synthetic antigens employed in experimental synthetic vaccines are generally small haptenic peptides. Therefore, effective immunization with these antigens usually requires the use of an immunogenic carrier. Tetanus toxoid has been proposed for use as a carrier in future synthetic vaccines due to its high immunogenicity and acceptance for human use. Previous studies employing standard hapten/carrier systems such as DNP/KLH have demonstrated, however, that an epitope-specific suppression occurs when mice previously primed with carrier are subsequently immunized with an haptenic epitope conjugated to the same carrier. These same studies have shown that Bordetella pertussis vaccine administered at the time of carrier priming abrogates epitopic suppression. In the present investigation, epitopic suppression was studied in a synthetic vaccine model employing tetanus toxoid as a carrier. Results from these studies indicated that mice primed with tetanus toxoid 1 month before immunization with a peptide-tetanus toxoid conjugate exhibited enhanced secondary anti-tetanus toxin responses but decreased anti-peptide responses. Furthermore, injection of pertussis vaccine or purified B. pertussis toxin or endotoxin at the time of carrier priming could block the establishment of epitopic suppression. Administration of B. pertussis components enhanced antibody responses to both the carrier and the synthetic peptides as compared with responses of control animals. In addition, administration of an adjuvant-active nonpyrogenic derivative of muramyl dipeptide. Murabutide, with carrier priming reduced epitopic suppression of anti-peptide responses. B. pertussis toxin or endotoxin administered to mice previously suppressed by carrier priming with the first injection of carrier-peptide conjugate overcame epitopic suppression with resultant titers of anti-peptide antibody equal to or greater than nonsuppressed controls. These results suggest that the use of adjuvants with future synthetic

  20. Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.

    PubMed

    Meiring, Hugo D; Soethout, Ernst C; Poelen, Martien C M; Mooibroek, Dennis; Hoogerbrugge, Ronald; Timmermans, Hans; Boog, Claire J; Heck, Albert J R; de Jong, Ad P J M; van Els, Cécile A C M

    2006-05-01

    Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

  1. Peptide induced crystallization of calcium carbonate on wrinkle patterned substrate: implications for chitin formation in molluscs.

    PubMed

    Ghatak, Anindita Sengupta; Koch, Marcus; Guth, Christina; Weiss, Ingrid M

    2013-06-04

    We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates.

  2. Peptide Induced Crystallization of Calcium Carbonate on Wrinkle Patterned Substrate: Implications for Chitin Formation in Molluscs

    PubMed Central

    Sengupta Ghatak, Anindita; Koch, Marcus; Guth, Christina; Weiss, Ingrid M.

    2013-01-01

    We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates. PMID:23736692

  3. Peptide induced crystallization of calcium carbonate on wrinkle patterned substrate: implications for chitin formation in molluscs.

    PubMed

    Ghatak, Anindita Sengupta; Koch, Marcus; Guth, Christina; Weiss, Ingrid M

    2013-01-01

    We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates. PMID:23736692

  4. Phospholipase C gamma mediates endogenous brain-derived neurotrophic factor-regulated calcitonin gene-related peptide expression in colitis-induced visceral pain

    PubMed Central

    Hashmi, Fiza; Liu, Miao; Shen, Shanwei

    2016-01-01

    Background Visceral hypersensitivity is a complex pathophysiological paradigm with unclear mechanisms. Primary afferent neuronal plasticity marked by alterations in neuroactive compounds such as calcitonin gene-related peptide is suggested to underlie the heightened sensory responses. Signal transduction that leads to calcitonin gene-related peptide expression thereby sensory neuroplasticity during colitis remains to be elucidated. Results In a rat model with colitis induced by 2,4,6-trinitrobenzene sulfonic acid, we found that endogenously elevated brain-derived neurotrophic factor elicited an up-regulation of calcitonin gene-related peptide in the lumbar L1 dorsal root ganglia. At seven days of colitis, neutralization of brain-derived neurotrophic factor with a specific brain-derived neurotrophic factor antibody reversed calcitonin gene-related peptide up-regulation in the dorsal root ganglia. Colitis-induced calcitonin gene-related peptide transcription was also inhibited by brain-derived neurotrophic factor antibody treatment. Signal transduction studies with dorsal root ganglia explants showed that brain-derived neurotrophic factor-induced calcitonin gene-related peptide expression was mediated by the phospholipase C gamma, but not the phosphatidylinositol 3-kinase/Akt or the mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway. Application of PLC inhibitor U73122 in vivo confirmed that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide up-regulation in the dorsal root ganglia was regulated by the phospholipase C gamma pathway. In contrast, suppression of the phosphatidylinositol 3-kinase activity in vivo had no effect on colitis-induced calcitonin gene-related peptide expression. During colitis, calcitonin gene-related peptide also co-expressed with phospholipase C gamma but not with p-Akt. Calcitonin gene-related peptide up-regulation during colitis correlated to the activation

  5. C-Type Natriuretic Peptide Induces Anti-contractile Effect Dependent on Nitric Oxide, Oxidative Stress, and NPR-B Activation in Sepsis

    PubMed Central

    Pernomian, Laena; Prado, Alejandro F.; Silva, Bruno R.; Azevedo, Aline; Pinheiro, Lucas C.; Tanus-Santos, José E.; Bendhack, Lusiane M.

    2016-01-01

    Aims: To evaluate the role of nitric oxide, reactive oxygen species (ROS), and natriuretic peptide receptor-B activation in C-type natriuretic peptide-anti-contractile effect on Phenylephrine-induced contraction in aorta isolated from septic rats. Methods and Results: Cecal ligation and puncture (CLP) surgery was used to induce sepsis in male rats. Vascular reactivity was conducted in rat aorta and resistance mesenteric artery (RMA). Measurement of survival rate, mean arterial pressure (MAP), plasma nitric oxide, specific protein expression, and localization were evaluated. Septic rats had a survival rate about 37% at 4 h after the surgery, and these rats presented hypotension compared to control-operated (Sham) rats. Phenylephrine-induced contraction was decreased in sepsis. C-type natriuretic peptide (CNP) induced anti-contractile effect in aortas. Plasma nitric oxide was increased in sepsis. Nitric oxide-synthase but not natriuretic peptide receptor-B expression was increased in septic rat aortas. C-type natriuretic peptide-anti-contractile effect was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation. Natriuretic peptide receptor-C, protein kinase-Cα mRNA, and basal nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were lower in septic rats. Phenylephrine and CNP enhanced ROS production. However, stimulated ROS production was low in sepsis. Conclusion: CNP induced anti-contractile effect on Phenylephrine contraction in aortas from Sham and septic rats that was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation. PMID:27445832

  6. Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria

    PubMed Central

    Herrera Diaz, Areli; Kovacs, Izabella; Lindermayr, Christian

    2016-01-01

    Antimicrobial peptides (AMPs) are small peptides with less than 50 amino acids and are part of the innate immune response in almost all organisms, including bacteria, vertebrates, invertebrates and plants. AMPs are active against a broad-spectrum of pathogens. The inducible expression of AMPs in plants is a promising approach to combat plant pathogens with minimal negative side effects, such as phytotoxicity or infertility. In this study, inducible expression of the de-novo designed AMP SP1-1 in Micro Tom tomato protected tomato fruits against bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria. The peptide SP1-1 was targeted to the apoplast which is the primary infection site for plant pathogens, by fusing SP1-1 peptide to the signal peptide RsAFP1 of radish (Raphanus sativus). The pathogen inducibility of the expression was enabled by using an optimized inducible 4XW2/4XS promoter. As a result, the tomato fruits of independently generated SP1-1 transgenic lines were significantly more resistant to X. campestris pv. vesicatoria than WT tomato fruits. In transgenic lines, bacterial infection was reduced up to 65% in comparison to the infection of WT plants. Our study demonstrates that the combination of the 4XW2/4XS cis-element from parsley with the synthetic antimicrobial peptide SP1-1 is a good alternative to protect tomato fruits against infections with X. campestris pv. vesicatoria. PMID:27706237

  7. Nascent Peptide Side-chains Induce Rearrangements in Distinct Locations of the Ribosomal Tunnel

    PubMed Central

    Lu, Jianli; Hua, Zhengmao; Kobertz, William R.; Deutsch, Carol

    2011-01-01

    Although we have numerous structures of ribosomes, none disclose side-chain rearrangements of the nascent peptide during chain elongation. This study reports for the first time that rearrangement of the peptide and/or tunnel occurs in distinct regions of the tunnel and is directed by the unique primary sequence of each nascent peptide. In the tunnel mid-region, the accessibility of an introduced cysteine to a series of novel hydrophilic maleimide reagents increases with increasing volume of the adjacent chain residue, a sensitivity not manifest at the constriction and exit port. This surprising result reveals molecular movements not yet resolvable from structural studies. These findings map solvent accessible volumes along the tunnel and provide novel insights critical to our understanding of allosteric communication within the ribosomal tunnel, translational arrest, chaperone interaction, folding, and rates of elongation. PMID:21663746

  8. Distal phenylalanine modification for enhancing cellular delivery of fluorophores, proteins and quantum dots by cell penetrating peptides.

    PubMed

    Sayers, E J; Cleal, K; Eissa, N G; Watson, P; Jones, A T

    2014-12-10

    For cell penetrating peptides (CPPs) to fulfil their promise as effective delivery vectors we need a better understanding of their mechanisms of cell binding and uptake. This is especially the case when they are linked to different types of cargo. Here we describe new studies based on our previous findings suggesting that, for peptide-CPP chimeras, distal hydrophobic residues upstream of the CPP sequence can have profound effects on the way they interact with cells. We studied peptides bearing an N-terminal Glycine or Phenylalanine linked via a neutral and flexible bridging group, SGSGSGSG, to three well-studied CPPs: octaarginine, penetratin and TP10. Using a combination of flow cytometry, live-cell imaging and image analysis we examined the effects of this single amino acid change on binding and uptake of Alexa488-fluorophore, bovine serum albumin and quantum dot cargoes. The influence of the glycine-phenylalanine switch for fluorophore delivery was most dramatic in TP10, increasing cellular uptake by 4.4 and 9.9 fold in non-adherent and adherent cells, respectively. Only penetratin showed effective uptake of bovine serum albumin with the phenylalanine variant showing an increase of 1.6 fold over the glycine variant. The uptake of quantum dots was most efficiently demonstrated by octaarginine, with the glycine variant increasing uptake 4.8 fold and the phenylalanine variant increasing uptake 9.5 fold over quantum dots alone. Overall the data demonstrate that hydrophobicity distal to the CPP could be utilised to enhance their capacity to bind to the cell membrane and deliver a range of macromolecules to the insides of cells.

  9. Serum amyloid A induces contrary immune responses via formyl peptide receptor-like 1 in human monocytes.

    PubMed

    Lee, Ha Young; Kim, Mi-Kyoung; Park, Kyoung Sun; Shin, Eun Ha; Jo, Seong Ho; Kim, Sang Doo; Jo, Eun Jin; Lee, Youl-Nam; Lee, Chuhee; Baek, Suk-Hwan; Bae, Yoe-Sik

    2006-07-01

    Although the level of serum amyloid A has been reported to be up-regulated during inflammatory response, the role of serum amyloid A on the regulation of inflammation and immune response has not been elucidated. We found that serum amyloid A stimulated the production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10, which are proinflammatory and anti-inflammatory cytokines, respectively, in human monocytes. Low concentrations of serum amyloid A stimulated TNF-alpha production with maximal activity at 6 h after stimulation, whereas high concentrations of serum amyloid A stimulated IL-10 production with maximal activity at 12 h. The activations of the two cytokines by serum amyloid A occurred at both the transcription and translational levels. Signaling events induced by serum amyloid A included the activation of two mitogen-activated protein kinases (extracellular signal-regulated kinase and p38 kinase), which were found to be required for TNF-alpha and IL-10 production, respectively. The stimulation of formyl peptide receptor-like-1-expressing RBL-2H3 cells, but not of vector-expressing RBL-2H3 cells with serum amyloid A, induced mitogen-activated protein kinases activation and the accumulation of the RNAs of these two cytokines. Together, our findings suggest that serum amyloid A modulates contrary immune responses via formyl peptide receptor-like 1, by inducing TNF-alpha or IL-10, and demonstrate that extracellular signal-regulated kinase and p38 kinase play counteracting roles in this process.

  10. Large-scale production of soluble recombinant amyloid-β peptide 1-42 using cold-inducible expression system.

    PubMed

    Kim, Eun-Kyung; Moon, Jeong Chan; Lee, Jeong Mi; Jeong, Min Seop; Oh, Choongseob; Ahn, Sung-Min; Yoo, Yung Joon; Jang, Ho Hee

    2012-11-01

    Amyloid-β peptide 1-42 (Aβ(1-42)), the predominant form in senile plaques, plays important roles in the pathogenesis of Alzheimer's disease. Because Aβ(1-42) has aggregation-prone nature, it has been difficult to produce in a soluble state in bacterial expression systems. In this study, we modified our expression system to increase the soluble fraction of Aβ(1-42) in Escherichia coli (E. coli) cells. The expression level and solubility of recombinant Aβ(1-42) induced at the low temperature (16°C) is highly increased compared to that induced at 37°C. To optimize expression temperature, the coding region of Aβ(1-42) was constructed in a pCold vector, pCold-TF, which has a hexahistidine-tagged trigger factor (TF). Recombinant Aβ(1-42) was expressed primarily as a soluble protein using pCold vector system and purified with a nickel-chelating resin. When the toxic effect of recombinant Aβ(1-42) examined on human neuroblastoma SH-SY5Y cells, the purified Aβ(1-42) induced cell toxicity on SH-SY5Y cells. In conclusion, the system developed in this study will provide a useful method for the production of aggregation prone-peptide such as Aβ(1-42).

  11. Calcitonin gene-related peptide (CGRP) levels during glyceryl trinitrate (GTN)-induced headache in healthy volunteers.

    PubMed

    Kruuse, C; Iversen, H K; Jansen-Olesen, I; Edvinsson, L; Olesen, J

    2010-04-01

    The role of nitric oxide (NO) in migraine has been studied in the experimental glyceryl trinitrate (GTN)-infusion headache model. We hypothesized that GTN-induced headache may activate the trigeminovascular system and be associated with increased levels of sensory neuropeptides, including calcitonin gene-related peptide (CGRP). CGRP, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and somatostatin plasma levels were measured before and after placebo/sumatriptan injection and during GTN-induced headache. Following a double-blind randomized cross-over design, 10 healthy volunteers received subcutaneous sumatriptan 6 mg or placebo. This was succeeded by 20 min of GTN (0.12 µg kg(-1) min(-1)) infusion. At baseline no subject reported headache (using verbal rating scale from 0 to 10) and the jugular CGRP-like immunoreactivity (-LI) level was 18.6 ± 2.5 pmol/l. After a 20-min intravenous infusion of GTN 0.12 µg kg(-1) min(-1), median peak headache intensity was 4 (range 2-6) (P < 0.05), while jugular CGRP-LI levels were unchanged (19.0 ± 2.8 pmol/l; P > 0.05). There were no changes in VIP-, NPY- or somatostatin-LI. In conclusion, the NO donor GTN appears not to induce headache via immediate CGRP release.

  12. Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia

    PubMed Central

    Carro, Lorena; Pujic, Petar; Alloisio, Nicole; Fournier, Pascale; Boubakri, Hasna; Hay, Anne E; Poly, Franck; François, Philippe; Hocher, Valerie; Mergaert, Peter; Balmand, Severine; Rey, Marjolaine; Heddi, Abdelaziz; Normand, Philippe

    2015-01-01

    Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family. PMID:25603394

  13. Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia.

    PubMed

    Carro, Lorena; Pujic, Petar; Alloisio, Nicole; Fournier, Pascale; Boubakri, Hasna; Hay, Anne E; Poly, Franck; François, Philippe; Hocher, Valerie; Mergaert, Peter; Balmand, Severine; Rey, Marjolaine; Heddi, Abdelaziz; Normand, Philippe

    2015-08-01

    Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family. PMID:25603394

  14. beta. -Endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes

    SciTech Connect

    Diamant, M.; Henricks, P.A.J.; Nijkamp, F.P.; de Wied, D. )

    1989-01-01

    In the present study, the immunomodulatory effect of {beta}-endorphin ({beta}-E) and shorter pro-opiomelancortin (POMC) fragments was evaluated by assessing their influence on respiratory burst in human polymorphonuclear leukocytes (PMN). The effect of the peptides on phorbol myristate acetate (PMA)-stimulated production of reactive oxygen metabolites was measured in a lucigenin-enhanced chemiluminescence (CL) assay. Both POMC peptides with opiate-like activity and their non-opioid derivatives were tested. With the exception of {alpha}-E, PMA-stimulated respiratory burst was suppressed by all POMC fragments tested. A U-shaped dose-response relation was observed. Doses lower than 10{sup {minus}17}M and higher than 10{sup {minus}8}M were without effect. {beta}-E and dT{beta}E both suppressed PMA-induced oxidative burst in human PMN at physiological concentrations. {gamma}-E and dT{gamma}E proved to be less potent inhibitors, reaching maximal effect at higher concentrations. DE{gamma}E exerted an even less pronounced but still significant suppressive effect at the concentration of 10{sup {minus}10}M. None of the endorphins tested was shown to affect resting oxidative metabolism in the PMN. The modulatory effects of the opioid peptides could not be blocked by the opioid antagonist naloxone.

  15. Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia.

    PubMed

    Carro, Lorena; Pujic, Petar; Alloisio, Nicole; Fournier, Pascale; Boubakri, Hasna; Hay, Anne E; Poly, Franck; François, Philippe; Hocher, Valerie; Mergaert, Peter; Balmand, Severine; Rey, Marjolaine; Heddi, Abdelaziz; Normand, Philippe

    2015-08-01

    Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.

  16. H2-M polymorphism in mice susceptible to collagen-induced arthritis involves the peptide binding groove

    SciTech Connect

    Walter, W.; Loos, M.; Maeurer, M.J.

    1996-12-31

    The ability to develop type II collagen (CII)-induced arthritis (CIA) in mice is associated with the major histocompatibility I-A gene and with as yet poorly defined regulatory molecules of the major histocompatibility complex (MHC) class II antigen processing and presentation pathway. H2-M molecules are thought to be involved in the loading of antigenic peptides into the MHC class II binding cleft. We sequenced H2-Ma, H2-Mb1, and H2-Mb2 genes from CIA-susceptible and -resistant mouse strains and identified four different Ma and Mb2 alleles, and three different Mb1 alleles defined by polymorphic residues within the predicted peptide binding groove. Most CIA-resistant mouse strains share common Ma, Mb1, and Mb2 alleles. In contrast, H2-M alleles designated Ma-III, Ma-IV, Mb1-III, and Mb2-IV could be exclusively identified in the CIA-susceptible H2{sup r} and H2{sup q} haplotypes, suggesting that allelic H2-M molecules may modulate the composition of different CII peptides loaded onto MHC class II molecules, presumably presenting {open_quotes}arthritogenic{close_quotes} epitopes to T lymphocytes. 42 refs., 4 figs., 3 tabs.

  17. αA-Crystallin Peptide 66SDRDKFVIFLDVKHF80 Accumulating in Aging Lens Impairs the Function of α-Crystallin and Induces Lens Protein Aggregation

    PubMed Central

    Santhoshkumar, Puttur; Raju, Murugesan; Sharma, K. Krishna

    2011-01-01

    Background The eye lens is composed of fiber cells that are filled with α-, β- and γ-crystallins. The primary function of crystallins is to maintain the clarity of the lens through ordered interactions as well as through the chaperone-like function of α-crystallin. With aging, the chaperone function of α-crystallin decreases, with the concomitant accumulation of water-insoluble, light-scattering oligomers and crystallin-derived peptides. The role of crystallin-derived peptides in age-related lens protein aggregation and insolubilization is not understood. Methodology/Principal Findings We found that αA-crystallin-derived peptide, 66SDRDKFVIFLDVKHF80, which accumulates in the aging lens, can inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. Age-related change in the concentration of αA-(66-80) peptide was estimated by mass spectrometry. The interaction of the peptide with native crystallin was studied by multi-angle light scattering and fluorescence methods. High molar ratios of peptide-to-crystallin were favourable for aggregation and precipitation. Time-lapse recordings showed that, in the presence of αA-(66-80) peptide, α-crystallin aggregates and functions as a nucleus for protein aggregation, attracting aggregation of additional α-, β- and γ-crystallins. Additionally, the αA-(66-80) peptide shares the principal properties of amyloid peptides, such as β-sheet structure and fibril formation. Conclusions/Significance These results suggest that crystallin-derived peptides such as αA-(66-80), generated in vivo, can induce age-related lens changes by disrupting the structure and organization of crystallins, leading to their insolubilization. The accumulation of such peptides in aging lenses may explain a novel mechanism for age-related crystallin aggregation and cataractogenesis. PMID:21552534

  18. CopA3 peptide prevents ultraviolet-induced inhibition of type-I procollagen and induction of matrix metalloproteinase-1 in human skin fibroblasts.

    PubMed

    Kim, Dong-Hee; Kim, Han-Hyuk; Kim, Hyeon-Jeong; Jung, Hyun-Gug; Yu, Jae-Myo; Lee, Eun-Su; Cho, Yong-Hun; Kim, Dong-In; An, Bong-Jeun

    2014-01-01

    Ultraviolet (UV) exposure is well-known to induce premature aging, which is mediated by matrix metalloproteinase-1 (MMP-1) activity. A 9-mer peptide, CopA3 (CopA3) was synthesized from a natural peptide, coprisin, which is isolated from the dung beetle Copris tripartitus. As part of our continuing search for novel bioactive natural products, CopA3 was investigated for its in vitro anti-skin photoaging activity. UV-induced inhibition of type-I procollagen and induction of MMP-1 were partially prevented in human skin fibroblasts by CopA3 peptide in a dose-dependent manner. At a concentration of 25 μM, CopA3 nearly completely inhibited MMP-1 expression. These results suggest that CopA3, an insect peptide, is a potential candidate for the prevention and treatment of skin aging. PMID:24853614

  19. A comparison of the effects of amide and acid groups at the C-terminus on the collision-induced dissociation of deprotonated peptides.

    PubMed

    Bokatzian-Johnson, Samantha S; Stover, Michele L; Dixon, David A; Cassady, Carolyn J

    2012-09-01

    The dissociative behavior of peptide amides and free acids was explored using low-energy collision-induced dissociation and high level computational theory. Both positive and negative ion modes were utilized, but the most profound differences were observed for the deprotonated species. Deprotonated peptide amides produce a characteristic c(m-2)(-) product ion (where m is the number of residues in the peptide) that is either absent or in low abundance in the analogous peptide acid spectrum. Peptide acids show an enhanced formation of c(m-3)(-); however, this is not generally as pronounced as c(m-2)(-) production from amides. The most notable occurrence of an amide-specific product ion is for laminin amide (YIGSR-NH(2)) and this case was investigated using several modified peptides. Mechanisms involving 6- and 9-membered ring formation were proposed, and their energetic properties were investigated using G3(MP2) molecular orbital theory calculations. For example, with C-terminal deprotonation of pentaglycine amide, formation of c(m-2)(-) and a 6-membered ring diketopiperazine neutral requires >31.6 kcal/mol, which is 26.1 kcal/mol less than the analogous process involving the peptide acid. The end group specific fragmentation of peptide amides in the negative ion mode may be useful for identifying such groups in proteomic applications.

  20. A Comparison of the Effects of Amide and Acid Groups at the C-Terminus on the Collision-Induced Dissociation of Deprotonated Peptides

    NASA Astrophysics Data System (ADS)

    Bokatzian-Johnson, Samantha S.; Stover, Michele L.; Dixon, David A.; Cassady, Carolyn J.

    2012-09-01

    The dissociative behavior of peptide amides and free acids was explored using low-energy collision-induced dissociation and high level computational theory. Both positive and negative ion modes were utilized, but the most profound differences were observed for the deprotonated species. Deprotonated peptide amides produce a characteristic cm-2 - product ion (where m is the number of residues in the peptide) that is either absent or in low abundance in the analogous peptide acid spectrum. Peptide acids show an enhanced formation of cm-3 -; however, this is not generally as pronounced as cm-2 - production from amides. The most notable occurrence of an amide-specific product ion is for laminin amide (YIGSR-NH2) and this case was investigated using several modified peptides. Mechanisms involving 6- and 9-membered ring formation were proposed, and their energetic properties were investigated using G3(MP2) molecular orbital theory calculations. For example, with C-terminal deprotonation of pentaglycine amide, formation of cm-2 - and a 6-membered ring diketopiperazine neutral requires >31.6 kcal/mol, which is 26.1 kcal/mol less than the analogous process involving the peptide acid. The end group specific fragmentation of peptide amides in the negative ion mode may be useful for identifying such groups in proteomic applications.

  1. Purification and characterization of a vaterite-inducing peptide, pelovaterin, from the eggshells of Pelodiscus sinensis (Chinese soft-shelled turtle).

    PubMed

    Lakshminarayanan, Rajamani; Chi-Jin, Emma Ooi; Loh, Xian Jun; Kini, R Manjunatha; Valiyaveettil, Suresh

    2005-01-01

    Proteins play a crucial role in the biomineralization of hard tissues such as eggshells. We report here the purification, characterization, and in vitro mineralization studies of a peptide, pelovaterin, extracted from eggshells of a soft-shelled turtle. It is a glycine-rich peptide with 42 amino acid residues and three disulfide bonds. When tested in vitro, the peptide induced the formation of a metastable vaterite phase. The floret-shaped morphology formed at a lower concentration ( approximately 1 microM) was transformed into spherical particles at higher concentrations (>500 microM). The solution properties of the peptide are investigated by circular dichroism (CD), fluorescence emission spectroscopy, and dynamic light scattering (DLS) experiments. The conformation of pelovaterin changed from an unordered state at a low concentration to a beta-sheet structure at high concentrations. Fluorescence emission studies indicated that the quantum yield is significantly decreased at higher concentrations, accompanied by a blue shift in the emission maximum. At higher concentrations a red-edge excitation shift was observed, indicating the restricted mobility of the peptide. On the basis of these observations, we discuss the presence of a peptide concentration-dependent monomer-multimer equilibrium in solution and its role in controlling the nucleation, growth, and morphology of CaCO(3) crystals. This is the first peptide known to induce the nucleation and stabilization of the vaterite phase in solution.

  2. Peptide-induced de novo bone formation after tooth extraction prevents alveolar bone loss in a murine tooth extraction model.

    PubMed

    Arai, Yuki; Aoki, Kazuhiro; Shimizu, Yasuhiro; Tabata, Yasuhiko; Ono, Takashi; Murali, Ramachandran; Mise-Omata, Setsuko; Wakabayashi, Noriyuki

    2016-07-01

    Tooth extraction causes bone resorption of the alveolar bone volume. Although recombinant human bone morphogenetic protein 2 (rhBMP-2) markedly promotes de novo bone formation after tooth extraction, the application of high-dose rhBMP-2 may induce side effects, such as swelling, seroma, and an increased cancer risk. Therefore, reduction of the necessary dose of rhBMP-2 which can still obtain sufficient bone mass is necessary by developing a new osteogenic reagent. Recently, we showed that the systemic administration of OP3-4 peptide, which was originally designed as a bone resorption inhibitor, had osteogenic ability both in vitro and in vivo. This study evaluated the ability of the local application of OP3-4 peptide to promote bone formation in a murine tooth extraction model with a very low-dose of BMP. The mandibular incisor was extracted from 10-week-old C57BL6/J male mice and a gelatin hydrogel containing rhBMP-2 with or without OP3-4 peptide (BMP/OP3-4) was applied to the socket of the incisor. Bone formation inside the socket was examined radiologically and histologically at 21 days after the extraction. The BMP/OP3-4-group showed significant bone formation inside the mandibular extraction socket compared to the gelatin-hydrogel-carrier-control group or rhBMP-2-applied group. The BMP/OP3-4-applied mice showed a lower reduction of alveolar bone and fewer osteoclast numbers, suggesting that the newly formed bone inside the socket may prevent resorption of the cortical bone around the extraction socket. Our data revealed that OP3-4 peptide promotes BMP-mediated bone formation inside the extraction socket of mandibular bone, resulting in preservation from the loss of alveolar bone. PMID:27118173

  3. Hypothalamic Agouti-Related Peptide mRNA is Elevated During Natural and Stress-Induced Anorexia.

    PubMed

    Dunn, I C; Wilson, P W; D'Eath, R B; Boswell, T

    2015-09-01

    As part of their natural lives, animals can undergo periods of voluntarily reduced food intake and body weight (i.e. animal anorexias) that are beneficial for survival or breeding, such as during territorial behaviour, hibernation, migration and incubation of eggs. For incubation, a change in the defended level of body weight or 'sliding set point' appears to be involved, although the neural mechanisms reponsible for this are unknown. We investigated how neuropeptide gene expression in the arcuate nucleus of the domestic chicken responded to a 60-70% voluntary reduction in food intake measured both after incubation and after an environmental stressor involving transfer to unfamiliar housing. We hypothesised that gene expression would not change in these circumstances because the reduced food intake and body weight represented a defended level in birds with free access to food. Unexpectedly, we observed increased gene expression of the orexigenic peptide agouti-related peptide (AgRP) in both incubating and transferred animals compared to controls. Also pro-opiomelanocortin (POMC) mRNA was higher in incubating hens and significantly increased 6 days after exposure to the stressor. Conversely expression of neuropeptide Y and cocaine- and amphetamine-regulated transcript gene was unchanged in both experimental situations. We conclude that AgRP expression remains sensitive to the level of energy stores during natural anorexias, which is of adaptive advantage, although its normal orexigenic effects are over-ridden by inhibitory signals. In the case of stress-induced anorexia, increased POMC may contribute to this inhibitory role, whereas, for incubation, reduced feeding may also be associated with increased expression in the hypothalamus of the anorexigenic peptide vasoactive intestinal peptide.

  4. Nucleation in mesoscopic systems under transient conditions: Peptide-induced pore formation in vesicles

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.; Höök, Fredrik

    2013-04-01

    Attachment of lytic peptides to the lipid membrane of virions or bacteria is often accompanied by their aggregation and pore formation, resulting eventually in membrane rupture and pathogen neutralization. The membrane rupture may occur gradually via formation of many pores or abruptly after the formation of the first pore. In academic studies, this process is observed during interaction of peptides with lipid vesicles. We present an analytical model and the corresponding Monte Carlo simulations focused on the pore formation in such situations. Specifically, we calculate the time of the first nucleation-limited pore-formation event and show the distribution of this time in the regime when the fluctuations of the number of peptides attached to a vesicle are appreciable. The results obtained are used to clarify the mechanism of the pore formation and membrane destabilization observed recently during interaction of highly active α-helical peptide with sub-100-nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature. The model proposed and the analysis presented are generic and may be applicable to other meso- and nanosystems.

  5. Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor

    SciTech Connect

    Dame, J.B.; Williams, J.L.; McCutchan, T.F.; Schneider, I.

    1987-11-17

    An antimalarial immunogenic stimulant is described comprising an immunogenic carrier and a peptide sequence of between 2 and 1000 consecutive repeats of a sequence Asn-X-Y-Pro, wherein X is Ala or Val and Y is Asn or Asp.

  6. Glucagon-like peptide-2 induces rapid digestive adaptation following intestinal resection in preterm neonates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Short bowel syndrome (SBS) is a frequent complication after intestinal resection in infants suffering from intestinal disease. We tested whether treatment with the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) increases intestinal volume and function in the period immediately following in...

  7. Inducible Resistance of Fish Bacterial Pathogens to the Antimicrobial Peptide Cecropin B

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cecropin B is a cationic antimicrobial peptide originally isolated from the diapausing pupae of the giant silk moth, Hylphora cecropia. Cecropin B elicits its antimicrobial effects through disruption of the anionic cell membranes of gram-negative bacteria. Previous work by our laboratory demonstra...

  8. 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.

  9. Amelioration of ovalbumin-induced allergic airway disease following Der p 1 peptide immunotherapy is not associated with induction of IL-35.

    PubMed

    Moldaver, D M; Bharhani, M S; Wattie, J N; Ellis, R; Neighbour, H; Lloyd, C M; Inman, M D; Larché, M

    2014-03-01

    In the present study, we show therapeutic amelioration of established ovalbumin (OVA)-induced allergic airway disease following house dust mite (HDM) peptide therapy. Mice were sensitized and challenged with OVA and HDM protein extract (Dermatophagoides species) to induce dual allergen sensitization and allergic airway disease. Treatment of allergic mice with peptides derived from the major allergen Der p 1 suppressed OVA-induced airway hyperresponsiveness, tissue eosinophilia, and goblet cell hyperplasia upon rechallenge with allergen. Peptide treatment also suppressed OVA-specific T-cell proliferation. Resolution of airway pathophysiology was associated with a reduction in recruitment, proliferation, and effector function of T(H)2 cells and decreased interleukin (IL)-17⁺ T cells. Furthermore, peptide immunotherapy induced the regulatory cytokine IL-10 and increased the proportion of Fox p3⁺ cells among those expressing IL-10. Tolerance to OVA was not associated with increased IL-35. In conclusion, our results provide in vivo evidence for the creation of a tolerogenic environment following HDM peptide immunotherapy, leading to the therapeutic amelioration of established OVA-induced allergic airway disease.

  10. Tuberoinfundibular Peptide of 39 Residues Is Activated during Lactation and Participates in the Suckling-Induced Prolactin Release in Rat

    PubMed Central

    Cservenák, Melinda; Bodnár, Ibolya; Usdin, Ted B.; Palkovits, Miklós; Nagy, György M.; Dobolyi, Arpád

    2010-01-01

    Tuberoinfundibular peptide of 39 residues (TIP39) and the PTH-2 receptor (PTH2R) constitute a peptide-receptor neuromodulator system. Based on the abundance of TIP39 fibers and axonal terminals as well as PTH2R-containing neurons and their processes in the hypothalamic para- and periventricular and arcuate nuclei TIP39 has been suggested to play a role in neuroendocrine regulation. We showed previously that TIP39 expression decreased dramatically by adulthood. In the present study, using in situ hybridization histochemistry, real-time RT-PCR, and immunohistochemistry, we found that TIP39 mRNA and peptide expression levels are markedly elevated in the posterior intralaminar complex of the thalamus (PIL) of lactating dams, one of the three locations of TIP39-containing cell bodies in the brain. In addition, in mother rats, these TIP39 neurons showed Fos expression in response to pup exposure. Transection of TIP39 fibers originating in the PIL resulted in an ipsilateral disappearance of TIP39 immunoreactivity throughout the mediobasal hypothalamus of mother rats, suggesting that TIP39 fibers there arise from the PIL. To elucidate the function of TIP39 activation in dams, mothers separated from their pups for 4 h on postpartum d 9 received injection of a PTH2R antagonist into the lateral ventricle 5 min before returning the pups. Blood samples were taken seven times during the experimental period through jugular cannulae. The PTH2R antagonist administered in two different concentrations markedly inhibited suckling-induced elevation of plasma prolactin levels in a dose-dependent manner. These results suggest that TIP39 neurons in the PIL may regulate suckling-induced prolactin release in rat dams. PMID:20861230

  11. C-peptide reverses TGF-beta1-induced changes in renal proximal tubular cells: implications for treatment of diabetic nephropathy.

    PubMed

    Hills, Claire E; Al-Rasheed, Nawal; Al-Rasheed, Nouf; Willars, Gary B; Brunskill, Nigel J

    2009-03-01

    The crucial pathology underlying progressive chronic kidney disease in diabetes is tubulointerstitial fibrosis. Central to this process is epithelial-mesenchymal transformation (EMT) of proximal tubular epithelial cells driven by maladaptive transforming growth factor-beta1 (TGF-beta1) signaling. Novel signaling roles for C-peptide have recently been discovered with evidence emerging that C-peptide may mitigate microvascular complications of diabetes. We studied the potential for C-peptide to interrupt injurious TGF-beta1 signaling pathways and thus block development of EMT in HK2 human kidney proximal tubular cells. Cells were incubated with TGF-beta1 either alone or with C-peptide in low or high glucose. Changes in cell morphology, TGF-beta1 receptor expression, vimentin, E-cadherin, and phosphorylated Smads were assessed. Luciferase reporters were used to assess Smad activity. The cytoskeleton was visualized by TRITC-phalloidin staining. The typical TGF-beta1-stimulated, EMT-associated morphological alterations of proximal tubular cells, including increased vimentin expression, decreased E-cadherin expression, and cytoskeletal rearrangements, were prevented by C-peptide treatment. C-peptide also blocked TGF-beta1-induced upregulation of expression of both type I and type II TGF-beta1 receptors and attenuated TGF-beta1-mediated Smad phosphorylation and Smad transcriptional activity. These effects of C-peptide were inhibited by pertussis toxin. The results demonstrate that C-peptide almost completely reversed the morphological changes in PT cells induced by TGF-beta1 and suggest a role or C-peptide as a renoprotective agent in diabetic nephropathy. PMID:19091788

  12. A PEPTIDE UNCOUPLING CRMP-2 FROM THE PRESYNAPTIC Ca(2+) CHANNEL COMPLEX DEMONSTRATES EFFICACY IN ANIMAL MODELS OF MIGRAINE AND AIDS THERAPY-INDUCED NEUROPATHY.

    PubMed

    Ripsch, Matthew S; Ballard, Carrie J; Khanna, May; Hurley, Joyce H; White, Fletcher A; Khanna, Rajesh

    2012-03-01

    Biological, genetic, and clinical data provide compelling proof for N-type voltage-gated calcium channels (CaV2.2) as therapeutic targets for chronic pain. While decreasing channel function is ultimately anti-nociceptive, directly targeting the channel can lead to multiple adverse effects. Targeting regulators of channel activity may facilitate improved analgesic properties associated with channel block and afford a broader therapeutic window. Towards this end, we recently identified a short peptide, designated CBD3, derived from collapsin response mediator protein 2 (CRMP-2) that suppressed inflammatory and neuropathic hypersensitivity by inhibiting CRMP-2 binding to CaV2.2 [Brittain et al., Nature Medicine 17:822-829 (2011)]. Rodents administered CBD3 intraperitoneally, fused to the HIV TAT protein cell penetrating domain, exhibited antinociception lasting ~4 hours highlighting potential instability, limited oral bioavailability, and/or rapid elimination of peptide. This report focuses on improving upon the parental CBD3 peptide. Using SPOTScan analysis of synthetic versions of the parental CBD3 peptide, we identified peptides harboring single amino acid mutations that bound with greater affinity to CaV2.2. One such peptide, harboring a phenylalanine instead of glycine (G14F), was tested in rodent models of migraine and neuropathic pain. In vivo laser Doppler blood flowmetry measure of capsaicin-induced meningeal vascular responses related to headache pain was almost completely suppressed by dural application of the G14F peptide. The G14F mutant peptide, administered intraperitoneally, also exhibited greater antinociception in Stavudine (2'-3'-didehydro-2'-3'-dideoxythymidine (d4T)/Zerit®) model of AIDS therapy-induced peripheral neuropathy compared to the parent CBD3 peptide. These results demonstrate the patent translational value of small biologic drugs targeting CaV2.2 for management of clinical pain. PMID:22662308

  13. A PEPTIDE UNCOUPLING CRMP-2 FROM THE PRESYNAPTIC Ca2+ CHANNEL COMPLEX DEMONSTRATES EFFICACY IN ANIMAL MODELS OF MIGRAINE AND AIDS THERAPY-INDUCED NEUROPATHY

    PubMed Central

    Ripsch, Matthew S.; Ballard, Carrie J.; Khanna, May; Hurley, Joyce H.; White, Fletcher A.; Khanna, Rajesh

    2012-01-01

    Biological, genetic, and clinical data provide compelling proof for N-type voltage-gated calcium channels (CaV2.2) as therapeutic targets for chronic pain. While decreasing channel function is ultimately anti-nociceptive, directly targeting the channel can lead to multiple adverse effects. Targeting regulators of channel activity may facilitate improved analgesic properties associated with channel block and afford a broader therapeutic window. Towards this end, we recently identified a short peptide, designated CBD3, derived from collapsin response mediator protein 2 (CRMP-2) that suppressed inflammatory and neuropathic hypersensitivity by inhibiting CRMP-2 binding to CaV2.2 [Brittain et al., Nature Medicine 17:822–829 (2011)]. Rodents administered CBD3 intraperitoneally, fused to the HIV TAT protein cell penetrating domain, exhibited antinociception lasting ~4 hours highlighting potential instability, limited oral bioavailability, and/or rapid elimination of peptide. This report focuses on improving upon the parental CBD3 peptide. Using SPOTScan analysis of synthetic versions of the parental CBD3 peptide, we identified peptides harboring single amino acid mutations that bound with greater affinity to CaV2.2. One such peptide, harboring a phenylalanine instead of glycine (G14F), was tested in rodent models of migraine and neuropathic pain. In vivo laser Doppler blood flowmetry measure of capsaicin-induced meningeal vascular responses related to headache pain was almost completely suppressed by dural application of the G14F peptide. The G14F mutant peptide, administered intraperitoneally, also exhibited greater antinociception in Stavudine (2'-3'-didehydro-2'-3'-dideoxythymidine (d4T)/Zerit®) model of AIDS therapy-induced peripheral neuropathy compared to the parent CBD3 peptide. These results demonstrate the patent translational value of small biologic drugs targeting CaV2.2 for management of clinical pain. PMID:22662308

  14. S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

    PubMed

    Cardoso, Ana M S; Trabulo, Sara; Cardoso, Ana L; Lorents, Annely; Morais, Catarina M; Gomes, Paula; Nunes, Cláudia; Lúcio, Marlene; Reis, Salette; Padari, Kärt; Pooga, Margus; Pedroso de Lima, Maria C; Jurado, Amália S

    2012-03-01

    The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

  15. Altered influenza virus haemagglutinin (HA)-derived peptide is potent therapy for CIA by inducing Th1 to Th2 shift.

    PubMed

    Sun, Jian; Jia, Yuan; Li, Ru; Guo, Jianping; Sun, Xiaolin; Liu, Yanying; Li, Yingni; Yao, Haihong; Liu, Xia; Zhao, Jing; Li, Zhanguo

    2011-07-01

    There has been an increase in interest in the use of altered peptides as antigen-specific therapeutic agents in autoimmune diseases. Here we investigated the inhibitory effect and possible mechanism of an altered influenza virus haemagglutinin (HA)-derived peptide in collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by immunisation with type II collagen (CII). Altered HA308-317, wild-type HA308-317 or irrelevant peptide was administered intranasally beginning from arthritis onset. Clinical and histological scores were assessed, and cytokine levels in the serum or supernatants from splenocytes were determined. The percentages of Th1 and Th2 cells in response to different peptides were analysed by FACS both in vivo and in vitro. Our results showed that intranasal administration of altered HA308-317 peptide significantly ameliorated CIA. The therapeutic effect of altered HA308-317 peptide was associated with a substantial decrease in production of interferon (IFN)-γ, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, anti-CII IgG, IgG1 and IgG2a antibodies, and an markedly increase in production of IL-10 and IL-4 in serum or supernatants from splenocytes treated with altered HA308-317 peptide. The percentage of Th2 (CD4(+)IL-4(+)) cells was upregulated significantly by altered HA308-317 peptide with a decreased percentage of Th1 (T helper 1; CD4(+)INF-γ(+)) cells both in vivo and in vitro. These findings suggest that altered HA308-317 peptide might be a promising candidate for rheumatoid arthritis (RA) treatment.

  16. Transition metals as electron traps. I. Structures, energetics, electron capture, and electron-transfer-induced dissociations of ternary copper-peptide complexes in the gas phase.

    PubMed

    Turecek, Frantisek; Jones, Jace W; Holm, Anne I S; Panja, Subhasis; Nielsen, Steen Brøndsted; Hvelplund, Preben

    2009-05-01

    Electron-induced dissociations of gas-phase ternary copper-2,2'-bipyridine complexes of Gly-Gly-Gly and Gly-Gly-Leu were studied on a time scale ranging from 130 ns to several milliseconds using a combination of charge-reversal ((+)CR(-)) and electron-capture-induced dissociation (ECID) measured on a beam instrument and electron capture dissociation (ECD) measured in a Penning trap. Charge-reduced intermediates were observed on the short time scale in the (+)CR(-) and ECID experiments but not in ECD. Ion dissociations following electron transfer or capture mostly occurred by competitive bpy or peptide ligand loss, whereas peptide backbone fragmentations were suppressed in the presence of the ligated metal ion. Extensive electron structure theory calculations using density functional theory and large basis sets provided optimized structures and energies for the precursor ions, charge-reduced intermediates, and dissociation products. The Cu complexes underwent substantial structure changes upon electron capture. Cu was calculated to be pentacoordinated in the most stable singly charged complexes of the [Cu(peptide-H)bpy](+*) type where it carried a approximately +1 atomic charge. Cu coordination in charge-reduced [Cu(peptide-H)bpy] intermediates depended on the spin state. The themodynamically more stable singlet states had tricoordinated Cu, whereas triplet states had a tetracoordinated Cu. Cu was tricoordinated in stable [Cu(peptide-H)bpy](-*) products of electron transfer. [Cu(peptide)bpy](2+*) complexes contained the peptide ligand in a zwitterionic form while Cu was tetracoordinated. Upon electron capture, Cu was tri- or tetracoordinated in the [Cu(peptide)bpy](+) charge-reduced analogs and the peptide ligands underwent prototropic isomerization to canonical forms. The role of excited singlet and triplet electronic states is assessed. PMID:19132713

  17. Enhanced transcription of pancreatic peptide YY by 1α-hydroxyvitamin D3 administration in streptozotocin-induced diabetic mice.

    PubMed

    Ozeki, Jun; Choi, Mihwa; Endo-Umeda, Kaori; Sakurai, Kenichi; Amano, Sadao; Makishima, Makoto

    2013-10-01

    Peptide YY (PYY) is a peptide hormone secreted from L cells in the intestine in response to food intake that regulates appetite and gastrointestinal function. PYY is also produced in the pancreatic islets. The vitamin D receptor (VDR) is a nuclear receptor for the active form of vitamin D3 that regulates numerous physiological processes. VDR is expressed in the pancreatic islets and pharmacological VDR activation increases PYY expression in mouse peripheral islet cells. Although VDR is present in insulin-producing β cells as well as non-β cells, the role of β cell VDR in Pyy transcription remains unknown. We treated mice with streptozotocin to ablate β cells in the pancreas. Pancreatic Vdr mRNA expression was decreased in streptozotocin-induced diabetic mice. Interestingly, streptozotocin-treated mice exhibited increased basal Pyy expression and 1α-hydroxyvitamin D3 treatment further increased expression. Moreover, 1α-hydroxyvitamin D3 increased mRNA expression of pancreatic polypeptide and decreased that of neuropeptide Y in streptozotocin-induced diabetic mice but not in control mice. 1α-Hydroxyvitamin D3 slightly increased mRNA expression of insulin but transcript levels were nearly undetectable in the pancreas of streptozotocin-treated mice. Thus, VDR in non-β islet cells is involved in Pyy expression in the mouse pancreas. The findings from this β cell ablation study suggest a hormone transcription regulatory network composed of β cells and non-β cells.

  18. Electron-Induced Dissociation of Peptides in a Triple Quadrupole Mass Spectrometer Retrofitted with an Electromagnetostatic Cell

    NASA Astrophysics Data System (ADS)

    Voinov, Valery G.; Bennett, Samuel E.; Barofsky, Douglas F.

    2015-05-01

    Dissociation of peptides induced by interaction with (free) electrons (electron-induced dissociation, EID) at electron energies ranging from near 0 to >30 eV was carried out using a radio-frequency-free electromagnetostatic (EMS) cell retrofitted into a triple quadrupole mass spectrometer. The product-ion mass spectra exhibited EID originating from electronically excited even-electron precursor ions, reduced radical cations formed by capture of low-energy electrons, and oxidized radical cations produced by interaction with high-energy electrons. The spectra demonstrate, within the limits of the triple quadrupole's resolving power, that high-energy EID product-ion spectra produced with an EMS cell exhibit essentially the same qualitative structural information, i.e., amino acid side-chain (SC) losses and backbone cleavages, as observed in high-energy EID spectra produced with a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. The levels of fragmentation efficiency evident in the product-ion spectra recorded in this study, as was the case for those recorded in earlier studies with FT ICR mass spectrometers, is currently at the margin of analytical utility. Given that this shortcoming can be remedied, EMS cells incorporated into QqQ or QqTOF mass spectrometers could make tandem high-energy EID mass spectrometry more widely accessible for analysis of peptides, small singly charged molecules, pharmaceuticals, and clinical samples.

  19. Electron-induced dissociation of peptides in a triple quadrupole mass spectrometer retrofitted with an electromagnetostatic cell.

    PubMed

    Voinov, Valery G; Bennett, Samuel E; Barofsky, Douglas F

    2015-05-01

    Dissociation of peptides induced by interaction with (free) electrons (electron-induced dissociation, EID) at electron energies ranging from near 0 to >30 eV was carried out using a radio-frequency-free electromagnetostatic (EMS) cell retrofitted into a triple quadrupole mass spectrometer. The product-ion mass spectra exhibited EID originating from electronically excited even-electron precursor ions, reduced radical cations formed by capture of low-energy electrons, and oxidized radical cations produced by interaction with high-energy electrons. The spectra demonstrate, within the limits of the triple quadrupole's resolving power, that high-energy EID product-ion spectra produced with an EMS cell exhibit essentially the same qualitative structural information, i.e., amino acid side-chain (SC) losses and backbone cleavages, as observed in high-energy EID spectra produced with a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. The levels of fragmentation efficiency evident in the product-ion spectra recorded in this study, as was the case for those recorded in earlier studies with FT ICR mass spectrometers, is currently at the margin of analytical utility. Given that this shortcoming can be remedied, EMS cells incorporated into QqQ or QqTOF mass spectrometers could make tandem high-energy EID mass spectrometry more widely accessible for analysis of peptides, small singly charged molecules, pharmaceuticals, and clinical samples. PMID:25652934

  20. Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

    PubMed Central

    Yang, Mu; Zhou, Mei; Bai, Bing; Ma, Chengbang; Wei, Le; Wang, Lei; Chen, Tianbao; Shaw, Chris

    2011-01-01

    Objectives: The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical –KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens. PMID:21687349

  1. Flurbiprofen inhibits capsaicin induced calcitonin gene related peptide release from rat spinal cord via an endocannabinoid dependent mechanism.

    PubMed

    Seidel, Kay; Hamza, May; Ates, Mehmet; Gühring, Hans

    2003-02-27

    Calcitonin gene related peptide (CGRP) is involved in nociceptive transmission and modulation at the spinal level. In the spinal superperfusion model, Delta(9) tetrahydrocannabinol inhibited capsaicin induced CGRP release in a concentration dependent manner. Similarly, flurbiprofen (3 microM) inhibited spinal CGRP release. This inhibition was reversed by the CB(1) antagonist AM-251 (1 microM), but not by co-administration of prostaglandin E(2) (PGE(2); 285 nM). AM-251 had no modulatory effect on flurbiprofen-induced cyclooxygenase (COX) inhibiting capacity as shown by PGE(2) levels. Furthermore, the phospholipase A(2) inhibitor palmityl trifluromethyl ketone (15 microM) reversed flurbiprofen's inhibitory effect. In conclusion the present work provides evidence on the shift of arachidonic acid metabolism towards endocannabinoids formation in response to COX inhibition as a mechanism for flurbiprofen inhibitory effect on spinal CGRP release.

  2. Synthesis and evaluation of amphiphilic peptides as nanostructures and drug delivery tools

    NASA Astrophysics Data System (ADS)

    Sayeh, Naser Ali

    conjugates although one limitation lies in the effort of controlling the rate of drug release. The encapsulated or complexed drugs tend to be released rapidly (before reaching the target site) and in the dendrimer--drug conjugates, it is the chemical linkage that controls the drug release. Thus, future studies in this field are urgently required to create more efficient and stable biomaterials. Peptides are considered as efficient vectors for achieving optimal cellular uptake. The potential use of peptides as drug delivery vectors received much attention by the discovery of several cell-penetrating peptides (CPPs). The first CPPs discovered in 1988, that were sequences from HIV-1 encoded TAT protein, TAT (48--60), and penetrated very efficiently through cell membranes of cultured mammalian cells. CPPs are a class of diverse peptides, typically with 8--25 amino acids, and unlike most peptides, they can cross the cellular membrane with more efficiency. CPPs have also shown to undergo self-assembly and generate nanostructures. The generation of self-assembled peptides and nanostructures occur through various types of interactions between functional groups of amino acid residues, such as electrostatic, hydrophobic, and hydrogen bonding. Appropriate design and functionalization of peptides are critical for generating nanostructures. Chemically CPPs are classified into two major groups: linear and cyclic peptides. It has been previously reported that linear peptides containing hydrophilic and hydrophobic amino acids could act as membrane protein stabilizers. These compounds are short hydrophilic or amphiphilic peptides that have positively charged amino acids, such as arginine, lysine or histidine, which can interact with the negative charge phospholipids layer on the cell membrane and translocate the cargo into the cells. Conjugation to cationic linear CPPs, such as TAT, penetratin, or oligoarginine efficiently improves the cellular uptake of large hydrophilic molecules, but the

  3. Whey peptides prevent chronic ultraviolet B radiation-induced skin aging in melanin-possessing male hairless mice.

    PubMed

    Kimura, Yoshiyuki; Sumiyoshi, Maho; Kobayashi, Toshiya

    2014-01-01

    Whey proteins or peptides exhibit various actions, including an antioxidant action, an anticancer action, and a protective action against childhood asthma and atopic syndrome. The effects of orally administered whey peptides (WPs) on chronic ultraviolet B (UVB) radiation-induced cutaneous changes, including changes in cutaneous thickness, elasticity, wrinkle formation, etc., have not been examined. In this study, we studied the preventive effects of WPs on cutaneous aging induced by chronic UVB irradiation in melanin-possessing male hairless mice (HRM). UVB (36-180 mJ/cm(2)) was irradiated to the dorsal area for 17 wk in HRM, and the measurements of cutaneous thickness and elasticity in UVB irradiated mice were performed every week. WPs (200 and 400 mg/kg, twice daily) were administered orally for 17 wk. WPs inhibited the increase in cutaneous thickness, wrinkle formation, and melanin granules and the reduction in cutaneous elasticity associated with photoaging. Furthermore, it has been reported that UVB irradiation-induced skin aging is closely associated with the increase in expression of matrix metalloproteinase (MMP), vascular endothelial growth factor (VEGF), Ki-67-, and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells. WPs also prevented increases in the expression of MMP-2 and pro-MMP-9, VEGF, and Ki-67- and 8-OHdG-positive cells induced by chronic UVB irradiation. It was found that WPs prevent type IV collagen degradation, angiogenesis, proliferation, and DNA damage caused by UVB irradiation. Overall, these results demonstrate the considerable benefit of WPs for protection against solar UV-irradiated skin aging as a supplemental nutrient.

  4. Natriuretic peptides induce weak VASP phosphorylation at Serine 239 in platelets.

    PubMed

    Borgognone, Alessandra; Lowe, Kate L; Watson, Stephen P; Madhani, Melanie

    2014-01-01

    Cyclic guanosine-3',5'-monophoshate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides (NP; e.g. atrial NP [ANP]), which activate soluble and particulate guanylyl cyclases, respectively. The role of NO in regulating cGMP and platelet function is well documented, whereas there is little evidence supporting a role for NPs in regulating platelet reactivity. By studying platelet aggregation and secretion in response to a PAR-1 peptide, collagen and ADP, and phosphorylation of the cGMP-dependent protein kinase (PKG) substrate vasodilator-stimulated phosphoprotein (VASP) at serine 239, we evaluated the effects of NPs in the absence or presence of the non-selective cGMP and cAMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Our results show that NPs, possibly through the clearance receptor (natriuretic peptide receptor-C) expressed on platelet membranes, increase VASP phosphorylation but only following PDE inhibition, indicating a small, localised cGMP synthesis. As platelet aggregation and secretion measured under the same conditions were not affected, we conclude that the magnitude of PKG activation achieved by NPs in platelets per se is not sufficient to exert functional inhibition of platelet involvement in haemostasis. PMID:23469931

  5. Direct Observation of Aggregation-Induced Backbone Conformational Changes in Tau Peptides.

    PubMed

    Jiji, A C; Shine, A; Vijayan, Vinesh

    2016-09-12

    In tau proteins, the hexapeptides in the R2 and R3 repeats are known to initiate tau fibril formation, which causes a class of neurodegenerative diseases called the taupathies. We show that in R3, in addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion-like fibrils that are capable of propagation. A time-dependent NMR aggregation assay of a slow fibril forming R3-S316P peptide revealed a trans to cis equilibrium shift in the peptide-bond conformation preceding P316 during the growth phase of the aggregation process. S316 was identified as the key residue in the turn that confers templating capacity on R3 fibrils to accelerate the aggregation of the R3-S316P peptide. These results on the specific interactions and conformational changes responsible for tau aggregation could prove useful for developing an efficient therapeutic intervention in Alzheimer's disease. PMID:27513615

  6. Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

    PubMed

    Poutiainen, Pekka K; Rönkkö, Teemu; Hinkkanen, Ari E; Palvimo, Jorma J; Närvänen, Ale; Turhanen, Petri; Laatikainen, Reino; Weisell, Janne; Pulkkinen, Juha T

    2014-01-15

    In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

  7. Administration of antioxidant peptide SS-31 attenuates transverse aortic constriction-induced pulmonary arterial hypertension in mice

    PubMed Central

    Lu, Hung-i; Huang, Tien-hung; Sung, Pei-hsun; Chen, Yung-lung; Chua, Sarah; Chai, Han-yan; Chung, Sheng-ying; Liu, Chu-feng; Sun, Cheuk-kwan; Chang, Hsueh-wen; Zhen, Yen-yi; Lee, Fan-yen; Yip, Hon-kan

    2016-01-01

    Aim: Antioxidant peptide SS-31 is a class of cell-permeable small peptides, which selectively resides on the inner mitochondrial membrane and possesses intrinsic mitochondrial protective capacities. In this study we investigated the therapeutic effects of antioxidant peptide SS-31 on transverse aortic constriction (TAC)-induced pulmonary arterial hypertension (PAH) in a murine model. Methods: Adult male mice were divided into 3 groups: sham-operated mice, TAC mice, and TAC+SS-31 mice that underwent TAC surgery and received SS-31 (2 mg/d, ip) for 60 d. The right ventricular systolic blood pressure (RVSBP) was measured on d 60 prior to sacrificing the mice; then their right heart and lung tissues were collected for histological and biochemical examinations. Lung injury scores were defined by the increased crowded area and decreased number of alveolar sacs. Results: TAC mice showed significantly higher RVSBP compared with sham-operated mice, the elevation was substantially suppressed in TAC+SS-31 mice. The same pattern of changes was found in pulmonary levels of oxidative stress proteins (NOX-1/NOX-2/oxidized proteins), cytosolic cytochrome c, biomarkers related to inflammation (MMP-9/TNF-α/iNOS), calcium overload index (TRPC1, 2, 4, 6), apoptosis (mitochondrial BAX, cleaved caspase 3/PARP), fibrosis (Smad3/TGF-β), hypoxic (HIF-1α), DNA damage (γ-H2AX) and endothelial function (eNOS/ET-1R), as well as in lung injury score, number of muscularized vessels in lungs, number of TRPC1+ and HIF-1α+ cells in pulmonary artery, and number of γ-H2AX+ and Ki-67+ cells in lung parenchyma. An opposite pattern of changes was observed in pulmonary anti-fibrotic markers (Smad1/5, BMP-2), number of small vessels, and number of alveolar sacs. In contrast, the levels of antioxidant proteins (HO-1/NQO-1/GR/GPx) in lung parenchyma were progressively and significantly increased from sham-operated mice, TAC mice to TAC+SS-31 mice. Conclusion: Antioxidant peptide SS-31 administration

  8. Enhanced mucosal immunoglobulin A response and solid protection against foot-and-mouth disease virus challenge induced by a novel dendrimeric peptide.

    PubMed

    Cubillos, Carolina; de la Torre, Beatriz G; Jakab, Annamaria; Clementi, Giorgia; Borrás, Eva; Bárcena, Juan; Andreu, David; Sobrino, Francisco; Blanco, Esther

    2008-07-01

    The successful use of a dendrimeric peptide to protect pigs against challenge with foot-and-mouth disease virus (FMDV), which causes the most devastating animal disease worldwide, is described. Animals were immunized intramuscularly with a peptide containing one copy of a FMDV T-cell epitope and branching out into four copies of a B-cell epitope. The four immunized pigs did not develop significant clinical signs upon FMDV challenge, neither systemic nor mucosal FMDV replication, nor was its transmission to contact control pigs observed. The dendrimeric construction specifically induced high titers of FMDV-neutralizing antibodies and activated FMDV-specific T cells. Interestingly, a potent anti-FMDV immunoglobulin A response (local and systemic) was observed, despite the parenteral administration of the peptide. On the other hand, peptide-immunized animals showed no antibodies specific of FMDV infection, which qualifies the peptide as a potential marker vaccine. Overall, the dendrimeric peptide used elicited an immune response comparable to that found for control FMDV-infected pigs that correlated with a solid protection against FMDV challenge. Dendrimeric designs of this type may hold substantial promise for peptide subunit vaccine development.

  9. Neuroprotective effect of peptides extracted from walnut (Juglans Sigilata Dode) proteins on Aβ25-35-induced memory impairment in mice.

    PubMed

    Zou, Juan; Cai, Pei-shan; Xiong, Chao-mei; Ruan, Jin-lan

    2016-02-01

    Alzheimer's disease (AD) is one of the major neurodegenerative disorders of the elderly, which is characterized by the accumulation and deposition of amyloid-beta (Aβ) peptide in human brains. Oxidative stress and neuroinflammation induced by Aβ in brain are increasingly considered to be responsible for the pathogenesis of AD. The present study aimed to determine the protective effects of walnut peptides against the neurotoxicity induced by Aβ25-35 in vivo. Briefly, the AD model was induced by injecting Aβ25-35 into bilateral hippocampi of mice. The animals were treated with distilled water or walnut peptides (200, 400 and 800 mg/kg, p.o.) for five consecutive weeks. Spatial learning and memory abilities of mice were investigated by Morris water maze test and step-down avoidance test. To further explore the underlying mechanisms of the neuroprotectivity of walnut peptides, the activities of superoxide dismutase (SOD), glutathione (GSH), acetylcholine esterase (AChE), and the content of malondialdehyde (MDA) as well as the level of nitric oxide (NO) in the hippocampus of mice were measured by spectrophotometric method. In addition, the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and IL-6 in the samples were determined using ELISA. The hippocampal expressions of inducible nitric oxide synthase (iNOS) and nuclear factor κB (NF-κB) were evaluated by Western blot analysis. The results showed that walnut peptides supplementation effectively ameliorated the cognitive deficits and memory impairment of mice. Meanwhile, our study also revealed effective restoration of levels of antioxidant enzymes as well as inflammatory mediators with supplementation of walnut peptides (400 or 800 mg/kg). All the above findings suggested that walnut peptides may have a protective effect on AD by reducing inflammatory responses and modulating antioxidant system.

  10. Therapeutic Effect of Ergotope Peptides on Collagen-Induced Arthritis by Downregulation of Inflammatory and Th1/Th17 Responses and Induction of Regulatory T Cells

    PubMed Central

    Niu, Xiaoyin; Deng, Shaohua; Li, Shan; Xi, Yebin; Li, Chengzhen; Wang, Li; He, Dongyi; Wang, Zhaojun; Chen, Guangjie

    2016-01-01

    Rheumatoid arthritis (RA) is a systemic autoimmune disease that results in a chronic and inflammatory disorder. Dynamic balance of helper T cells (Th) 1 and 17 and regulatory T cells (Treg) is broken in RA. Since there is no cure for RA at present, it is necessary to find a truly effective and convenient treatment. Several studies have intended to induce ergotopic regulation to treat autoimmune diseases. This study was undertaken to find potential ergotope peptides and investigate their effects in treating the animal model of RA and their underlying regulatory mechanisms. First, we selected functional ergotope peptides from 25 overlapping peptides derived from the interleukin 2 receptor (IL-2R) α chain, and then used these peptides to treat collagen-induced arthritis (CIA). We showed ergotope peptides as immunomodulatory factors with great benefits at the clinical and pathologic levels. This effect was associated with inhibition of type II collagen (CII)-specific proliferation and autoantibody production as well as induction of antiergotypic immune response, downregulation of both Th1 and Th17 cells and their related components, and emergence of Treg cells that had suppressive action on autoreactive T cells. We also proved that cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) and IL-10 are two important mediators that are critical to Treg suppressive function. Inhibition of Th1 and Th17 in established CIA could be attributed to ergotope-induced Treg cells. Our findings reveal that ergotope peptides induce regulatory immune responses and restore immune tolerance, suggesting that treatment with ergotope peptides may be a novel approach to therapy for RA patients and has good application prospects, with cheap, effective, convenient, wide-spectrum features. PMID:27579476

  11. Vaccination with Lipid Core Peptides Fails to Induce Epitope-Specific T Cell Responses but Confers Non-Specific Protective Immunity in a Malaria Model

    PubMed Central

    Apte, Simon H.; Groves, Penny L.; Skwarczynski, Mariusz; Fujita, Yoshio; Chang, Chenghung; Toth, Istvan; Doolan, Denise L.

    2012-01-01

    Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP) vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4+ and/or CD8+ T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8+ T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8+ T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge. PMID:22936972

  12. Topical application of superoxide dismutase mediated by HIV-TAT peptide attenuates UVB-induced damages in human skin.

    PubMed

    Chen, Xiaochao; Liu, Shutao; Rao, Pingfan; Bradshaw, Jeremy; Weller, Richard

    2016-10-01

    The purpose of this study was to evaluate whether topical application of superoxide dismutase with cell penetrating peptide (HIV-TAT) could protect against skin damage induced by UVB irradiation in humans. The permeability through stratum corneum of large proteins linked to TAT peptide was firstly confirmed by confocal microscopy and tape stripping. Ten healthy volunteers with either Fitzpatrick skin type II or III were recruited in this clinical study. TAT-SOD (300units/cm(2)) and vehicle cream were applied on two symmetric areas of both inner upper arms 1h prior to UVB irradiation. After one hour of pretreatment, subjects received 10 incremental doses of UVB on pretreated areas. 24h later, erythema, blood flow and apoptotic cells were measured. Pretreatment with TAT-SOD 1h prior to UVB radiation promoted a mean minimal erythema dose (MED) increase of 36.6±18.4% (p=0.013<0.05. n=10) compared to vehicle control. The median blood flow values of all subjects following 2 and 3-MED of UVB were 107.8±51.0units and 239.5±88.0units respectively, which account for 26% and 25% decrease with respect to vehicle groups. These data suggest that TAT-SOD significantly suppresses UVB induced erythema formation and blood flow rise. Furthermore, pretreatment with TAT-SOD 1h prior to 2-MED of UVB irradiation reduced the apoptotic sunburn cell formation by 47.6±8.6% (p<0.0001) in all subjects. Evaluating results generated from all measurements, we conclude that topical application of TAT-SOD significantly attenuates UVB-induced skin damage in man. These biological effects of TAT-SOD are probably mediated via its free radical scavenging properties, clearly differentiating it from other physical sunscreen agents. PMID:27460952

  13. Topical application of superoxide dismutase mediated by HIV-TAT peptide attenuates UVB-induced damages in human skin.

    PubMed

    Chen, Xiaochao; Liu, Shutao; Rao, Pingfan; Bradshaw, Jeremy; Weller, Richard

    2016-10-01

    The purpose of this study was to evaluate whether topical application of superoxide dismutase with cell penetrating peptide (HIV-TAT) could protect against skin damage induced by UVB irradiation in humans. The permeability through stratum corneum of large proteins linked to TAT peptide was firstly confirmed by confocal microscopy and tape stripping. Ten healthy volunteers with either Fitzpatrick skin type II or III were recruited in this clinical study. TAT-SOD (300units/cm(2)) and vehicle cream were applied on two symmetric areas of both inner upper arms 1h prior to UVB irradiation. After one hour of pretreatment, subjects received 10 incremental doses of UVB on pretreated areas. 24h later, erythema, blood flow and apoptotic cells were measured. Pretreatment with TAT-SOD 1h prior to UVB radiation promoted a mean minimal erythema dose (MED) increase of 36.6±18.4% (p=0.013<0.05. n=10) compared to vehicle control. The median blood flow values of all subjects following 2 and 3-MED of UVB were 107.8±51.0units and 239.5±88.0units respectively, which account for 26% and 25% decrease with respect to vehicle groups. These data suggest that TAT-SOD significantly suppresses UVB induced erythema formation and blood flow rise. Furthermore, pretreatment with TAT-SOD 1h prior to 2-MED of UVB irradiation reduced the apoptotic sunburn cell formation by 47.6±8.6% (p<0.0001) in all subjects. Evaluating results generated from all measurements, we conclude that topical application of TAT-SOD significantly attenuates UVB-induced skin damage in man. These biological effects of TAT-SOD are probably mediated via its free radical scavenging properties, clearly differentiating it from other physical sunscreen agents.

  14. Glucagon-like peptide-1 inhibits angiotensin II-induced mesangial cell damage via protein kinase A.

    PubMed

    Ishibashi, Yuji; Matsui, Takanori; Ojima, Ayako; Nishino, Yuri; Nakashima, Sae; Maeda, Sayaka; Yamagishi, Sho-ichi

    2012-11-01

    There is a growing body of evidence that renin-angiotensin system plays a role in diabetic nephropathy. Recently, we have found that glucagon-like peptide-1 (GLP-1), one of the incretins, a gut hormone secreted from L cells in the intestine in response to food intake, inhibits advanced glycation end product-induced monocyte chemoattractant protein-1 gene expression in mesangial cells thorugh the interaction with the receptor of GLP-1. However, effects of GLP-1 on angiotensin II-exposed mesangial cells are unknown. This study investigated whether and how GLP-1 blocked the angiotensin II-induced mesangial cell damage in vitro. GLP-1 completely blocked the angiotensin II-induced superoxide generation, NF-κB activation, up-regulation of mRNA levels of intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 in mesangial cells, all of which were prevented by the treatments with H-89, an inhibitor of protein kinase A. The present results demonstrated for the first time that GLP-1 blocked the angiotensin II-induced mesangial cell injury by inhibiting superoxide-mediated NF-κB activation via protein kinase C pathway. Our present study suggests that strategies to enhance the biological actions of GLP-1 may be a promising strategy for the treatment of diabetic nephropathy.

  15. Apparent involvement of opioid peptides in stress-induced enhancement of tumor growth.

    PubMed

    Lewis, J W; Shavit, Y; Terman, G W; Nelson, L R; Gale, R P; Liebeskind, J C

    1983-01-01

    Exposure to stress has been associated with alterations in both immune function and tumor development in man and laboratory animals. In the present study, we investigated the effect of a particular type of inescapable footshock stress, known to cause an opioid mediated form of analgesia, on survival time of female Fischer 344 rats injected with a mammary ascites tumor. Rats subjected to inescapable footshock manifested an enhanced tumor growth indicated by a decreased survival time and decreased percent survival. This tumor enhancing effect of stress was prevented by the opiate antagonist, naltrexone, suggesting a role for endogenous opioid peptides in this process. In the absence of stress, naltrexone did not affect tumor growth.

  16. Lipid polymorphism induced by surfactant peptide SP-B(1-25).

    PubMed

    Farver, R Suzanne; Mills, Frank D; Antharam, Vijay C; Chebukati, Janetricks N; Fanucci, Gail E; Long, Joanna R

    2010-09-22

    Pulmonary surfactant protein B (SP-B) is an essential protein for lowering surface tension in the alveoli. SP-B(1-25), a peptide comprised of the N-terminal 25 amino-acid residues of SP-B, is known to retain much of the biological activity of SP-B. Circular dichroism has shown that when SP-B(1-25) interacts with negatively charged lipid vesicles, it contains significant helical structure for the lipid compositions and peptide/lipid ratios studied here. The effect of SP-B(1-25) on lipid organization and polymorphisms was investigated via DSC, dynamic light scattering, transmission electron microscopy, and solid-state NMR spectroscopy. At 1-3 mol% peptide and physiologic temperature, SP-B(1-25) partitions at the interface of negatively charged PC/PG lipid bilayers. In lipid mixtures containing 1-5 mol% peptide, the structure of SP-B(1-25) remains constant, but (2)H and (31)P NMR spectra show the presence of an isotropic lipid phase in exchange with the lamellar phase below the T(m) of the lipids. This behavior is observed for both DPPC/POPG and POPC/POPG lipid mixtures as well as for both the PC and PG components of the mixtures. For 1-3 mol% SP-B(1-25), a return to a single lamellar phase above the lipid mixture T(m) is observed, but for 5 mol% SP-B(1-25) a significant isotropic component is observed at physiologic temperatures for DPPC and exchange broadening is observed in (2)H and (31)P NMR spectra of the other lipid components in the two mixtures. DLS and TEM rule out the formation of micellar structures and suggest that SP-B(1-25) promotes the formation of a fluid isotropic phase. The ability of SP-B(1-25) to fuse lipid lamellae via this mechanism, particularly those enriched in DPPC, suggests a specific role for the highly conserved N-terminus of SP-B in the packing of lipid lamellae into surfactant lamellar bodies or in stabilizing multilayer structures at the air-liquid interface. Importantly, this behavior has not been seen for the other SP-B fragments of

  17. Delivery of RANKL-Binding Peptide OP3-4 Promotes BMP-2-Induced Maxillary Bone Regeneration.

    PubMed

    Uehara, T; Mise-Omata, S; Matsui, M; Tabata, Y; Murali, R; Miyashin, M; Aoki, K

    2016-06-01

    Although bone morphogenetic protein 2 (BMP-2) is known to stimulate osteogenesis, there is evidence that high doses of BMP-2 can lead to side effects, including inflammation and carcinogenesis. The supplementation of other bone-augmenting agents is considered helpful in preventing such side effects by reducing the amount of BMP-2 required to obtain a sufficient amount of bone. We recently showed that a receptor activator of nuclear factor κB ligand (RANKL)-binding peptide promotes osteoblast differentiation. In the present study, we aimed to investigate whether OP3-4, a RANKL-binding peptide, promotes BMP-2-induced bone formation in the murine maxilla using an injectable gelatin hydrogel (GH) carrier. A GH carrier containing OP3-4 with BMP-2 was subperiosteally injected into the murine maxillary right diastema between the incisor and the first molar. The mice were sacrificed 28 d after the injections. The local bone formation in the OP3-4-BMP-2-injected group was analyzed in comparison to the carrier-injected, BMP-2-injected, and control-peptide-BMP-2-injected groups. The GH carrier containing OP3-4 with BMP-2 enlarged the radio-opaque area and increased the bone mineral content and density in the radiological analyses in comparison to the other experimental groups. Interestingly, fluorescence-based histological analyses revealed that the mineralization had started from the outside, then proceeded inward, suggesting that the size of the newly formed bone had already been set before calcification started and that the effects of OP3-4 might be involved in accelerating the early steps of osteogenesis. Actually, OP3-4 enhanced the BMP-2-induced 5-bromo-2'-deoxyuridine (BrdU)-positive cell numbers at the injected site on day 7 and the expression of Runx2 and Col1a1, which are early osteogenic cell markers, on day 10 after the subperiosteal injections. In summary, we demonstrated, for the first time, that the application of OP3-4 by subperiosteal injection promoted BMP

  18. Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production

    PubMed Central

    Szarka, Eszter; Neer, Zsuzsa; Balogh, Péter; Ádori, Monika; Angyal, Adrienn; Prechl, József; Kiss, Endre; Kövesdi, Dorottya; Sármay, Gabriella

    2012-01-01

    Antibodies specific for bovine type II collagen (CII) and Fcγ receptors play a major role in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA) and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv) fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2). These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro-inflammatory cytokines. Taken together, these results suggest that the in vivo emerging immune complexes formed with autoantigen(s) may trigger the IL-12/23 dependent pathways, escalating the inflammation in RA. Thus blockade of these cytokines may be beneficial to downregulate immune complex-induced inflammation in autoimmune arthritis. PMID:22532778

  19. Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production.

    PubMed

    Szarka, Eszter; Neer, Zsuzsa; Balogh, Péter; Adori, Monika; Angyal, Adrienn; Prechl, József; Kiss, Endre; Kövesdi, Dorottya; Sármay, Gabriella

    2012-01-01

    Antibodies specific for bovine type II collagen (CII) and Fcγ receptors play a major role in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA) and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv) fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2). These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro-inflammatory cytokines. Taken together, these results suggest that the in vivo emerging immune complexes formed with autoantigen(s) may trigger the IL-12/23 dependent pathways, escalating the inflammation in RA. Thus blockade of these cytokines may be beneficial to downregulate immune complex-induced inflammation in autoimmune arthritis. PMID:22532778

  20. Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes.

    PubMed

    Ochoa-Zarzosa, Alejandra; Villarreal-Fernández, Edith; Cano-Camacho, Horacio; López-Meza, Joel E

    2009-07-01

    A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25-0.5mM) reduces approximately 50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), beta-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC. PMID:19393738

  1. Sera from Children with Autism Induce Autistic Features Which Can Be Rescued with a CNTF Small Peptide Mimetic in Rats

    PubMed Central

    Kazim, Syed Faraz; Cardenas-Aguayo, Maria del Carmen; Arif, Mohammad; Blanchard, Julie; Fayyaz, Fatima; Grundke-Iqbal, Inge; Iqbal, Khalid

    2015-01-01

    Autism is a neurodevelopmental disorder characterized clinically by impairments in social interaction and verbal and non-verbal communication skills as well as restricted interests and repetitive behavior. It has been hypothesized that altered brain environment including an imbalance in neurotrophic support during early development contributes to the pathophysiology of autism. Here we report that sera from children with autism which exhibited abnormal levels of various neurotrophic factors induced cell death and oxidative stress in mouse primary cultured cortical neurons. The effects of sera from autistic children were rescued by pre-treatment with a ciliary neurotrophic factor (CNTF) small peptide mimetic, Peptide 6 (P6), which was previously shown to exert its neuroprotective effect by modulating CNTF/JAK/STAT pathway and LIF signaling and by enhancing brain derived neurotrophic factor (BDNF) expression. Similar neurotoxic effects and neuroinflammation were observed in young Wistar rats injected intracerebroventricularly with autism sera within hours after birth. The autism sera injected rats demonstrated developmental delay and deficits in social communication, interaction, and novelty. Both the neurobiological changes and the behavioral autistic phenotype were ameliorated by P6 treatment. These findings implicate the involvement of neurotrophic imbalance during early brain development in the pathophysiology of autism and a proof of principle of P6 as a potential therapeutic strategy for autism. PMID:25769033

  2. Sera from children with autism induce autistic features which can be rescued with a CNTF small peptide mimetic in rats.

    PubMed

    Kazim, Syed Faraz; Cardenas-Aguayo, Maria Del Carmen; Arif, Mohammad; Blanchard, Julie; Fayyaz, Fatima; Grundke-Iqbal, Inge; Iqbal, Khalid

    2015-01-01

    Autism is a neurodevelopmental disorder characterized clinically by impairments in social interaction and verbal and non-verbal communication skills as well as restricted interests and repetitive behavior. It has been hypothesized that altered brain environment including an imbalance in neurotrophic support during early development contributes to the pathophysiology of autism. Here we report that sera from children with autism which exhibited abnormal levels of various neurotrophic factors induced cell death and oxidative stress in mouse primary cultured cortical neurons. The effects of sera from autistic children were rescued by pre-treatment with a ciliary neurotrophic factor (CNTF) small peptide mimetic, Peptide 6 (P6), which was previously shown to exert its neuroprotective effect by modulating CNTF/JAK/STAT pathway and LIF signaling and by enhancing brain derived neurotrophic factor (BDNF) expression. Similar neurotoxic effects and neuroinflammation were observed in young Wistar rats injected intracerebroventricularly with autism sera within hours after birth. The autism sera injected rats demonstrated developmental delay and deficits in social communication, interaction, and novelty. Both the neurobiological changes and the behavioral autistic phenotype were ameliorated by P6 treatment. These findings implicate the involvement of neurotrophic imbalance during early brain development in the pathophysiology of autism and a proof of principle of P6 as a potential therapeutic strategy for autism.

  3. Heterologous Prime-Boost Oral Immunization with GK-1 Peptide from Taenia crassiceps Cysticerci Induces Protective Immunity▿

    PubMed Central

    Fragoso, Gladis; Esquivel-Guadarrama, Fernando; Santana, M. Angélica; Bobes, Raul J.; Hernández, Beatriz; Cervantes, Jacquelynne; Segura, René; Goldbaum, Fernando A.; Sciutto, Edda; Rosas, Gabriela

    2011-01-01

    Oral immunization is a goal in vaccine development, particularly for pathogens that enter the host through the mucosal system. This study was designed to explore the immunogenic properties of the Taenia crassiceps protective peptide GK-1 administered orally. Mice were orally immunized with the synthetic GK-1 peptide in its linear form with or without the Brucella lumazine synthase (BLS) protein adjuvant or as a chimera recombinantly bound to BLS (BLS-GK-1). Mice were boosted twice with GK-1 only at 15-day intervals. A significant rate of protection of 64.7% was achieved in GK-1-immunized mice, and that rate significantly increased to 91.8 and 96% when mice were primed with GK-1 coadministered with BLS as an adjuvant and BLS as a carrier, respectively. Specific antibodies and T cell activation and proliferation accompanied the protection induced, revealing the potent immunogenicity of GK-1. Through immunohistochemical studies, GK-1 was detected in T and B cell zones of the Peyer's patches (PP) and mesenteric lymph nodes. In the latter, abundant proliferating cells were detected by 5′-bromo-2′-deoxyuridine incorporation. No proliferation was detected in PP. Altogether, these results portray the potent immunogenic properties of GK-1 administered orally and reinforce the usefulness of BLS as an adjuvant and adequate vaccine delivery system for oral vaccines. PMID:21593234

  4. Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes.

    PubMed

    Ochoa-Zarzosa, Alejandra; Villarreal-Fernández, Edith; Cano-Camacho, Horacio; López-Meza, Joel E

    2009-07-01

    A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25-0.5mM) reduces approximately 50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), beta-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC.

  5. Snakin-2, an Antimicrobial Peptide from Potato Whose Gene Is Locally Induced by Wounding and Responds to Pathogen Infection1

    PubMed Central

    Berrocal-Lobo, Marta; Segura, Ana; Moreno, Manuel; López, Gemma; García-Olmedo, Francisco; Molina, Antonio

    2002-01-01

    The peptide snakin-2 (StSN2) has been isolated from potato (Solanum tuberosum cv Jaerla) tubers and found to be active (EC50 = 1–20 μm) against fungal and bacterial plant pathogens. It causes a rapid aggregation of both Gram-positive and Gram-negative bacteria. The corresponding StSN2 cDNA encodes a signal sequence followed by a 15-residue acidic sequence that precedes the mature StSN2 peptide, which is basic (isoelectric point = 9.16) and 66 amino acid residues long (molecular weight of 7,025). The StSN2 gene is developmentally expressed in tubers, stems, flowers, shoot apex, and leaves, but not in roots, or stolons, and is locally up-regulated by wounding and by abscisic acid treatment. Expression of this gene is also up-regulated after infection of potato tubers with the compatible fungus Botritys cinerea and down-regulated by the virulent bacteria Ralstonia solanacearum and Erwinia chrysanthemi. These observations are congruent with the hypothesis that the StSN2 is a component of both constitutive and inducible defense barriers. PMID:11891250

  6. Mechanisms of peptide fragmentation from time- and energy-resolved surface-induced dissociation studies: Dissociation of angiotensin analogs

    NASA Astrophysics Data System (ADS)

    Laskin, Julia; Bailey, Thomas H.; Futrell, Jean H.

    2006-03-01

    Energetics and mechanism of dissociation of singly protonated angiotensin III (RVYIHPF) and its analogs RVYIFPF, RVYIYPF, RVYIHAF and RVYIHDF was studied using surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially configured for studying ion activation by collisions with surfaces. The energetics and dynamics of peptide fragmentation were deduced by modeling the time- and energy-resolved survival curves for each precursor ion using an RRKM-based approach developed in our laboratory. Fragmentation mechanisms were inferred from comparison of time- and energy-resolved fragmentation efficiency curves (TFECs) of different fragment ions followed by RRKM modeling of dissociation of angiotensin III into six major families of fragment ions. Detailed modeling demonstrated that dissociation of these peptides is dominated by loss of ammonia from the precursor ion and characterized by a high-energy barrier of 1.6 eV. Loss of NH3 and subsequent rearrangement of the MH+-NH3 ion results in proton mobilization and release of ca. 30 kcal/mol into internal excitation of the MH+-NH3 ion. The resulting highly excited ion accesses a variety of non-specific dissociation pathways with very high rate constants. Fast fragmentation of excited MH+-NH3 ion forms a variety of abundant bn-NH3 and an-NH3 fragment ions. Abundant XH and HX internal fragments are also formed, reflecting the stability of histidine-containing diketopiperazine structures.

  7. Superior Cervical Ganglia Neurons Induce Foxp3+ Regulatory T Cells via Calcitonin Gene-Related Peptide

    PubMed Central

    Szklany, Kirsten; Ruiter, Evelyn; Mian, Firoz; Kunze, Wolfgang; Bienenstock, John; Forsythe, Paul; Karimi, Khalil

    2016-01-01

    The nervous and immune systems communicate bidirectionally, utilizing diverse molecular signals including cytokines and neurotransmitters to provide an integrated response to changes in the body’s internal and external environment. Although, neuro-immune interactions are becoming better understood under inflammatory circumstances and it has been evidenced that interaction between neurons and T cells results in the conversion of encephalitogenic T cells to T regulatory cells, relatively little is known about the communication between neurons and naïve T cells. Here, we demonstrate that following co-culture of naïve CD4+ T cells with superior cervical ganglion neurons, the percentage of Foxp3 expressing CD4+CD25+ cells significantly increased. This was mediated in part by immune-regulatory cytokines TGF-β and IL-10, as well as the neuropeptide calcitonin gene-related peptide while vasoactive intestinal peptide was shown to play no role in generation of T regulatory cells. Additionally, T cells co-cultured with neurons showed a decrease in the levels of pro-inflammatory cytokine IFN-γ released upon in vitro stimulation. These findings suggest that the generation of Tregs may be promoted by naïve CD4+ T cell: neuron interaction through the release of neuropeptide CGRP. PMID:27022966

  8. In cellulo phosphorylation induces pharmacological reprogramming of maurocalcin, a cell-penetrating venom peptide

    PubMed Central

    Ronjat, Michel; Feng, Wei; Dardevet, Lucie; Dong, Yao; Al Khoury, Sawsan; Chatelain, Franck C.; Vialla, Virginie; Chahboun, Samir; Lesage, Florian; Darbon, Hervé; Pessah, Isaac N.; De Waard, Michel

    2016-01-01

    The venom peptide maurocalcin (MCa) is atypical among toxins because of its ability to rapidly translocate into cells and potently activate the intracellular calcium channel type 1 ryanodine receptor (RyR1). Therefore, MCa is potentially subjected to posttranslational modifications within recipient cells. Here, we report that MCa Thr26 belongs to a consensus PKA phosphorylation site and can be phosphorylated by PKA both in vitro and after cell penetration in cellulo. Unexpectedly, phosphorylation converts MCa from positive to negative RyR1 allosteric modulator. Thr26 phosphorylation leads to charge neutralization of Arg24, a residue crucial for MCa agonist activity. The functional effect of Thr26 phosphorylation is partially mimicked by aspartyl mutation. This represents the first case, to our knowledge, of both ex situ posttranslational modification and pharmacological reprogramming of a small natural cystine-rich peptide by target cells. So far, phosphorylated MCa is the first specific negative allosteric modulator of RyR1, to our knowledge, and represents a lead compound for further development of phosphatase-resistant analogs. PMID:27071086

  9. Vitamin D receptor activation induces peptide YY transcription in pancreatic islets.

    PubMed

    Choi, Mihwa; Ozeki, Jun; Hashizume, Masami; Kato, Shigeaki; Ishihara, Hisamitsu; Makishima, Makoto

    2012-11-01

    Peptide YY (PYY) is a peptide hormone secreted from L cells in the intestine after food intake and regulates appetite and intestinal function. PYY is also expressed in the pancreas, but the mechanisms of regulation of pancreatic PYY expression have not been elucidated. The vitamin D receptor (VDR) is a nuclear receptor for the active form of vitamin D(3) and regulates numerous physiological processes. Because VDR is expressed in the pancreas, we investigated the role of pancreatic VDR activation and found that Pyy is a VDR target gene in the mouse pancreas. Treatment of mice with 1α-hydroxyvitamin D(3) increased plasma PYY levels. VDR activation increased mRNA and protein expression of PYY in the pancreatic islets of mice and pancreatic endocrine cell lines but did not change intestinal PYY expression. 1α-Hydroxyvitamin D(3)-dependent induction of pancreatic and plasma PYY was abolished in VDR-null mice. We identified a functional vitamin D-responsive element in the mouse Pyy promoter using chromatin immunoprecipitation assay, EMSA, and luciferase promoter assay. Thus, Pyy is a tissue-specific VDR target gene. The pancreatic VDR-PYY pathway may mediate a regulatory function of vitamin D in the neuroendocrine system.

  10. Growth Hormone Releasing Peptide-2 Attenuation of Protein Kinase C-Induced Inflammation in Human Ovarian Granulosa Cells

    PubMed Central

    Chao, Yi-Ning; Sun, David; Peng, Yen-Chun; Wu, Yuh-Lin

    2016-01-01

    Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two important inflammatory mediators in ovulation. Ghrelin may modulate inflammatory signaling via growth hormone secretagogue receptors. We investigated the role of ghrelin in KGN human ovarian granulosa cells using protein kinase C (PKC) activator phorbol 12, 13-didecanoate (PDD) and synthetic ghrelin analog growth hormone releasing peptide-2 (GHRP-2). GHRP-2 attenuated PDD-induced expression of protein and mRNA, the promoter activity of COX-2 and IL-8 genes, and the secretion of prostaglandin E2 (PGE2) and IL-8. GHRP-2 promoted the degradation of PDD-induced COX-2 and IL-8 proteins with the involvement of proteasomal and lysosomal pathways. PDD-mediated COX-2 production acts via the p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways; PDD-mediated IL-8 production acts via the p38, JNK and ERK pathways. GHRP-2 reduced the PDD-induced phosphorylation of p38 and JNK and activator protein 1 (AP-1) reporter activation and PDD-induced NF-κB nuclear translocation and reporter activation. The inhibitors of mitogen-activated protein kinase phosphatase-1 (MKP-1) and protein phosphatase 2 (PP2A) reduced the inhibitory effect of GHRP-2 on PDD-induced COX-2 and IL-8 expression. Our findings demonstrate an anti-inflammatory role for ghrelin (GHRP-2) in PKC-mediated inflammation of granulosa cells, at least in part, due to its inhibitory effect on PKC-induced activation of p38, JNK and NF-κB, possibly by targeting to MKP-1 and PP2A. PMID:27548147

  11. Synthesis of CNS active thyrotropin-releasing hormone (TRH)-like peptides: Biological evaluation and effect on cognitive impairment induced by cerebral ischemia in mice.

    PubMed

    Meena, Chhuttan L; Thakur, Avinash; Nandekar, Prajwal P; Sangamwar, Abhay T; Sharma, Shyam S; Jain, Rahul

    2015-09-01

    Thyrotropin-releasing hormone (TRH)-like peptides were synthesized by replacing critical histidine and pGlu residues in the native peptide. The peptides were evaluated in vitro for receptor binding activity assay and in the cell functional assay; the peptides exhibit selective basal signaling agonist behavior toward TRH-R2. For example, peptides 8a, 8b, 8c, 8 f, 8 h, 8 l and 12 d activated TRH-R2 with potency (EC50) of 0.53 μM, 0.048 μM, 0.05 μM, 0.006 μM, 0.31 μM, 0.034 μM and 0.004 μM, respectively. In contrast for signaling activation of TRH-R1, the same peptide required higher concentration of 19.35 μM, 3.98 μM, 2.54 μM, 0.287 μM, 11.28 μM, 0.986 μM and 0.944 μM, respectively. The results showed that peptides were 36.5, 82.9, 50.8, 47.8, 36.3, 32.6 and 235-fold selective to TRH-R2 receptor subtype. The peptides were investigated for CNS activity at 10 μmol/kg in pentobarbital-induced sleep assay study. Peptides 8c (16.5 ± 1.4 min) and 8l (16.5 ± 2.1 min) displayed excellent CNS activity. In an in vivo study, peptide 8c did not cause significant change in the rat plasma TSH levels. The peptide 8c was further investigated for neuroprotective potential, and significantly reduced infracts volume and neurological score in the focal cerebral ischemia model in mice. Peptide 8c also significantly lowered MDA levels, indicating reduction of oxidative and enhanced percentage cell survival in CA1 region, when compared to ischemic brain. PMID:26216015

  12. A rare complication of transitional cell carcinoma of the renal pelvis: parathyroid hormone-related peptide-induced hypercalcaemia.

    PubMed

    O Sullivan, Eoin; Plant, William

    2014-01-01

    We describe a rare occurrence of parathyroid hormone-related peptide (PTHrp) associated hypercalcaemia with a recurrence of transitional cell carcinoma of the renal pelvis. Our patient presented with serum calcium of 3.9 mmol/L, PTH of 5 ng/L and a PTHrp of 9.8 pmol/L (<2 pmol/L). He had no evidence of metastatic disease. His hypercalcaemia responded to bisphosphonate therapy. He chose to be treated conservatively and died 5 weeks after presentation. This is the seventh such case described in the literature. PTHrp-induced hypercalcaemia is associated with a grave prognosis, with a mean survival of 65 days from presentation. PMID:24951595

  13. Inhibitory effect of oatmeal extract oligomer on vasoactive intestinal peptide-induced inflammation in surviving human skin.

    PubMed

    Boisnic, S; Branchet-Gumila, M C; Coutanceau, C

    2003-01-01

    The aim of this study was to evaluate the antiinflammatory effect of oatmeal extract oligomer on skin fragments stimulated by a neuromediator, vasoactive intestinal peptide (VIP). Skin fragments (from plastic surgery) were maintained in survival conditions for 6 h. To induce inflammation, VIP was placed in contact with dermis by culture medium. Histological analysis was then performed on hematoxylin- and eosin-stained slides. Edema was evaluated with semiquantitative scores. Vasodilation was studied by quantifying the percentage of dilated vessels according to scores and by measuring their surface by morphometrical image analysis. TNF-alpha dosage was made on culture supernatants. Vasodilation was significantly increased after application of VIP. After treatment with oatmeal extract oligomer, the mean surface of dilated vessels and edema were significantly decreased compared with VIP-treated skin. Moreover, treatment with this extract decreased TNF-alpha.

  14. RasGAP-derived peptide GAP159 enhances cisplatin-induced cytotoxicity and apoptosis in HCT116 cells

    PubMed Central

    Zhang, Hao; Zhang, Shenghua; He, Hongwei; Zhang, Caixia; Chen, Yi; Yu, Dongke; Chen, Jianhua; Shao, Rongguang

    2014-01-01

    To increase the efficacy of currently used anti-cancer genotoxins, one of the current efforts is to find agents that can sensitize cancer cells to genotoxins so that the efficacious doses of genotoxins can be lowered to reduce deleterious side-effects. In this study, we reported that a synthetic RasGAP-derived peptide GAP159 could enhance the effect of chemotherapeutic agent cisplatin (CDDP) in human colon carcinoma HCT116 cells. Our results showed that GAP159 significantly increased the CDDP-induced cytotoxicity and apoptosis in HCT116 cells. This synergistic effect was associated with the inhibitions of phospho-AKT, phospho-ERK and NF-κB. In mouse colon tumor CT26 animal models, GAP159 combined with CDDP significantly suppressed CT26 tumor growth, and GAP159 alone showed slight inhibitory effect. Our data suggests that co-treatment of GAP159 and chemotherapeutics will become a potential therapeutic strategy for colon cancers. PMID:26579374

  15. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

    PubMed

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-01-01

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies. PMID:26561805

  16. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

    PubMed

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-11-06

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

  17. Fragmentation of singly, doubly, and triply charged hydrogen deficient peptide radical cations in infrared multiphoton dissociation and electron induced dissociation.

    PubMed

    Kalli, Anastasia; Hess, Sonja

    2012-02-01

    Gas phase fragmentation of hydrogen deficient peptide radical cations continues to be an active area of research. While collision induced dissociation (CID) of singly charged species is widely examined, dissociation channels of singly and multiply charged radical cations in infrared multiphoton dissociation (IRMPD) and electron induced dissociation (EID) have not been, so far, investigated. Here, we report on the gas phase dissociation of singly, doubly and triply charged hydrogen deficient peptide radicals, [M + nH]((n+1)+·) (n=0, 1, 2), in MS(3) IRMPD and EID and compare the observed fragmentation pathways to those obtained in MS(3) CID. Backbone fragmentation in MS(3) IRMPD and EID was highly dependent on the charge state of the radical precursor ions, whereas amino acid side chain cleavages were largely independent of the charge state selected for fragmentation. Cleavages at aromatic amino acids, either through side chain loss or backbone fragmentation, were significantly enhanced over other dissociation channels. For singly charged species, the MS(3) IRMPD and EID spectra were mainly governed by radical-driven dissociation. Fragmentation of doubly and triply charged radical cations proceeded through both radical- and charge-driven processes, resulting in the formation of a wide range of backbone product ions including, a-, b-, c-, y-, x-, and z-type. While similarities existed between MS(3) CID, IRMPD, and EID of the same species, several backbone product ions and side chain losses were unique for each activation method. Furthermore, dominant dissociation pathways in each spectrum were dependent on ion activation method, amino acid composition, and charge state selected for fragmentation.

  18. 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. PMID:25616869

  19. A naturally occurring α(s1)-casein-derived peptide in bovine milk inhibits apoptosis of granulosa cells induced by serum-free conditions.

    PubMed

    Shimizu, T; Ganzorig, K; Miyamoto, A; Ishii, T; Urashima, T; Fukuda, K

    2014-03-01

    Several naturally occurring peptides in bovine milk were characterized by tandem mass spectrometry and Edman degradation. Chromatograms of peptide fractions (passed through an ultra-filtration membrane, nominal molecular weight limit 3000) prepared from colostrum (collected immediately after parturition) and transitional milk (collected 5 days postpartum) showed that they were almost identical. In total, six peptides, α(s1)-CN (f16-23) (RPKHPIKH), α(s1)-CN (f16-24) (RPKHPIKHQ), α(s1)-CN (f17-25) (PKHPIKHQG), α(s1)-CN (f46-52) (VFGKEKV), α(s1)-CN (f94-105) (HIQKEDVPSER), and β-CN (f121-128) (HKEMPFPK), were identified. One of the major peptides, the N-terminal fragment of αs1 -casein, varied structurally during early lactation: α(s1)-CN (f17-25) (PKHPIKHQG) and α(s1)-CN (f16-23) (RPKHPIKH)/α(s1)-CN (f16-24) (RPKHPIKHQ) were found in colostrum and transitional milk, respectively. A chemically synthesized peptide, α(s1)-CN (f16-23) (RPKHPIKH), inhibited apoptosis of bovine granulosa cells induced by serum-free conditions in a dose-dependent manner, in consequence of caspase-3 and caspase-9 suppressions. The physiological function of the peptide remains unclear, but it may have potential use as pharmaceutical agent and as an anti-apoptotic agent in cell culture medium. PMID:24399684

  20. Intra-accumbal administration of shRNAs against CART peptides cause increases in body weight and cocaine-induced locomotor activity in rats.

    PubMed

    Job, M O; Licata, J; Hubert, G W; Kuhar, M J

    2012-10-30

    In order to examine the effect of cocaine and amphetamine regulated transcript (CART) peptide depletion in adult rats, CART shRNAs or scrambled control shRNAs were administered bilaterally into the nucleus accumbens (NAc). There was an increase in body weight of the shRNA injected rats compared with the rats injected with the scrambled RNA. This is compatible with the data showing a role for the peptide in body weight and food intake. Also at this time, there was about a two-and-a-half fold increase in cocaine-mediated locomotion in the shRNA injected rats compared to the control rats. This finding is critical support for the hypothesis that endogenous CART peptides in the NAc inhibit the actions of cocaine and other psychostimulants. In immunohistochemical experiments on these same animals, there was a decrease in the staining density of CART peptide in the NAc of the shRNA injected rats. These data show that shRNA can reduce CART peptides in the NAc and that endogenous CART peptides influence body weight and cocaine-induced locomotor activity (LMA).

  1. Influence of ETR-p1/f1 antisense peptide on endothelin-induced constriction in rat renal arcuate arteries

    PubMed Central

    Wu, Xiaochun; Richards, Nicholas T; Johns, Edward J; Kohsaka, Takeo; Nakamura, Akio; Okada, Hidechika

    1997-01-01

    This study set out to examine the endothelin receptor subtypes mediating vasoconstriction in the rat renal arcuate artery. This was done in isolated vessels 120–200 μm in diameter, incubated with a selective agonist and the novel ‘antisense' peptide to part of the human endothelinA receptor. Groups of vessels (n=6) were incubated with increasing concentrations of endothelin-1 (ET-1), from 1 to 100 nM, which caused a 65% maximal contraction at the highest dose with an pEC50 of 8.16±0.11 M. By contrast, in six other vessels sarafotoxin 6c over the same dose range gave a minimal contraction (around 5% of maximum). Preincubation of six vessels with the antisense peptide ETR p1/f1 at 1 μM had no effect on the ET-1 induced vasoconstriction, in terms of displacement of the concentration-response curve or the maximal tension achieved by the agonist. In the six vessels exposed to 4 μM ETR p1/f1, there was a significant shift of the concentration-response curve and a lower pEC50 at 7.78±0.09 M (P<0.05). At the highest concentrations of ETR p1/f1, there was a marked suppression of all responses to ET-1, which at the maximal concentrations tested, 0.1 μM, only reached some 10% of the maximal achievable contraction. Increasing ET-1 concentrations up to 2 μM in vessels incubated with 40 μM ETR-p1/f1 showed that the blockade could be overcome and that the relationship was shifted to the right (P<0.001) by approximately one log unit with a pEC50 of 7.13±0.11 M. A Schild plot of the data indicated the antagonist to be acting competitively at a single population of receptors. At the highest concentrations tested, 40 μM, ETR-p1/f1 had no effect on noradrenaline-induced contractions, indicating a lack of non-specific actions. Together, these data suggest that at the rat renal arcuate artery the endothelinA receptor is the predominant functional receptor mediating contraction. Furthermore, this study has shown the potential usefulness of this novel

  2. Antimicrobial peptides.

    PubMed

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  3. Amyloid β Peptide Enhances RANKL-Induced Osteoclast Activation through NF-κB, ERK, and Calcium Oscillation Signaling

    PubMed Central

    Li, Shangfu; Yang, Bu; Teguh, Dian; Zhou, Lin; Xu, Jiake; Rong, Limin

    2016-01-01

    Osteoporosis and Alzheimer’s disease (AD) are common chronic degenerative disorders which are strongly associated with advanced age. We have previously demonstrated that amyloid beta peptide (Aβ), one of the pathological hallmarks of AD, accumulated abnormally in osteoporotic bone specimens in addition to having an activation effect on osteoclast (Bone 2014,61:164-75). However, the underlying molecular mechanisms remain unclear. Activation of NF-κB, extracellular signal-regulated kinase (ERK) phosphorylates, and calcium oscillation signaling pathways by receptor activator NF-κB ligand (RANKL) plays a pivotal role in osteoclast activation. Targeting this signaling to modulate osteoclast function has been a promising strategy for osteoclast-related diseases. In this study, we investigated the effects of Aβ on RANKL-induced osteoclast signaling pathways in vitro. In mouse bone marrow monocytes (BMMs), Aβ exerted no effect on RANKL-induced osteoclastogenesis but promoted osteoclastic bone resorption. In molecular levels, Aβ enhanced NF-κB activity and IκB-α degradation, activated ERK phosphorylation and stimulated calcium oscillation, thus leading to upregulation of NFAT-c1 expression during osteoclast activation. Taken together, our data demonstrate that Aβ enhances RANKL-induced osteoclast activation through IκB-α degradation, ERK phosphorylation, and calcium oscillation signaling pathways and that Aβ may be a promising agent in the treatment of osteoclast-related disease such as osteoporosis. PMID:27735865

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

    PubMed

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

    2016-06-01

    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

  5. Protective Effect of Cod (Gadus macrocephalus) Skin Collagen Peptides on Acetic Acid-Induced Gastric Ulcer in Rats.

    PubMed

    Niu, Huina; Wang, Zhicong; Hou, Hu; Zhang, Zhaohui; Li, Bafang

    2016-07-01

    This research was performed to explore the protective effect of cod skin collagen peptides (CCP) on gastric ulcer induced by acetic acid. The CCP were fractionated into low molecular CCP (LMCCP, Mw < 3 kDa) and high molecular CCP (HMCCP, Mw > 3 kDa). In HMCCP and LMCCP, glycine of accounted for about one-third of the total amino acids without cysteine and tryptophan, and hydrophobic amino acids accounted for about 50%. After 21 d CCP treatment (60 or 300 mg/kg, p.o./daily), the healing effects on acetic acid-induced gastric ulcers were evaluated by macroscopic measure, microscopic measure, and immune histochemistry. Moreover, the expression levels of the growth factors, such as vascular endothelial growth factor, epidermal growth factor, transforming growth factor β1 (TGFβ1), and the heat shock protein 70 (HSP70) was detected. The results showed that both LMCCP and HMCCP could significantly decrease the ulcer areas and promote the healing of the lesions. They also could improve the levels of hexosamine, glutathione, superoxide dismutase, and glutathione peroxidase, and reduce the content of malondialdehyde and inducible nitric oxide synthase. In addition, the expression level of TGFβ1 gene and HSP70 mRNA was significantly improved by the treatment. It suggested that CCP could be able to improve symptoms of gastric ulcer and probably be used in the treatment of gastric ulcer. PMID:27219644

  6. Role of Notch-1 signaling pathway in PC12 cell apoptosis induced by amyloid beta-peptide (25–35)

    PubMed Central

    Liang, Huimin; Zhang, Yaozhou; Shi, Xiaoyan; Wei, Tianxiang; Lou, Jiyu

    2014-01-01

    Recent studies have demonstrated that Notch-1 expression is increased in the hippocampus of Alzheimer's disease patients. We speculate that Notch-1 signaling may be involved in PC12 cell apoptosis induced by amyloid beta-peptide (25–35) (Aβ25–35). In the present study, PC12 cells were cultured with different doses (0, 0.1, 1.0, 10 and 100 nmol/L) of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a Notch-1 signaling pathway inhibitor, for 30 minutes. Then cultured cells were induced with Aβ25–35 for 48 hours. Pretreatment of PC12 cells with high doses of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (> 10 nmol/L) prolonged the survival of PC12 cells after Aβ25–35 induction, decreased the expression of apoptosis-related proteins caspase-3, -8, -9, increased the activity of oxidative stress-related superoxide dismutase and catalase, inhibited the production of active oxygen, and reduced nuclear factor kappa B expression. This study indicates that the Notch-1 signaling pathway plays a pivotal role in Aβ25–35-induced PC12 apoptosis. PMID:25221582

  7. Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells

    PubMed Central

    Chin, Joanna Y; Reza, Faisal; Glazer, Peter M

    2013-01-01

    Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes. PMID:23337982

  8. Metformin ameliorates lipotoxicity-induced mesangial cell apoptosis partly via upregulation of glucagon like peptide-1 receptor (GLP-1R).

    PubMed

    Kim, Dong-il; Park, Min-jung; Heo, Young-ran; Park, Soo-hyun

    2015-10-15

    Glucagon like peptide-1 receptor (GLP-1R), known to be expressed in pancreatic beta cells, is also expressed in glomerular mesangial cells and its agonist has protective effects in diabetic nephropathy. However, its regulatory mechanisms by lipotoxicity in glomerular mesangial cells are not understood. We found that palmitate-mediated lipotoxicity increased apoptosis and decreased GLP-1R expression in a rat mesangial cell line. Silencing GLP-1R expression also increased mesangial cell apoptosis. Interestingly, metformin, one of the biguanide drugs that has anti-diabetic effects, attenuated lipotoxicity-induced mesangial cell apoptosis and restored GLP-1R expression. Moreover, this treatment alleviated GLP-1R knockdown-induced mesangial cell apoptosis. To further evaluate in vivo, diabetic obese db/db mice were administered metformin. Glomerular GLP-1R expression was diminished in db/db mice, as compared with db/m control mice. However, this decrease significantly recovered on metformin administration. Together, these data provide novel evidence that lipotoxicity decreases the mesangial GLP-1R expression in intact cells and in vivo. The decrease induced mesangial cell apoptosis. Furthermore, we provided the evidence that metformin treatment has a renal protective effect partly via increased mesangial GLP-1R expression. Our data suggested that regulation of GLP-1R expression could be a promising approach to treat diabetic nephropathy and the novel mechanism of metformin mediated GLP-1R regulation.

  9. IL-2 induces T cell adherence to extracellular matrix: inhibition of adherence and migration by IL-2 peptides generated by leukocyte elastase.

    PubMed

    Ariel, A; Yavin, E J; Hershkoviz, R; Avron, A; Franitza, S; Hardan, I; Cahalon, L; Fridkin, M; Lider, O

    1998-09-01

    Migration of inflammatory cells requires cell adhesion and their subsequent detachment from the extracellular matrix (ECM). Leukocyte activation and migration must be terminated to stop inflammation. Here, we report that IL-2 enhances human T cell adherence to laminin, collagen type IV, and fibronectin (FN). In contrast, neutrophil elastase, an enzyme activated during inflammation, degrades IL-2 to yield IL-2 fractions that inhibit IL-2-induced T cell adhesion to FN. The amino acid composition of two of these IL-2 fractions, which appear to block T cell adherence to FN, were analyzed, and three peptides were consequently synthesized. The three peptides IVL, RMLT, and EFLNRWIT, but not the corresponding inversely synthesized peptides, inhibited T cell adhesion to FN induced by a variety of activators: IL-2, IL-7, macrophage inflammatory protein (MIP)-1beta, and PMA, as well as anti-CD3 and anti-beta1 integrin-activating mAb. Moreover, these IL-2 peptides inhibited T cell chemotaxis via FN-coated membranes induced by IL-2 and MIP-1beta. Inhibition of T cell adherence and migration apparently involves abrogation of the rearrangement of the T cell actin cytoskeleton. Thus, the migrating immune cells, the cytokines, and the ECM can create a functional relationship in which both inflammation-inducing signals and inhibitory molecules of immune responses can coexist; the enzymatic products of IL-2 may serve as natural feedback inhibitors of inflammation. PMID:9725245

  10. Induced Bacterial Cross-Resistance toward Host Antimicrobial Peptides: A Worrying Phenomenon.

    PubMed

    Fleitas, Osmel; Franco, Octávio L

    2016-01-01

    Bacterial resistance to conventional antibiotics has reached alarming levels, threatening to return to the pre-antibiotic era. Therefore, the search for new antimicrobial compounds that overcome the resistance phenomenon has become a priority. Antimicrobial peptides (AMPs) appear as one of the most promising antibiotic medicines. However, in recent years several AMP-resistance mechanisms have been described. Moreover, the AMP-resistance phenomenon has become more complex due to its association with cross-resistance toward AMP effectors of the host innate immune system. In this context, the use of AMPs as a therapeutic option could be potentially hazardous, since bacteria could develop resistance toward our innate immune system. Here, we review the findings of major studies that deal with the AMP cross-resistance phenomenon.

  11. ``Cold Denaturation'' induces inversion of dipole and spin transfer in chiral peptide monolayers

    NASA Astrophysics Data System (ADS)

    Sarkar, Soumyajit; Eckshtain-Levi, Meital; Capua, Eyal; Refaely-Abramson, Sivan; Gavrilov, Yulian; Mathew, Shinto; Paltiel, Yossi; Levy, Yaakov; Kronik, Leeor; Naaman, Ron

    Using a combination of several experimental and computational techniques, we show that the α-helix structure of oligopeptides based on alanine and aminoisobutyric acid is transformed to a more linear conformation upon cooling, due to interaction with neighboring molecules in a self-assembled monolayer (SAM) structure. This process is similar to the known ``cold denaturation'' in peptides, but here the SAM plays the role of the solvent. Our DFT-based first principles calculations show that the structural change results in a flip in the direction of the electrical dipole moment of the adsorbed molecules. The dipole flip is accompanied by an associated change in the spin channel that is preferred in electron transfer through the molecules. This is also experimentally observed via a new solid state hybrid organic-inorganic device that is based on the Hall effect, but operates with no external magnetic field or magnetic material.

  12. Peptide F (pro-enkephalin fragment): radioimmunoassay, and stress-induced changes in adrenal

    SciTech Connect

    Alessi, N.; Taylor, L.; Akil, H.

    1982-10-18

    Utilizing a nine amino-acid (Asp-Glu-Leu-Tyr-Pro-Leu-Glu-Val-Glu) non-enkephalin containing fragment of Peptide F from the pro-enkephalin molecule, a radioimmunoassay was developed. Extraction of bovine, rat, and guinea pig adrenomedullary preparations demonstrated this fragment to be present and apparently partially conserved across species. In rats, acute inescapable foot-shock stress led to a significant decrease of the immunoreactive material in the adrenal medulla. Chronic daily stress for two weeks resulted in an inability of the adrenals to alter F levels upon subsequent stress. The existence of F-like immunoreactivity and its alteration by environmental manipulation, suggest that it may play a unique physiological role.

  13. Induced Bacterial Cross-Resistance toward Host Antimicrobial Peptides: A Worrying Phenomenon

    PubMed Central

    Fleitas, Osmel; Franco, Octávio L.

    2016-01-01

    Bacterial resistance to conventional antibiotics has reached alarming levels, threatening to return to the pre-antibiotic era. Therefore, the search for new antimicrobial compounds that overcome the resistance phenomenon has become a priority. Antimicrobial peptides (AMPs) appear as one of the most promising antibiotic medicines. However, in recent years several AMP-resistance mechanisms have been described. Moreover, the AMP-resistance phenomenon has become more complex due to its association with cross-resistance toward AMP effectors of the host innate immune system. In this context, the use of AMPs as a therapeutic option could be potentially hazardous, since bacteria could develop resistance toward our innate immune system. Here, we review the findings of major studies that deal with the AMP cross-resistance phenomenon. PMID:27047486

  14. Effects of a polar amino acid substitution on helix formation and aggregate size along the detergent-induced peptide folding pathway.

    PubMed

    Alvares, Rohan D A; Tulumello, David V; Macdonald, Peter M; Deber, Charles M; Prosser, R Scott

    2013-02-01

    Membrane proteins constitute a significant fraction of the proteome and are important drug targets. While the transmembrane (TM) segments of these proteins are primarily composed of hydrophobic residues, the inclusion of polar residues-either naturally occurring or as a consequence of a disease-related mutation-places a significant folding burden in this environment, potentially impacting bilayer insertion and/or association of neighboring TM helices. Here we investigate the role of an anionic detergent, sodium dodecylsulfate (SDS), and a zwitterionic detergent, dodecylphosphocholine (DPC), in the folding process, and the effects induced by a single polar substitution, on structure and topology of model α-helical TM segments. The peptides, represented by KK-YAAAIAAIAWAXAAIAAAIAA-KKK-NH(2), where X is I or N, are designed with high aqueous solubilities, through poly-lysine tags. Circular dichroism (CD) and NMR were used to monitor peptide secondary structure and diffusional mobility of both peptide and the detergent hosts. For both peptides, SDS binding commenced at a concentration below its CMC, due to Coulombic attraction of anionic SDS to cationic Lys residues. Increasing SDS binding correlated with increasing peptide helicity. Pulsed field gradient (PFG) NMR diffusion measurements revealed that the Asn-containing peptide bound four fewer detergent molecules, corresponding to ca. 20% less SDS than bound by the Ile peptide. Conversely, zwitterionic DPC binding to either peptide was not observed until the DPC concentration approached its CMC. Our findings confirm quantitatively that a single polar residue within a TM segment may have a significant influence on its local membrane environment.

  15. A thermodynamic signature of lipid segregation in biomembranes induced by a short peptide derived from glycoprotein gp36 of feline immunodeficiency virus.

    PubMed

    Oliva, Rosario; Del Vecchio, Pompea; Stellato, Marco Ignazio; D'Ursi, Anna Maria; D'Errico, Gerardino; Paduano, Luigi; Petraccone, Luigi

    2015-02-01

    The interactions between proteins/peptides and lipid bilayers are fundamental in a variety of key biological processes, and among these, the membrane fusion process operated by viral glycoproteins is one of the most important, being a fundamental step of the infectious event. In the case of the feline immunodeficiency virus (FIV), a small region of the membrane proximal external region (MPER) of the glycoprotein gp36 has been demonstrated to be necessary for the infection to occur, being able to destabilize the membranes to be fused. In this study, we report a physicochemical characterization of the interaction process between an eight-residue peptide, named C8, modeled on that gp36 region and some biological membrane models (liposomes) by using calorimetric and spectroscopic measurements. CD studies have shown that the peptide conformation changes upon binding to the liposomes. Interestingly, the peptide folds from a disordered structure (in the absence of liposomes) to a more ordered structure with a low but significant helix content. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) results show that C8 binds with high affinity the lipid bilayers and induces a significant perturbation/reorganization of the lipid membrane structure. The type and the extent of such membrane reorganization depend on the membrane composition. These findings provide interesting insights into the role of this short peptide fragment in the mechanism of virus-cell fusion, demonstrating its ability to induce lipid segregation in biomembranes.

  16. Vascular Relaxation Induced by C-Type Natriuretic Peptide Involves the Ca2+/NO-Synthase/NO Pathway

    PubMed Central

    Andrade, Fernanda A.; Restini, Carolina B. A.; Grando, Marcella D.; Ramalho, Leandra N. Z.; Bendhack, Lusiane M.

    2014-01-01

    Aims C-type natriuretic peptide (CNP) and nitric oxide (NO) are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3) contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery. Main Methods Concentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser1177 of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry. Key Findings CNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO0 scavenger. CNP did not change the phosphorylation of Ser1177, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca2+]c in endothelial cells and a decrease in [Ca2+]c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas. Significance These results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca2+]c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP. PMID:24787693

  17. Identification of Novel Formyl Peptide Receptor-Like 1 Agonists that Induce Macrophage Tumor Necrosis Factor α Production

    PubMed Central

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

    2008-01-01

    Development of immunomodulatory agents that enhance innate immune responses represents a promising strategy for combating infectious diseases. In the present studies, we screened a series of 71 arylcarboxylic acid hydrazide derivatives for their ability to induce macrophage tumor necrosis factor α (TNF-α) production and identified 6 such compounds, including one compound previously shown to be a formyl peptide receptor (FPR/FPRL1) agonist. The two most potent compounds [Compound 1: nicotinic acid [5-(3-bromophenyl)-2-furyl]methylene-hydrazide; Compound 2: 4-fluoro-benzoic acid [5-(3-trifluoromethyl-phenyl)-2-furyl]methylene-hydrazide] were selected for further analysis. These compounds induced de novo production of TNF-α in a dose- and time-dependent manner in human and murine monocyte/macrophage cell lines and in primary macrophages. These compounds also induced mobilization of intracellular Ca2+, production of reactive oxygen species, and chemotaxis in human and murine phagocytes. Induction of macrophage TNF-α production was pertussis toxin-sensitive, and analysis of the cellular target of these compounds showed that they were FPRL1-specific agonists and that this response was blocked by FPR/FPRL1 and FPRL1-specific antagonists. Additionally, pharmacophore modeling showed a high degree of similarity for low-energy conformations of these two compounds to the current pharmacophore model for FPR ligands (Edwards et al., 2005). Overall, these compounds represent novel FPRL1 agonists that induce TNF-α, a response distinct from those induced by other known FPR and FPRL1 agonists. PMID:18458054

  18. Hinokitiol protects primary neuron cells against prion peptide-induced toxicity via autophagy flux regulated by hypoxia inducing factor-1.

    PubMed

    Moon, Ji-Hong; Lee, Ju-Hee; Lee, You-Jin; Park, Sang-Youel

    2016-05-24

    Prion diseases are fatal neurodegenerative disorders that are derived from structural changes of the native PrPc. Recent studies indicated that hinokitiol induced autophagy known to major function that keeps cells alive under stressful conditions. We investigated whether hinokitiol induces autophagy and attenuates PrP (106-126)-induced neurotoxicity. We observed increase of LC3-II protein level, GFP-LC3 puncta by hinokitiol in neuronal cells. Addition to, electron microscopy showed that hinokitiol enhanced autophagic vacuoles in neuronal cells. We demonstrated that hinokitiol protects against PrP (106-126)-induced neurotoxicity via autophagy by using autophagy inhibitor, wortmannin and 3MA, and ATG5 small interfering RNA (siRNA). We checked hinokitiol activated the hypoxia-inducible factor-1α (HIF-1α) and identified that hinokitiol-induced HIF-1α regulated autophagy. Taken together, this study is the first report demonstrating that hinokitiol protected against prion protein-induced neurotoxicity via autophagy regulated by HIF-1α. We suggest that hinokitiol is a possible therapeutic strategy in neuronal disorders including prion disease.

  19. Leader peptides of inducible chloramphenicol resistance genes from gram-positive and gram-negative bacteria bind to yeast and Archaea large subunit rRNA.

    PubMed Central

    Harrod, R; Lovett, P S

    1997-01-01

    catA86 is the second gene in a constitutively transcribed, two-gene operon cloned from Bacillus pumilus . The region that intervenes between the upstream gene, termed the leader, and the catA86 coding sequence contains a pair of inverted repeat sequences which cause sequestration of the catA86 ribosome binding site in mRNA secondary structure. As a consequence, the catA86 coding sequence is untranslatable in the absence of inducer. Translation of the catA86 coding sequence is induced by chloramphenicol in Gram-positives and induction requires a function of the leader coding sequence. The leader-encoded peptide has been proposed to instruct its translating ribosome to pause at leader codon 6, enabling chloramphenicol to stall the ribosome at that site. Ribosome stalling causes destabilization of the RNA secondary structure, exposing the catA86 ribosome binding site, allowing activation of its translation. A comparable mechanism of induction by chloramphenicol has been proposed for the regulated cmlA gene from Gram-negative bacteria. The catA86 and cmlA leader-encoded peptides are in vitro inhibitors of peptidyl transferase, which is thought to be the basis for selection of the site of ribosome stalling. Both leader-encoded peptides have been shown to alter the secondary structure of Escherichia coli 23S rRNA in vitro. All peptide-induced changes in rRNA conformation are within domains IV and V, which contains the peptidyl transferase center. Here we demonstrate that the leader peptides alter the conformation of domains IV and V of large subunit rRNA from yeast and a representative of the Archaea. The rRNA target for binding the leader peptides is therefore conserved across kingdoms. PMID:9108153

  20. Adaptive Evolution of Escherichia coli to an α-Peptide/β-Peptoid Peptidomimetic Induces Stable Resistance

    PubMed Central

    Hein-Kristensen, Line; Franzyk, Henrik; Holch, Anne; Gram, Lone

    2013-01-01

    Antimicrobial peptides (AMPs) and synthetic analogues thereof target conserved structures of bacterial cell envelopes and hence, development of resistance has been considered an unlikely event. However, recently bacterial resistance to AMPs has been observed, and the aim of the present study was to determine whether bacterial resistance may also evolve against synthetic AMP analogues, e.g. α-peptide/β-peptoid peptidomimetics. E. coli ATCC 25922 was exposed to increasing concentrations of a peptidomimetic (10 lineages), polymyxin B (10 lineages), or MilliQ water (4 lineages) in a re-inoculation culturing setup covering approx. 500 generations. All 10 lineages exposed to the peptidomimetic adapted to 32×MIC while this occurred for 8 out of 10 of the polymyxin B-exposed lineages. All lineages exposed to 32×MIC of either the peptidomimetic or polymyxin B had a significantly increased MIC (16–32×) to the selection agent. Five transfers (∼35 generations) in unsupplemented media did not abolish resistance indicating that resistance was heritable. Single isolates from peptidomimetic-exposed lineage populations displayed MICs against the peptidomimetic from wild-type MIC to 32×MIC revealing heterogeneous populations. Resistant isolates showed no cross-resistance against a panel of membrane-active AMPs. These isolates were highly susceptible to blood plasma antibacterial activity and were killed when plasma concentrations exceeded ∼30%. Notably, MIC of the peptidomimetic against resistant isolates returned to wild-type level upon addition of 25% plasma. Whole-genome sequencing of twenty isolates from four resistant lineages revealed mutations, in murein transglycosylase D (mltD) and outer-membrane proteins, which were conserved within and between lineages. However, no common resistance-conferring mutation was identified. We hypothesise that alterations in cell envelope structure result in peptidomimetic resistance, and that this may occur via several distinct

  1. Onset and Regression of Pregnancy-Induced Cardiac Alterations in Gestationally Hypertensive Mice: The Role of the Natriuretic Peptide System.

    PubMed

    Ventura, Nicole M; Li, Terry Y; Tse, M Yat; Andrew, R David; Tayade, Chandrakant; Jin, Albert Y; Pang, Stephen C

    2015-12-01

    Pregnancy induces cardiovascular adaptations in response to increased volume overload. Aside from the hemodynamic changes that occur during pregnancy, the maternal heart also undergoes structural changes. However, cardiac modulation in pregnancies complicated by gestational hypertension is incompletely understood. The objectives of the current investigation were to determine the role of the natriuretic peptide (NP) system in pregnancy and to assess alterations in pregnancy-induced cardiac hypertrophy between gestationally hypertensive and normotensive dams. Previously we have shown that mice lacking the expression of atrial NP (ANP; ANP(-/-)) exhibit a gestational hypertensive phenotype. In the current study, female ANP(+/+) and ANP(-/-) mice were mated with ANP(+/+) males. Changes in cardiac size and weight were evaluated across pregnancy at Gestational Days 15.5 and 17.5 and Postnatal Days 7, 14, and 28. Nonpregnant mice were used as controls. Physical measurement recordings and histological analyses demonstrated peak cardiac hypertrophy occurring at 14 days postpartum in both ANP(+/+) and ANP(-/-) dams with little to no change during pregnancy. Additionally, left ventricular expression of the renin-angiotensin system (RAS) and NP system was quantified by real-time quantitative polymerase chain reaction. Up-regulation of Agt and AT(1a) genes was observed late in pregnancy, while Nppa and Nppb genes were significantly up-regulated postpartum. Our data suggest that pregnancy-induced cardiac hypertrophy may be influenced by the RAS throughout gestation and by the NP system postpartum. Further investigations are required to gain a complete understanding of the mechanistic aspects of pregnancy-induced cardiac hypertrophy.

  2. Altered vasoactive intestinal peptides expression in irritable bowel syndrome patients and rats with trinitrobenzene sulfonic acid-induced colitis

    PubMed Central

    Del Valle-Pinero, Arseima Y; Sherwin, LeeAnne B; Anderson, Ethan M; Caudle, Robert M; Henderson, Wendy A

    2015-01-01

    AIM: To investigate the vasoactive intestinal peptides (VIP) expression in irritable bowel syndrome (IBS) and trinitrobenzene sulfonic acid (TNBS) induced colitis. METHODS: The VIP gene expression and protein plasma levels were measured in adult participants (45.8% male) who met Rome III criteria for IBS for longer than 6 mo and in a rat model of colitis as induced by TNBS. Plasma and colons were collected from naïve and inflamed rats. Markers assessing inflammation (i.e., weight changes and myeloperoxidase levels) were assessed on days 2, 7, 14 and 28 and compared to controls. Visceral hypersensitivity of the rats was assessed with colo-rectal distension and mechanical threshold testing on hind paws. IBS patients (n = 12) were age, gender, race, and BMI-matched with healthy controls (n = 12). Peripheral whole blood and plasma from fasting participants was collected and VIP plasma levels were assayed using a VIP peptide-enzyme immunoassay. Human gene expression of VIP was analyzed using a custom PCR array. RESULTS: TNBS induced colitis in the rats was confirmed with weight loss (13.7 ± 3.2 g) and increased myeloperoxidase activity. Visceral hypersensitivity to colo-rectal distension was increased in TNBS treated rats up to 21 d and resolved by day 28. Somatic hypersensitivity was also increased up to 14 d post TNBS induction of colitis. The expression of an inflammatory marker myeloperoxidase was significantly elevated in the intracellular granules of neutrophils in rat models following TNBS treatment compared to naïve rats. This confirmed the induction of inflammation in rats following TNBS treatment. VIP plasma concentration was significantly increased in rats following TNBS treatment as compared to naïve animals (P < 0.05). Likewise, the VIP gene expression from peripheral whole blood was significantly upregulated by 2.91-fold in IBS patients when compared to controls (P < 0.00001; 95%CI). VIP plasma protein was not significantly different when compared with

  3. Short-days induce weight loss in Siberian hamsters despite overexpression of the agouti-related peptide gene.

    PubMed

    Jethwa, P H; Warner, A; Fowler, M J; Murphy, M; de Backer, M W; Adan, R A H; Barrett, P; Brameld, J M; Ebling, F J P

    2010-06-01

    Many vertebrates express profound annual cycles of body fattening, although it is not clear whether these represent differential activity of the central pathways known to mediate homeostatic control of food intake and energy expenditure, or whether the recent discovery of a major role for pars tuberalis-ependymal signalling points towards novel mechanisms. We examined this in the Siberian hamster (Phodopus sungorus) by using gene transfection to up-regulate a major orexigenic peptide, agouti-related peptide (AgRP), and then determined whether this increased anabolic drive could prevent the short-day induced winter catabolic state. Infusions of a recombinant adeno-associated virus encoding an AgRP construct into the hypothalamus of hamsters in the long-day obese phase of their seasonal cycle produced a 20% gain in body weight over 6 weeks compared to hamsters receiving a control reporter construct, reflecting a significant increase in food intake and a significant decrease in energy expenditure. However, all hamsters showed a significant, prolonged decrease in body weight when exposed to short photoperiods, despite the hamsters expressing the AgRP construct maintaining a higher food intake and lower energy expenditure relative to the control hamsters. Visualisation of the green fluorescent protein reporter and analysis of AgRP-immunoreactivity confirmed widespread expression of the construct in the hypothalamus, which was maintained for the 21-week duration of the study. In conclusion, the over-expression of AgRP in the hypothalamus produced a profoundly obese state but did not block the seasonal catabolic response, suggesting a separation of rheostatic mechanisms in seasonality from those maintaining homeostasis of energy metabolism.

  4. Methylglyoxal produced by amyloid-β peptide-induced nitrotyrosination of triosephosphate isomerase triggers neuronal death in Alzheimer's disease.

    PubMed

    Tajes, Marta; Eraso-Pichot, Abel; Rubio-Moscardó, Fanny; Guivernau, Biuse; Ramos-Fernández, Eva; Bosch-Morató, Mònica; Guix, Francesc Xavier; Clarimón, Jordi; Miscione, Gian Pietro; Boada, Mercé; Gil-Gómez, Gabriel; Suzuki, Toshiharu; Molina, Henrik; Villà-Freixa, Jordi; Vicente, Rubén; Muñoz, Francisco J

    2014-01-01

    Amyloid-β peptide (Aβ) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimer's disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aβ42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aβ42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aβ action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aβ oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center.

  5. Apoptosis induced by Aβ25-35 peptide is Ca(2+) -IP3 signaling-dependent in murine astrocytes.

    PubMed

    Oseki, K T; Monteforte, P T; Pereira, G J S; Hirata, H; Ureshino, R P; Bincoletto, C; Hsu, Y-T; Smaili, S S

    2014-08-01

    Although the accumulation of the neurotoxic peptide β-amyloid (Aβ) in the central nervous system is a hallmark of Alzheimer's disease, whether Aβ acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca(2+) dysregulation, induced by a neurotoxic fragment (Aβ25-35), caused apoptosis in a concentration-dependent manner, leading to cytoplasmic Ca(2+) mobilization from extra- and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP3 receptor activation. This mechanism was related to Aβ-mediated apoptosis by the intrinsic pathway because the expression of pro-apoptotic Bax was accompanied by its translocation in cells transfected with GFP-Bax. Aβ-mediated apoptosis was reduced by BAPTA-AM, a fast Ca(2+) chelator, indicating that an increase in intracellular Ca(2+) was involved in cell death. Interestingly, the Bax translocation was dependent on Ca(2+) mobilization from IP3 receptors because pre-incubation with xestospongin C, a selective IP3 receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aβ dysregulation of Ca(2+) homeostasis induces ER depletion of Ca(2+) stores and leads to apoptosis; this mechanism plays a significant role in Aβ apoptotic cell death and might be a new target for neurodegeneration treatments.

  6. A peptide antagonist of CD28 signaling attenuates toxic shock and necrotizing soft-tissue infection induced by Streptococcus pyogenes.

    PubMed

    Ramachandran, Girish; Tulapurkar, Mohan E; Harris, Kristina M; Arad, Gila; Shirvan, Anat; Shemesh, Ronen; Detolla, Louis J; Benazzi, Cinzia; Opal, Steven M; Kaempfer, Raymond; Cross, Alan S

    2013-06-15

    Staphylococcus aureus and group A Streptococcus pyogenes (GAS) express superantigen (SAg) exotoxin proteins capable of inducing lethal shock. To induce toxicity, SAgs must bind not only to the major histocompatibility complex II molecule of antigen-presenting cells and the variable β chain of the T-cell receptor but also to the dimer interface of the T-cell costimulatory receptor CD28. Here, we show that the CD28-mimetic peptide AB103 (originally designated "p2TA") protects mice from lethal challenge with streptococcal exotoxin A, as well as from lethal GAS bacterial infection in a murine model of necrotizing soft-tissue infection. Administration of a single dose of AB103 increased survival when given up to 5 hours after infection, reduced inflammatory cytokine expression and bacterial burden at the site of infection, and improved muscle inflammation in a dose-dependent manner, without compromising cellular and humoral immunity. Thus, AB103 merits further investigation as a potential therapeutic in SAg-mediated necrotizing soft-tissue infection.

  7. Suppression of ischaemia-induced injuries in rat brain by protease-activated receptor-1 (PAR-1) activating peptide.

    PubMed

    Zhen, Xia; Ng, Ethel Sau Kuen; Lam, Francis Fu Yuen

    2016-09-01

    Ischaemic stroke has become one of the leading causes of death and disability worldwide. The role of protease activated receptor-1 (PAR-1) in this disease is uncertain. In the present study, the actions of a protease activated receptor-1 activating peptide (PAR-1 AP) SFLLRN-NH2 were investigated in an in vivo rat model of ischaemic stroke induced by middle cerebral artery occlusion (MCAO) and in an in vitro model induced by oxygen and glucose deprivation (OGD) in primary cultured rat embryonic cortical neurones. Rats subjected to MCAO exhibited increased brain infarct volume, oedema, and neurological deficit. Rat cortical neurones subjected to OGD showed increased lactate dehydrogenase, caspase-3 activity and TUNEL positive cells, whereas, mitochondrial membrane potential and cell viability were decreased. Furthermore, both models had elevated levels of reactive oxygen species, nitrite, and malondialdehyde, while anti-oxidant enzymes and bcl-2/bax ratio were decreased. These detrimental changes were suppressed by SFLLRN-NH2, and its protective actions were inhibited by a PAR-1 antagonist (BMS-200261). In summary, SFLLRN-NH2 was found to possess anti-oxidant and anti-apoptotic properties, and it produced marked inhibition on the detrimental effects of ischaemia in in vivo and in vitro models of ischaemic stroke. The present findings suggest PAR-1 is a promising target for development of novel treatments of ischaemic brain disease. PMID:27238976

  8. Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15

    PubMed Central

    Yang, Zhilin; Weisshaar, James C.

    2015-01-01

    Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1–7 of cecropin A (from moth) with residues 2–9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O2−, H2O2, and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency. PMID:25561551

  9. Methylglyoxal produced by amyloid-β peptide-induced nitrotyrosination of triosephosphate isomerase triggers neuronal death in Alzheimer's disease.

    PubMed

    Tajes, Marta; Eraso-Pichot, Abel; Rubio-Moscardó, Fanny; Guivernau, Biuse; Ramos-Fernández, Eva; Bosch-Morató, Mònica; Guix, Francesc Xavier; Clarimón, Jordi; Miscione, Gian Pietro; Boada, Mercé; Gil-Gómez, Gabriel; Suzuki, Toshiharu; Molina, Henrik; Villà-Freixa, Jordi; Vicente, Rubén; Muñoz, Francisco J

    2014-01-01

    Amyloid-β peptide (Aβ) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimer's disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aβ42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aβ42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aβ action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aβ oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center. PMID:24614897

  10. Changes in the Mechanical Properties of Pseudomonas aeruginosa Bacterial Cells Induced by Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Dutcher, John

    2011-03-01

    In our research group, we have developed an atomic force microscopy nano-creep technique to study the mechanical properties of individual Pseudomonas aeruginosa bacterial cells in a liquid environment. In the present study, we have used this technique to measure changes to the mechanical properties of the cells produced by exposing the cells to well-studied antimicrobial peptides: polymyxin B (PMB) and its derivative polymyxin B nonapeptide (PMBN). We find that the creep response of cells under a fixed applied load is very different after exposure of the cells to PMB and PMBN, which is possibly due to the disruption of its outer membrane. To describe the viscoelastic properties of the cells exposed to PMB and PMBN, we found that it was necessary to use a four element spring and dashpot model, instead of the three element standard linear solid model that describes the viscoelastic properties of cells in Millipore water. We also found that PMB and PMBN have qualitatively different effects on the stiffness of the cell membrane. These measurements provide a first step towards understanding the different mechanisms of action of PMB and PMBN on bacterial cells.

  11. Electrospun Scaffolds for Osteoblast Cells: Peptide-Induced Concentration-Dependent Improvements of Polycaprolactone

    PubMed Central

    Dettin, Monica; Zamuner, Annj; Roso, Martina; Gloria, Antonio; Iucci, Giovanna; Messina, Grazia M. L.; D'Amora, Ugo; Marletta, Giovanni; Modesti, Michele; Castagliuolo, Ignazio; Brun, Paola

    2015-01-01

    The design of hybrid poly-ε-caprolactone (PCL)-self-assembling peptides (SAPs) matrices represents a simple method for the surface functionalization of synthetic scaffolds, which is essential for cell compatibility. This study investigates the influence of increasing concentrations (2.5%, 5%, 10% and 15% w/w SAP compared to PCL) of three different SAPs on the physico-chemical/mechanical and biological properties of PCL fibers. We demonstrated that physico-chemical surface characteristics were slightly improved at increasing SAP concentrations: the fiber diameter increased; surface wettability increased with the first SAP addition (2.5%) and slightly less for the following ones; SAP-surface density increased but no change in the conformation was registered. These results could allow engineering matrices with structural characteristics and desired wettability according to the needs and the cell system used. The biological and mechanical characteristics of these scaffolds showed a particular trend at increasing SAP concentrations suggesting a prevailing correlation between cell behavior and mechanical features of the matrices. As compared with bare PCL, SAP enrichment increased the number of metabolic active h-osteoblast cells, fostered the expression of specific osteoblast-related mRNA transcripts, and guided calcium deposition, revealing the potential application of PCL-SAP scaffolds for the maintenance of osteoblast phenotype. PMID:26361004

  12. Electrospun Scaffolds for Osteoblast Cells: Peptide-Induced Concentration-Dependent Improvements of Polycaprolactone.

    PubMed

    Dettin, Monica; Zamuner, Annj; Roso, Martina; Gloria, Antonio; Iucci, Giovanna; Messina, Grazia M L; D'Amora, Ugo; Marletta, Giovanni; Modesti, Michele; Castagliuolo, Ignazio; Brun, Paola

    2015-01-01

    The design of hybrid poly-ε-caprolactone (PCL)-self-assembling peptides (SAPs) matrices represents a simple method for the surface functionalization of synthetic scaffolds, which is essential for cell compatibility. This study investigates the influence of increasing concentrations (2.5%, 5%, 10% and 15% w/w SAP compared to PCL) of three different SAPs on the physico-chemical/mechanical and biological properties of PCL fibers. We demonstrated that physico-chemical surface characteristics were slightly improved at increasing SAP concentrations: the fiber diameter increased; surface wettability increased with the first SAP addition (2.5%) and slightly less for the following ones; SAP-surface density increased but no change in the conformation was registered. These results could allow engineering matrices with structural characteristics and desired wettability according to the needs and the cell system used. The biological and mechanical characteristics of these scaffolds showed a particular trend at increasing SAP concentrations suggesting a prevailing correlation between cell behavior and mechanical features of the matrices. As compared with bare PCL, SAP enrichment increased the number of metabolic active h-osteoblast cells, fostered the expression of specific osteoblast-related mRNA transcripts, and guided calcium deposition, revealing the potential application of PCL-SAP scaffolds for the maintenance of osteoblast phenotype. PMID:26361004

  13. Cytosolic amyloid-{beta} peptide 42 escaping from degradation induces cell death

    SciTech Connect

    Lee, Eun Kyung; Park, Yong Wook; Shin, Dong Yeon; Mook-Jung, Inhee; Yoo, Yung Joon . E-mail: yjyoo@gist.ac.kr

    2006-06-02

    Accumulating evidence suggests that intracellular amyloid-{beta} (A{beta}) peptide triggers the early pathological events in Alzheimer's disease (AD). However, little is known about the consequence of cytosolic A{beta}. In this study, we ectopically expressed A{beta}42 in the cytoplasm of SH-SY5Y neuroblastoma cells by expressing a fusion protein of GFP-tagged ubiquitin and A{beta}42 (GFPUb-A{beta}42). Although GFPUb and A{beta}42 are stochastically produced with the same molar ratio in the cytoplasm, A{beta}42 was completely degraded in more than 50% of the GFPUb-expressing cells. However, if A{beta}42 was not degraded in their cytoplasm, then A{beta}42-expressing cells underwent apoptosis. The number of A{beta}42-expressing cells is significantly increased by the inhibition of proteasome with MG132. Cytosolic A{beta}42 which has escaped degradation inhibits proteasome and thereby may accelerate the accumulation of A{beta}42 and its detrimental effects. Our findings suggest that cells have the potential to degrade A{beta}42 in their cytoplasm but if A{beta}42 appears in the cytoplasm due to its incomplete degradation, it accumulates and may trigger the fatal cascade of pathology of AD.

  14. Immunization with antigenic peptides complexed with β-glucan induces potent cytotoxic T-lymphocyte activity in combination with CpG-ODNs.

    PubMed

    Mochizuki, Shinichi; Morishita, Hiromi; Kobiyama, Kouji; Aoshi, Taiki; Ishii, Ken J; Sakurai, Kazuo

    2015-12-28

    The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that β-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases. PMID:26562685

  15. AC-1001 H3 CDR peptide induces apoptosis and signs of autophagy in vitro and exhibits antimetastatic activity in a syngeneic melanoma model.

    PubMed

    Rabaça, Aline N; Arruda, Denise C; Figueiredo, Carlos R; Massaoka, Mariana H; Farias, Camyla F; Tada, Dayane B; Maia, Vera C; Silva Junior, Pedro I; Girola, Natalia; Real, Fernando; Mortara, Renato A; Polonelli, Luciano; Travassos, Luiz R

    2016-09-01

    Antibody-derived peptides modulate functions of the immune system and are a source of anti-infective and antitumor substances. Recent studies have shown that they comprise amino acid sequences of immunoglobulin complementarity-determining regions, but also fragments of constant regions. VH CDR3 of murine mAb AC-1001 displays antimetastatic activities using B16F10-Nex2 murine melanoma cells in a syngeneic model. The peptide was cytotoxic in vitro in murine and human melanoma cells inducing reactive oxygen species (ROS) and apoptosis by the intrinsic pathway. Signs of autophagy were also suggested by the increased expression of LC3/LC3II and Beclin 1 and by ultrastructural evidence. AC-1001 H3 bound to both G- and F-actin and inhibited tumor cell migration. These results are important evidence of the antitumor activity of Ig CDR-derived peptides. PMID:27642552

  16. Regulatory function of the P295-T311 motif of the estrogen receptor α - does proteasomal degradation of the receptor induce emergence of peptides implicated in estrogenic responses?

    PubMed Central

    Gallo, Dominique; Haddad, Iman; Laurent, Guy; Vinh, Joëlle; Jacquemotte, Françoise; Jacquot, Yves; Leclercq, Guy

    2008-01-01

    The way in which estrogen receptor α (ERα) mediates gene transcription and hormone-dependent cancer cell proliferation is now being largely reconsidered in view of several recent discoveries. ERα-mediated transcription appears to be a cyclic and transient process where the proteasome - and thus receptor degradation - plays a pivotal role. In view of our recent investigations, which demonstrate the estrogenic activity of a synthetic peptide corresponding to a regulatory motif of the receptor (ERα17p), we propose that ERα proteasomal degradation could induce the emergence of regulatory peptide(s). The latter would function as a signal and contribute to the ERα activation process, amplifying the initial hormonal stimulation and giving rise to sustained estrogenic response. PMID:18432312

  17. Pigment Epithelium Derived Factor Peptide Protects Murine Hepatocytes from Carbon Tetrachloride-Induced Injury

    PubMed Central

    Shih, Shou-Chuan; Ho, Tsung-Chuan; Chen, Show-Li; Tsao, Yeou-Ping

    2016-01-01

    Fibrogenesis is induced by repeated injury to the liver and reactive regeneration and leads eventually to liver cirrhosis. Pigment epithelium derived factor (PEDF) has been shown to prevent liver fibrosis induced by carbon tetrachloride (CCl4). A 44 amino acid domain of PEDF (44-mer) was found to have a protective effect against various insults to several cell types. In this study, we investigated the capability of synthetic 44-mer to protect against liver injury in mice and in primary cultured hepatocytes. Acute liver injury, induced by CCl4, was evident from histological changes, such as cell necrosis, inflammation and apoptosis, and a concomitant reduction of glutathione (GSH) and GSH redox enzyme activities in the liver. Intraperitoneal injection of the 44-mer into CCl4-treated mice abolished the induction of AST and ALT and markedly reduced histological signs of liver injury. The 44-mer treatment can reduce hepatic oxidative stress as evident from lower levels of lipid hydroperoxide, and higher levels of GSH. CCl4 caused a reduction of Bcl-xL, PEDF and PPARγ, which was markedly restored by the 44-mer treatment. Consequently, the 44-mer suppressed liver fibrosis induced by repeated CCl4 injury. Furthermore, our observations in primary culture of rat hepatocytes showed that PEDF and the 44-mer protected primary rat hepatocytes against apoptosis induced by serum deprivation and TGF-β1. PEDF/44-mer induced cell protective STAT3 phosphorylation. Pharmacological STAT3 inhibition prevented the antiapoptotic action of PEDF/44-mer. Among several PEDF receptor candidates that may be responsible for hepatocyte protection, we demonstrated that PNPLA2 was essential for PEDF/44-mer-mediated STAT3 phosphorylation and antiapoptotic activity by using siRNA to selectively knockdown PNPLA2. In conclusion, the PEDF 44-mer protects hepatocytes from single and repeated CCl4 injury. This protective effect may stem from strengthening the counter oxidative stress capacity and

  18. The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74-103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice.

    PubMed

    Vanheule, Vincent; Janssens, Rik; Boff, Daiane; Kitic, Nikola; Berghmans, Nele; Ronsse, Isabelle; Kungl, Andreas J; Amaral, Flavio Almeida; Teixeira, Mauro Martins; Van Damme, Jo; Proost, Paul; Mortier, Anneleen

    2015-08-28

    The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.

  19. The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74–103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice*

    PubMed Central

    Vanheule, Vincent; Janssens, Rik; Boff, Daiane; Kitic, Nikola; Berghmans, Nele; Ronsse, Isabelle; Kungl, Andreas J.; Amaral, Flavio Almeida; Teixeira, Mauro Martins; Van Damme, Jo; Proost, Paul; Mortier, Anneleen

    2015-01-01

    The ELR−CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74–103)) being the most potent. The COOH-terminal peptide CXCL9(74–103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74–103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74–103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74–103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions. PMID:26183778

  20. Characterization of the humoral response induced by a synthetic peptide of the major outer membrane protein of Chlamydia trachomatis serovar B.

    PubMed Central

    Villeneuve, A; Brossay, L; Paradis, G; Hébert, J

    1994-01-01

    The major outer membrane protein of Chlamydia trachomatis has been extensively studied and is still considered one of the most promising candidates for development of a synthetic vaccine. Neutralizing epitopes in variable domains I, II, and IV have already been reported. In variable domain I, residues 69 to 78 have been identified as a neutralizing epitope for some of the C- and C-related complex serovars (A, C, I, J, L3, and K). It is not known whether epitopes located at the same position in B-complex serovars are neutralizing. To clarify this point, rabbit polyclonal antibodies directed against the peptide 69TTTGNAVAPS78 from the B serovar were produced. Rabbit antisera were further rendered peptide specific by purification on a peptide-bovine serum albumin-Sepharose affinity column. Peptide-specific rabbit immunoglobulin reacted with five of the B-complex serovars (B, Ba, E, L1, and L2) by immunoblot and by direct-binding enzyme-linked immunosorbent assay. Furthermore, this peptide-specific rabbit immunoglobulin neutralized the chlamydial infectivity of both serovars B and E for HaK cells in a complement-independent in vitro assay. The importance of these results stems from the fact that peptide 69TTTGNAVAPS78 was able to induce an antibody response directed against B- and B-related complex serovars, including serovar E, which is responsible for a high proportion of genital infections. This peptide could therefore be considered for the construction of a multivalent synthetic vaccine. Images PMID:8039926

  1. Amide-I relaxation-induced hydrogen bond distortion: An intermediate in electron capture dissociation mass spectrometry of alpha-helical peptides?

    PubMed

    Pouthier, Vincent; Tsybin, Yury O

    2008-09-01

    Electron capture dissociation (ECD) of peptides and proteins in the gas phase is a powerful tool in tandem mass spectrometry whose current description is not sufficient to explain many experimental observations. Here, we attempt to bridge the current understanding of the vibrational dynamics in alpha-helices with the recent experimental results on ECD of alpha-helical peptides through consideration of amide-I relaxation-induced hydrogen bond distortion. Based on a single spine of H-bonded peptide units, we assume that charge neutralization upon electron capture by a charged alpha-helix excites a nearby amide-I mode, which relaxes over a few picoseconds due to Fermi resonances with intramolecular normal modes. The amide-I population plays the role of an external force, which drives the displacements of each peptide unit. It induces a large immobile contraction of the H bonds surrounding the excited site whose lifetime is about the amide-I lifetime. In addition, it creates two lattice deformations describing H bond stretchings, which propagate from the excited region toward both termini of the alpha-helix, get reflected at the termini and yield H bond contractions which move back to the excited region. Consequently, we show that H bonds experience rather large contractions whose amplitude depends on general features such as the position of the amide-I mode, the peptide length and the H bond force constants. When an H bond contraction is sufficiently large, it may promote a hydrogen atom transfer between two neighboring peptide units leading to the formation of a radical at charge site remote carbonyl carbon which is known to be a precursor to the rupture of the corresponding N[Single Bond]C(alpha) bond. The introduced here way of excitation energy generation and transfer may significantly advance ECD understanding and complement existing ECD mechanisms.

  2. Soy peptide lunasin induces pten-mediated apoptosis in human breast cancer cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The tumor suppressor PTEN inhibits the AKT signaling pathway whose unrestrained activity underlies many human malignancies. Previously we showed that dietary intake of soy protein isolate (SPI) enhanced PTEN expression in mammary tissue of rats with lower NMU-induced mammary tumor incidence relative...

  3. Antimicrobial Peptides and Complement in Neonatal Hypoxia-Ischemia Induced Brain Damage

    PubMed Central

    Rocha-Ferreira, Eridan; Hristova, Mariya

    2015-01-01

    Hypoxic-ischemic encephalopathy (HIE) is a clinical condition in the neonate, resulting from oxygen deprivation around the time of birth. HIE affects 1–5/1000 live births worldwide and is associated with the development of neurological deficits, including cerebral palsy, epilepsy, and cognitive disabilities. Even though the brain is considered as an immune-privileged site, it has innate and adaptive immune response and can produce complement (C) components and antimicrobial peptides (AMPs). Dysregulation of cerebral expression of AMPs and C can exacerbate or ameliorate the inflammatory response within the brain. Brain ischemia triggers a prolonged inflammatory response affecting the progression of injury and secondary energy failure and involves both innate and adaptive immune systems, including immune-competent and non-competent cells. Following injury to the central nervous system (CNS), including neonatal hypoxia-ischemia (HI), resident microglia, and astroglia are the main cells providing immune defense to the brain in a stimulus-dependent manner. They can express and secrete pro-inflammatory cytokines and therefore trigger prolonged inflammation, resulting in neurodegeneration. Microglial cells express and release a wide range of inflammation-associated molecules including several components of the complement system. Complement activation following neonatal HI injury has been reported to contribute to neurodegeneration. Astrocytes can significantly affect the immune response of the CNS under pathological conditions through production and release of pro-inflammatory cytokines and immunomodulatory AMPs. Astrocytes express β-defensins, which can chemoattract and promote maturation of dendritic cells (DC), and can also limit inflammation by controlling the viability of these same DC. This review will focus on the balance of complement components and AMPs within the CNS following neonatal HI injury and the effect of that balance on the subsequent brain damage

  4. Caspase inhibition therapy abolishes brain trauma-induced increases in Abeta peptide: implications for clinical outcome.

    PubMed

    Abrahamson, Eric E; Ikonomovic, Milos D; Ciallella, John R; Hope, Caroline E; Paljug, William R; Isanski, Barbara A; Flood, Dorothy G; Clark, Robert S B; DeKosky, Steven T

    2006-02-01

    The detrimental effects of traumatic brain injury (TBI) on brain tissue integrity involve progressive axonal damage, necrotic cell loss, and both acute and delayed apoptotic neuronal death due to activation of caspases. Post-injury accumulation of amyloid precursor protein (APP) and its toxic metabolite amyloid-beta peptide (Abeta) has been implicated in apoptosis as well as in increasing the risk for developing Alzheimer's disease (AD) after TBI. Activated caspases proteolyze APP and are associated with increased Abeta production after neuronal injury. Conversely, Abeta and related APP/Abeta fragments stimulate caspase activation, creating a potential vicious cycle of secondary injury after TBI. Blockade of caspase activation after brain injury suppresses apoptosis and improves neurological outcome, but it is not known whether such intervention also prevents increases in Abeta levels in vivo. The present study examined the effect of caspase inhibition on post-injury levels of soluble Abeta, APP, activated caspase-3, and caspase-cleaved APP in the hippocampus of nontransgenic mice expressing human Abeta, subjected to controlled cortical injury (CCI). CCI produced brain tissue damage with cell loss and elevated levels of activated caspase-3, Abeta(1-42) and Abeta(1-40), APP, and caspase-cleaved APP fragments in hippocampal neurons and axons. Post-CCI intervention with intracerebroventricular injection of 100 nM Boc-Asp(OMe)-CH(2)F (BAF, a pan-caspase inhibitor) significantly reduced caspase-3 activation and improved histological outcome, suppressed increases in Abeta and caspase-cleaved APP, but showed no significant effect on overall APP levels in the hippocampus after CCI. These data demonstrate that after TBI, caspase inhibition can suppress elevations in Abeta. The extent to which Abeta suppression contributes to improved outcome following inhibition of caspases after TBI is unclear, but such intervention may be a valuable therapeutic strategy for preventing the

  5. Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers.

    PubMed

    Magzoub, Mazin; Pramanik, Aladdin; Gräslund, Astrid

    2005-11-15

    Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.

  6. Microbial challenge promotes the regenerative process of the injured central nervous system of the medicinal leech by inducing the synthesis of antimicrobial peptides in neurons and microglia.

    PubMed

    Schikorski, David; Cuvillier-Hot, Virginie; Leippe, Matthias; Boidin-Wichlacz, Céline; Slomianny, Christian; Macagno, Eduardo; Salzet, Michel; Tasiemski, Aurélie

    2008-07-15

    Following trauma, the CNS of the medicinal leech, unlike the mammalian CNS, has a strong capacity to regenerate neurites and synaptic connections that restore normal function. In this study, we show that this regenerative process is enhanced by a controlled bacterial infection, suggesting that induction of regeneration of normal CNS function may depend critically upon the coinitiation of an immune response. We explore the interaction between the activation of a neuroimmune response and the process of regeneration by assaying the potential roles of two newly characterized antimicrobial peptides. Our data provide evidence that microbial components differentially induce the transcription, by microglial cells, of both antimicrobial peptide genes, the products of which accumulate rapidly at sites in the CNS undergoing regeneration following axotomy. Using a preparation of leech CNS depleted of microglial cells, we also demonstrate the production of antimicrobial peptides by neurons. Interestingly, in addition to exerting antibacterial properties, both peptides act as promoters of the regenerative process of axotomized leech CNS. These data are the first to report the neuronal synthesis of antimicrobial peptides and their participation in the immune response and the regeneration of the CNS. Thus, the leech CNS appears as an excellent model for studying the implication of immune molecules in neural repair. PMID:18606660

  7. Inducible ASABF-Type Antimicrobial Peptide from the Sponge Suberites domuncula: Microbicidal and Hemolytic Activity in Vitro and Toxic Effect on Molluscs in Vivo†

    PubMed Central

    Wiens, Matthias; Schröder, Heinz C.; Korzhev, Michael; Wang, Xiao-Hong; Batel, Renato; Müller, Werner E. G.

    2011-01-01

    Since sponges, as typical filter-feeders, are exposed to a high load of attacking prokaryotic and eukaryotic organisms, they are armed with a wide arsenal of antimicrobial/cytostatic low-molecular-weight, non-proteinaceous bioactive compounds. Here we present the first sponge agent belonging to the group of ASABF-type antimicrobial peptides. The ASABF gene was identified and cloned from the demosponge Suberites domuncula. The mature peptide, with a length of 64 aa residues has a predicted pI of 9.24, and comprises the characteristic CSα β structural motif. Consequently, the S. domuncula ASABF shares high similarity with the nematode ASABFs; it is distantly related to the defensins. The recombinant peptide was found to display besides microbicidal activity, anti-fungal activity. In addition, the peptide lyses human erythrocytes. The expression of ASABF is upregulated after exposure to the apoptosis-inducing agent 2,2′-dipyridyl. During the process of apoptosis of surface tissue of S. domuncula, grazing gastropods (Bittium sp.) are attracted by quinolinic acid which is synthesized through the kynurenine pathway by the enzyme 3-hydroxyanthranilate 3,4-dioxygenase (HAD). Finally, the gastropods are repelled from the sponge tissue by the ASABF. It is shown that the effector peptide ASABF is sequentially expressed after the induction of the HAD gene and a caspase, as a central enzyme executing apoptosis. PMID:22073005

  8. Defective stretch-induced release of atrial natriuretic peptide from aging hypertensive rat heart: possible role of phosphatidylinositol pathway.

    PubMed

    Brunner, F; Mouton, R; Lochner, A; Opie, L H

    1995-01-01

    Because the phosphatidylinositol pathway may be part of the signaling system associated with stretch-induced release of atrial natriuretic peptide (ANP), we tested the hypothesis that formation of the intermediate inositol-1,4,5-trisphosphate (IP3) is impaired when ANP release is decreased in response to atrial stretch in hearts from aging genetically hypertensive (GH) rats. Immunoreactive ANP release into the coronary effluent and IP3 levels were studied in cardiac tissues of isolated perfused hearts from normotensive control (WAG) or GH rats aged 4, 11, or 16 months. Left atria were repeatedly distended and released with a latex balloon. ANP was measured in coronary effluent, and IP3 was measured in cardiac tissues. In all age groups, stretch and relief of stretch evoked considerably less ANP release in spontaneously beating hearts from GH than from WAG rats. Hearts from GH rats aged 16 months released no ANP, but electrical pacing restored some stretch-induced ANP secretion. With repeated stretch and release of stretch of the left atrium for 2 min, IP3 levels increased in left atrial tissue in WAG but not in GH hearts of all age groups. IP3 levels in (unstretched) left ventricles were much lower than in left atria and were unaltered by atrial stretch. In aging GH rats, the capacity to release ANP on atrial stretch is largely lost, in association with complete suppression of stimulus-induced increase in IP3 levels. These data support a role for IP3 in stretch-mediated atrial ANP secretion and suggest a progressive uncoupling of this signaling pathway in aging hypertensive rats. PMID:7723347

  9. Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes

    PubMed Central

    Bordicchia, Marica; Liu, Dianxin; Amri, Ez-Zoubir; Ailhaud, Gerard; Dessì-Fulgheri, Paolo; Zhang, Chaoying; Takahashi, Nobuyuki; Sarzani, Riccardo; Collins, Sheila

    2012-01-01

    The ability of mammals to resist body fat accumulation is linked to their ability to expand the number and activity of “brown adipocytes” within white fat depots. Activation of β-adrenergic receptors (β-ARs) can induce a functional “brown-like” adipocyte phenotype. As cardiac natriuretic peptides (NPs) and β-AR agonists are similarly potent at stimulating lipolysis in human adipocytes, we investigated whether NPs could induce human and mouse adipocytes to acquire brown adipocyte features, including a capacity for thermogenic energy expenditure mediated by uncoupling protein 1 (UCP1). In human adipocytes, atrial NP (ANP) and ventricular NP (BNP) activated PPARγ coactivator-1α (PGC-1α) and UCP1 expression, induced mitochondriogenesis, and increased uncoupled and total respiration. At low concentrations, ANP and β-AR agonists additively enhanced expression of brown fat and mitochondrial markers in a p38 MAPK–dependent manner. Mice exposed to cold temperatures had increased levels of circulating NPs as well as higher expression of NP signaling receptor and lower expression of the NP clearance receptor (Nprc) in brown adipose tissue (BAT) and white adipose tissue (WAT). NPR-C–/– mice had markedly smaller WAT and BAT depots but higher expression of thermogenic genes such as Ucp1. Infusion of BNP into mice robustly increased Ucp1 and Pgc-1α expression in WAT and BAT, with corresponding elevation of respiration and energy expenditure. These results suggest that NPs promote “browning” of white adipocytes to increase energy expenditure, defining the heart as a central regulator of adipose tissue biology. PMID:22307324

  10. Estrogen receptors regulate the estrous behavior induced by progestins, peptides, and prostaglandin E2.

    PubMed

    Lima-Hernández, F J; Gómora-Arrati, P; García-Juárez, M; Blaustein, J D; Etgen, A M; Beyer, C; González-Flores, O

    2014-07-01

    The role of classical estrogen receptors (ERs) in priming female reproductive behavior has been studied previously; however, the participation of this receptor during activation of estrous behavior has not been extensively studied. The purpose of this work was to test the possibility that the facilitation of lordosis behavior in estrogen-primed rats by progesterone (P) and its 5α- and 5β-reduced metabolites, gonadotropin-releasing hormone (GnRH), leptin, prostaglandin E2 (PGE2) and vagino-cervical stimulation (VCS) involves interactions with classical ERs by using the selective ER modulator, tamoxifen. To further assess the role of ERs, we also explored the effects of the pure ER antagonist, ICI182780 (ICI), on estrous behavior induced by P and GnRH. Ovariectomized, estrogen-primed rats (5μg estradiol benzoate 40h earlier) were injected intraventricularly with the above-mentioned compounds, or they received VCS. All compounds and VCS effectively facilitated estrous behavior when tested at 60, 120 or 240min after infusion or application of VCS. Intraventricular infusion of tamoxifen (5μg), 30min before, significantly attenuated estrous behaviors induced in estradiol-primed rats by P, most of its 5α- and 5β-reduced metabolites, GnRH, and PGE2, but not by VCS. Although there was a trend for reduction, tamoxifen did not significantly decrease lordosis in females treated with 5β-pregnan-3,20-dione. ICI also inhibited lordosis behavior induced by P and GnRH at some testing intervals. These results suggest that activation of classical ERs participates in the triggering effects on estrous behavior induced by agents with different chemical structures that do not bind directly to ERs.

  11. Cellular immune responses induced in vitro by Ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides.

    PubMed

    Thema, Nontobeko; Pretorius, Alri; Tshilwane, Selaelo I; Liebenberg, Junita; Steyn, Helena; Van Kleef, Mirinda

    2016-01-01

    Secreted proteins are reported to induce cell-mediated immunity characterised by the production of interferon-gamma (IFN)-γ. In this study three open reading frames (ORFs) (Erum8060, Erum7760, Erum5000) encoding secreted proteins were selected from the Ehrlichia ruminantium (Welgevonden) genome sequence using bioinformatics tools to determine whether they induce a cellular immune response in vitro with mononuclear cells from needle and tick infected animals. The whole recombinant protein of the three ORFs as well as four adjacent fragments of the Erum5000 protein (Erum5000A, Erum5000B, Erum5000C, Erum5000D) were successfully expressed in a bacterial expression system which was confirmed by immunoblots using anti-His antibodies and sheep sera. These recombinant proteins were assayed with immune sheep and cattle peripheral blood mononuclear cells (PBMCs), spleen and lymph node (LN) cells to determine whether they induce recall cellular immune responses in vitro. Significant proliferative responses and IFN-γ production were evident for all recombinant proteins, especially Erum5000A, in both ruminant species tested. Thus overlapping peptides spanning Erum5000A were synthesised and peptides that induce proliferation of memory CD4+ and CD8+ T cells and production of IFN-γ were identified. These results illustrate that a Th1 type immune response was elicited and these recombinant proteins and peptides may therefore be promising candidates for development of a heartwater vaccine. PMID:27608502

  12. Human Leukocyte Antigen (HLA) Peptides Derived from Tumor Antigens Induced by Inhibition of DNA Methylation for Development of Drug-facilitated Immunotherapy.

    PubMed

    Shraibman, Bracha; Kadosh, Dganit Melamed; Barnea, Eilon; Admon, Arie

    2016-09-01

    Treatment of cancer cells with anticancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor's gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anticancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (Decitabine) has limited antitumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The proteomics and HLA peptidomics data are available via ProteomeXchange with identifier PXD003790 and the transcriptomics data are available via GEO with identifier GSE80137.

  13. Cyclotriveratrylene (CTV) as a new chiral triacid scaffold capable of inducing triple helix formation of collagen peptides containing either a native sequence or Pro-Hyp-Gly repeats.

    PubMed

    Rump, Erik T; Rijkers, Dirk T S; Hilbers, Hans W; de Groot, Philip G; Liskamp, Rob M J

    2002-10-18

    A new triacid scaffold is described based on the cone-shaped cyclotriveratrylene (CTV) molecule that facilitates the triple helical folding of peptides containing either a unique blood platelet binding collagen sequence or collagen peptides composed of Pro-Hyp-Gly repeats. The latter were synthesized by segment condensation using Fmoc-Pro-Hyp-Gly-OH. Peptides were coupled to this CTV scaffold and also coupled to the Kemp's triacid (KTA) scaffold. After assembly of peptide H-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu(OAll)-Arg-Gly-Val-Glu (OAll)-Gly-[Pro-Hyp-Gly]2-NH2 (13) by an orthogonal synthesis strategy to both triacid scaffolds, followed by deprotection of the allyl groups, the molecular constructs spontaneously folded into a triple helical structure. In contrast, the non-assembled peptides did not. The melting temperature (Tm) of (+/-) CTV[CH2C(O)N(H)Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly- [Pro-Hyp-Gly]2-NH2]3 (14) is 19 degrees C, whereas KTA[Gly-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly- [Pro-Hyp-Gly]2-NH2]3 (15) has a Tm of 20 degrees C. Thus, it was shown for the first time that scaffolds were also effective in stabilizing the triple helix of native collagen sequences. The different stabilizing properties of the two CTV enantiomers could be measured after coupling of racemic CTV triacid to the collagen peptide, and subsequent chromatographic separation of the diastereomers. After assembly of the two chiral CTV scaffolds to the model peptide H-Gly-Gly-(Pro-Hyp-Gly)5-NH2 (24), the (+)-enantiomer of CTV 28b was found to serve as a better triple helix-inducing scaffold than the (-)-enantiomer 28a. In addition to an effect of the chirality of the CTV scaffold, a certain degree of flexibility between the CTV cone and the folded peptide was also shown to be of importance. Restricting the flexibility from two to one glycine residues resulted in a significant difference between the two collagen mimics 20a and 20b, whereas the difference was

  14. Sea cucumber peptides exert anti-inflammatory activity through suppressing NF-κB and MAPK and inducing HO-1 in RAW264.7 macrophages.

    PubMed

    Song, Jiajia; Li, Tiange; Cheng, Xue; Ji, Xiaomin; Gao, Dongxiao; Du, Min; Jiang, Naiyi; Liu, Xueling; Mao, Xueying

    2016-06-15

    The anti-inflammatory effect of sea cucumber peptides (SCP) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages was tested. SCP significantly reduced LPS-induced nitric oxide release by inhibiting the inducible nitric oxide synthase mRNA expression without affecting the cell viability. The mRNA expression of LPS-induced inflammatory cytokines including tumour necrosis factor-α, interleukin (IL)-1β and IL-6 was suppressed. SCP inhibited LPS-induced degradation of the inhibitor of κBα (IκBα) and nuclear transposition of NF-κB p65, resulting in decreased NF-κB transactivation. Moreover, SCP suppressed the LPS-induced phosphorylation of JNK, ERK and p38. In addition, the expression of heme oxygenase (HO)-1 in macrophages was up-regulated by SCP in a dose-dependent manner. The inhibition effect of SCP on the mRNA expression of LPS-induced inflammatory cytokines was partially reversed by co-treatment with a HO-1 inhibitor. The SCP with anti-inflammatory activity was made up of low-molecular-weight peptides rich in glycine, glutamic acid and aspartic acid. Collectively, these results demonstrate that SCP exerts anti-inflammatory function through inhibiting NF-κB and MAPK activation and inducing HO-1 expression in macrophages.

  15. Sea cucumber peptides exert anti-inflammatory activity through suppressing NF-κB and MAPK and inducing HO-1 in RAW264.7 macrophages.

    PubMed

    Song, Jiajia; Li, Tiange; Cheng, Xue; Ji, Xiaomin; Gao, Dongxiao; Du, Min; Jiang, Naiyi; Liu, Xueling; Mao, Xueying

    2016-06-15

    The anti-inflammatory effect of sea cucumber peptides (SCP) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages was tested. SCP significantly reduced LPS-induced nitric oxide release by inhibiting the inducible nitric oxide synthase mRNA expression without affecting the cell viability. The mRNA expression of LPS-induced inflammatory cytokines including tumour necrosis factor-α, interleukin (IL)-1β and IL-6 was suppressed. SCP inhibited LPS-induced degradation of the inhibitor of κBα (IκBα) and nuclear transposition of NF-κB p65, resulting in decreased NF-κB transactivation. Moreover, SCP suppressed the LPS-induced phosphorylation of JNK, ERK and p38. In addition, the expression of heme oxygenase (HO)-1 in macrophages was up-regulated by SCP in a dose-dependent manner. The inhibition effect of SCP on the mRNA expression of LPS-induced inflammatory cytokines was partially reversed by co-treatment with a HO-1 inhibitor. The SCP with anti-inflammatory activity was made up of low-molecular-weight peptides rich in glycine, glutamic acid and aspartic acid. Collectively, these results demonstrate that SCP exerts anti-inflammatory function through inhibiting NF-κB and MAPK activation and inducing HO-1 expression in macrophages. PMID:27220344

  16. Inhibition of phosphate-induced apoptosis in resting zone chondrocytes by thrombin peptide 508.

    PubMed

    Zhong, Ming; Carney, Darrell H; Ryaby, James T; Schwartz, Zvi; Boyan, Barbara D

    2009-01-01

    Growth plate chondrocytes are susceptible to apoptosis. Terminally differentiated chondrocytes are deleted via apoptosis, which primes the growth plate to vascular invasion and subsequent bone formation. Whether less differentiated resting zone chondrocytes are subject to the same mechanism that governs the apoptotic pathway of more differentiated growth zone chondrocytes is not known. In our current study, we demonstrated that inorganic phosphate, a key inducer of growth plate chondrocyte apoptosis, also causes apoptosis in resting zone chondrocytes, via a pathway similar to the one in growth zone chondrocytes. Our results demonstrated that the conditions that cause growth plate chondrocyte apoptosis lie in the external environment, instead of the differences in differentiation state.

  17. Peptide nanotubes.

    PubMed

    Hamley, Ian W

    2014-07-01

    The self-assembly of different classes of peptide, including cyclic peptides, amyloid peptides and surfactant-like peptides into nanotube structures is reviewed. The modes of self-assembly are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized.

  18. Effects of pyrenebutyrate on the translocation of arginine-rich cell-penetrating peptides through artificial membranes: recruiting peptides to the membranes, dissipating liquid-ordered phases, and inducing curvature.

    PubMed

    Katayama, Sayaka; Nakase, Ikuhiko; Yano, Yoshiaki; Murayama, Tomo; Nakata, Yasushi; Matsuzaki, Katsumi; Futaki, Shiroh

    2013-09-01

    Arginine-rich cell-penetrating peptides, including octaarginine (R8) and HIV-1 TAT peptides, have the ability to translocate through cell membranes and transport exogenous bioactive molecules into cells. Hydrophobic counteranions such as pyrenebutyrate (PyB) have been reported to markedly promote the membrane translocation of these peptides. In this study, using model membranes having liquid-ordered (Lo) and liquid-disordered (Ld) phases, we explored the effects of PyB on the promotion of R8 translocation. Confocal microscopic observations of giant unilamellar vesicles (GUVs) showed that PyB significantly accelerated the accumulation of R8 on membranes containing negatively charged lipids, leading to the internalization of R8 without collapse of the GUV structures. PyB displayed an alternative activity, increasing the fluidity of the negatively charged membranes, which diminished the distinct Lo/Ld phase separation on GUVs. This was supported by the decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Additionally, PyB induced membrane curvature, which has been suggested as a possible mechanism of membrane translocation for R8. Taken together, our results indicate that PyB may have multiple effects that promote R8 translocation through cell membranes.

  19. Interplay of histidine residues of the Alzheimer’s disease Aβ peptide governs its Zn-induced oligomerization

    PubMed Central

    Istrate, Andrey N.; Kozin, Sergey A.; Zhokhov, Sergey S.; Mantsyzov, Alexey B.; Kechko, Olga I.; Pastore, Annalisa; Makarov, Alexander A.; Polshakov, Vladimir I.

    2016-01-01

    Conformational changes of Aβ peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer’s disease (AD). Interactions of zinc ions with Aβ are mediated by the N-terminal Aβ1–16 domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aβ plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aβ variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6HDSGYEVHH14. The residue H6 and segment 11EVHH14, which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aβ. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aβ aggregation. PMID:26898943

  20. Spergularia marina Induces Glucagon-Like Peptide-1 Secretion in NCI-H716 Cells Through Bile Acid Receptor Activation

    PubMed Central

    Kim, Kyong; Lee, Yu Mi; Rhyu, Mee-Ra

    2014-01-01

    Abstract Spergularia marina Griseb. (SM) is a halophyte that grows in mud flats. The aerial portions of SM have been eaten as vegetables and traditionally used to prevent chronic diseases in Korea. However, there has been no scientific report that demonstrates the pharmacological effects of SM. Glucagon-like peptide-1 (GLP-1) is important for the maintenance of glucose and energy homeostasis through acting as a signal in peripheral and neural systems. To discover a functional food for regulating glucose and energy homeostasis, we evaluated the effect of an aqueous ethanolic extract (AEE) of SM on GLP-1 release from enteroendocrine NCI-H716 cells. In addition, we explored the Takeda G-protein-coupled receptor 5 (TGR5) agonist activity of AEE-SM in Chinese hamster ovary (CHO)-K1 cells transiently transfected with human TGR5. As a result, treatment of NCI-H716 cells with AEE-SM increased GLP-1 secretion and intracellular Ca2+ and cyclic AMP (cAMP) levels in a dose-dependent manner. Transfection of NCI-H716 cells with TGR5-specific small interference RNA inhibited AEE-SM-induced GLP-1 secretion and the increase in Ca2+ and cAMP levels. Moreover, AEE-SM showed that the TGR5 agonist activity in CHO-K1 cells transiently transfected with TGR5. The results suggest that AEE-SM might be a candidate for a functional food to regulate glucose and energy homeostasis. PMID:25260089

  1. Rational design of thermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions.

    PubMed

    Wang, Guangchuan; Cao, Rui-Yuan; Chen, Rong; Mo, Lijuan; Han, Jian-Feng; Wang, Xiaoyu; Xu, Xurong; Jiang, Tao; Deng, Yong-Qiang; Lyu, Ke; Zhu, Shun-Ya; Qin, E-De; Tang, Ruikang; Qin, Cheng-Feng

    2013-05-01

    The development of vaccines against infectious diseases represents one of the most important contributions to medical science. However, vaccine-preventable diseases still cause millions of deaths each year due to the thermal instability and poor efficacy of vaccines. Using the human enterovirus type 71 vaccine strain as a model, we suggest a combined, rational design approach to improve the thermostability and immunogenicity of live vaccines by self-biomineralization. The biomimetic nucleating peptides are rationally integrated onto the capsid of enterovirus type 71 by reverse genetics so that calcium phosphate mineralization can be biologically induced onto vaccine surfaces under physiological conditions, generating a mineral exterior. This engineered self-biomineralized virus was characterized in detail for its unique structural, virological, and chemical properties. Analogous to many exteriors, the mineral coating confers some new properties on enclosed vaccines. The self-biomineralized vaccine can be stored at 26 °C for more than 9 d and at 37 °C for approximately 1 wk. Both in vitro and in vivo experiments demonstrate that this engineered vaccine can be used efficiently after heat treatment or ambient temperature storage, which reduces the dependence on a cold chain. Such a combination of genetic technology and biomineralization provides an economic solution for current vaccination programs, especially in developing countries that lack expensive refrigeration infrastructures. PMID:23589862

  2. C-type Natriuretic Peptide: a novel biomarker of steroid induced bone toxicity in children with acute lymphoblastic leukemia (ALL).

    PubMed

    Prickett, Timothy C R; Lyver, Amanda; Wilson, Rachel; Espiner, Eric A; Sullivan, Michael J

    2012-07-01

    Impaired bone growth and mineralization, and osteonecrosis are significant and common long-term sequelae of chemotherapy for childhood acute lymphoblastic leukemia (ALL). Here we have evaluated the relationship between linear bone growth during chemotherapy for ALL and bone derived C-type Natriuretic Peptide (CNP). CNP is known to be critical to normal endochondral bone growth in both rodents and humans, and plasma concentration of the amino terminal pro CNP (NTproCNP) is strongly correlated with concurrent height velocity in children. Plasma NTproCNP and CNP were measured by radio-immunoassay in 12 children aged 2-9 years during induction and maintenance chemotherapy for children with ALL. Height velocity was calculated from stadiometer readings at intervals of 3-12 months and related to plasma NTproCNP during each growth interval. Plasma NTproCNP was markedly suppressed in all subjects during induction chemotherapy. Brief periods of NTproCNP decline and rapid rebound during maintenance treatment coincided with the use of dexamethasone but not with other chemotherapeutics. Height velocity was markedly reduced during ALL induction but unaffected in maintenance phase, and these changes in growth were strongly correlated with plasma NTproCNP concentration. Plasma NTproCNP has potential use as a biomarker of glucocorticoid-induced bone toxicity.

  3. Degradation and aggregation of delta sleep-inducing peptide (DSIP) and two analogs in plasma and serum

    SciTech Connect

    Graf, M.V.; Saegesser, B.; Schoenenberger, G.A.

    1987-07-01

    The biostability of DSIP (delta sleep-inducing peptide) and two analogs in blood was investigated in order to determine if rates of inactivation contribute to variable effects in vivo. Incubation of DSIP in human or rat blood led to release of products having retention times on a gel filtration column equivalent to Trp. Formation of products was dependent on temperature, time, and species. Incubation of /sup 125/I-N-Tyr-DSIP and /sup 125/I-N-Tyr-P-DSIP, a phosphorylated analog, revealed slower degradation and, in contrast to DSIP, produced complex formation. An excess of unlabeled material did not displace the radioactivity supporting the assumption of non-specific binding/aggregation. It was concluded that the rapid disappearance of injected DSIP in blood was due to degradation, whereas complex formation together with slower degradation resulted in longer persistence of apparently intact analogs. Whether this could explain the sometimes stronger and more consistent effects of DSIP-analogs remains to be examined.

  4. Interplay of histidine residues of the Alzheimer’s disease Aβ peptide governs its Zn-induced oligomerization

    NASA Astrophysics Data System (ADS)

    Istrate, Andrey N.; Kozin, Sergey A.; Zhokhov, Sergey S.; Mantsyzov, Alexey B.; Kechko, Olga I.; Pastore, Annalisa; Makarov, Alexander A.; Polshakov, Vladimir I.

    2016-02-01

    Conformational changes of Aβ peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer’s disease (AD). Interactions of zinc ions with Aβ are mediated by the N-terminal Aβ1-16 domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aβ plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aβ variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6HDSGYEVHH14. The residue H6 and segment 11EVHH14, which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aβ. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aβ aggregation.

  5. Platelet-released growth factors induce the antimicrobial peptide human beta-defensin-2 in primary keratinocytes.

    PubMed

    Bayer, Andreas; Lammel, Justus; Rademacher, Franziska; Groß, Justus; Siggelkow, Markus; Lippross, Sebastian; Klüter, Tim; Varoga, Deike; Tohidnezhad, Mersedeh; Pufe, Thomas; Cremer, Jochen; Gläser, Regine; Harder, Jürgen

    2016-06-01

    Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds. PMID:26843467

  6. Brain natriuretic peptide levels in six basic underwater demolitions/SEAL recruits presenting with swimming induced pulmonary edema (SIPE).

    PubMed

    Shearer, Damon; Mahon, Richard

    2009-01-01

    Swimming induced pulmonary edema (SIPE) is associated with both SCUBA diving and strenuous surface swimming; however, the majority of reported cases and clinically observed cases tend to occur during or after aggressive surface swimming. Capillary stress failure appears to be central to the pathophysiology of this disorder. Regional pulmonary capillaries are exposed to relatively high pressures secondary to increased vascular volume, elevation of pulmonary vascular resistance, and regional differences in perfusion secondary to forces of gravity and high cardiac output. Acute pulmonary edema can be classified as either cardiogenic or noncardiogenic or both. Cardiogenic pulmonary edema occurs when the pulmonary capillary hydrostatic pressure exceeds plasma oncotic pressure. Noncardiogenic pulmonary edema occurs when pulmonary capillary permeability is increased. Given the pathophysiology noted above, SIPE can be described as a cardiogenic pulmonary edema, at least in part, since an increased transalveolar pressure gradient has been implicated in the pathogenesis of SIPE. Brain natriuretic peptide (BNP) is used in the clinical setting to differentiate cardiac from pulmonary sources of dyspnea, specifically to diagnose cardiogenic pulmonary edema. During clinical management, BNP levels were drawn on six BUD/S recruits simultaneously presenting with pulmonary complaints consistent with SIPE, after an extended surface bay swim. This paper analyzes that data after de-identification and reviews the pathophysiology and clinical management of SIPE.

  7. Phosphatidylinositol 3-kinase regulation of gastrin-releasing peptide-induced cell cycle progression in neuroblastoma cells.

    PubMed

    Ishola, Titilope A; Kang, JungHee; Qiao, Jingbo; Evers, B Mark; Chung, Dai H

    2007-06-01

    Gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin (BBS), is an autocrine growth factor for neuroblastoma; its receptor is up-regulated in undifferentiated neuroblastomas. Phosphatidylinositol 3-kinase (PI3K) is a critical cell survival pathway; it is negatively regulated by the PTEN tumor suppressor gene. We have recently found that poorly differentiated neuroblastomas express decreased PTEN protein levels. Moreover, overexpression of the GRP receptor, a member of the G-protein coupled receptor family, down-regulates PTEN expression, resulting in increased neuroblastoma cell growth. Therefore, we sought to determine whether GRP or BBS activates PI3K in neuroblastoma cells (BE(2)-C, LAN-1, SK-N-SH). GRP or BBS treatment rapidly increased phosphorylation of Akt and GSK-3beta in neuroblastoma cells. Inhibition of GRP receptor, with antagonist GRP-H2756 or siRNA, attenuated BBS-induced phosphorylation of Akt. LY294002, a PI3K inhibitor, also abrogated BBS-stimulated phospho-Akt as well as its cell cycle targets. GRP increased G1/S phase progression in SK-N-SH cells. BBS-mediated BrdU incorporation was blocked by LY294002. Our findings identify PI3K as an important signaling pathway for GRP-mediated neuroblastoma cell growth. A novel therapy targeted at GRP/GRP receptor may prove to be an effective treatment option to inhibit PI3K in neuroblastomas.

  8. Interplay of histidine residues of the Alzheimer’s disease Aβ peptide governs its Zn-induced oligomerization

    NASA Astrophysics Data System (ADS)

    Istrate, Andrey N.; Kozin, Sergey A.; Zhokhov, Sergey S.; Mantsyzov, Alexey B.; Kechko, Olga I.; Pastore, Annalisa; Makarov, Alexander A.; Polshakov, Vladimir I.

    2016-02-01

    Conformational changes of Aβ peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer’s disease (AD). Interactions of zinc ions with Aβ are mediated by the N-terminal Aβ1–16 domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aβ plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aβ variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6HDSGYEVHH14. The residue H6 and segment 11EVHH14, which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aβ. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aβ aggregation.

  9. Novel Nuclear Factor-KappaB Targeting Peptide Suppresses β-Amyloid Induced Inflammatory and Apoptotic Responses in Neuronal Cells

    PubMed Central

    Srinivasan, Mythily; Bayon, Baindu; Chopra, Nipun; Lahiri, Debomoy K.

    2016-01-01

    In the central nervous system (CNS), activation of the transcription factor nuclear factor-kappa B (NF-κβ) is associated with both neuronal survival and increased vulnerability to apoptosis. The mechanisms underlying these dichotomous effects are attributed to the composition of NF-κΒ dimers. In Alzheimer’s disease (AD), β-amyloid (Aβ) and other aggregates upregulate activation of p65:p50 dimers in CNS cells and enhance transactivation of pathological mediators that cause neuroinflammation and neurodegeneration. Hence selective targeting of activated p65 is an attractive therapeutic strategy for AD. Here we report the design, structural and functional characterization of peptide analogs of a p65 interacting protein, the glucocorticoid induced leucine zipper (GILZ). By virtue of binding the transactivation domain of p65 exposed after release from the inhibitory IκΒ proteins in activated cells, the GILZ analogs can act as highly selective inhibitors of activated p65 with minimal potential for off-target effects. PMID:27764084

  10. HYPERPHAGIA INDUCED BY SUCROSE: RELATION TO CIRCULATING AND CSF GLUCOSE AND CORTICOSTERONE AND OREXIGENIC PEPTIDES IN THE ARCUATE NUCLEUS

    PubMed Central

    Gaysinskaya, V. A.; Karatayev, O.; Shuluk, J.; Leibowitz, S. F.

    2010-01-01

    Sucrose-rich diets compared to starch-rich diets are known to stimulate overeating under chronic conditions. The present study in normal-weight rats established an acute “preload-to-test meal” paradigm for demonstrating sucrose-induced hyperphagia and investigating possible mechanisms that mediate this behavioral phenomenon. In this acute paradigm, the rats were first given a small (15 kcals) sucrose preload (30% sucrose) for 30 min compared to an equicaloric, starch preload (25% starch with 5% sucrose) and then allowed to freely consume a subsequent test meal of lab chow. The sucrose preload, when compared to a starch preload equal in energy density and palatability, consistently increased food intake in the subsequent test meal occurring between 60–120 min after the end of the preload. Measurements of hormones, metabolites and hypothalamic peptides immediately preceding this hyperphagia revealed marked differences between the sucrose vs starch groups that could contribute to the increase in food intake. Whereas the sucrose group compared to starch group immediately after the preload (at 10 min) had elevated levels of glucose in serum and cerebrospinal fluid (CSF) along with reduced expression of neuropeptide Y (NPY) and agouti-related protein (AgRP) in the arcuate nucleus (ARC), the subsequent effects (at 30–60 min) just preceding the test meal hyperphagia were the reverse. Along with lower levels of glucose, they included markedly elevated serum and CSF levels of corticosterone and mRNA levels of NPY and AgRP in the ARC. In addition to establishing an animal model for sucrose-induced hyperphagia, these results demonstrate peripheral and central mechanisms that may mediate this behavioral phenomenon. PMID:21036188

  11. Astrocytic adenosine A2A receptors control the amyloid-β peptide-induced decrease of glutamate uptake.

    PubMed

    Matos, Marco; Augusto, Elisabete; Machado, Nuno J; dos Santos-Rodrigues, Alexandre; Cunha, Rodrigo A; Agostinho, Paula

    2012-01-01

    Alzheimer's disease (AD) is characterized by a progressive cognitive impairment tightly correlated with the accumulation of amyloid-β (Aβ) peptides (mainly Aβ(1-42)). There is a precocious disruption of glutamatergic synapses in AD, in line with an ability of Aβ to decrease astrocytic glutamate uptake. Accumulating evidence indicates that caffeine prevents the burden of AD, likely through the antagonism of A(2A) receptors (A(2A)R) which attenuates Aβ-induced memory impairment and synaptotoxicity. Since A(2A)R also modulate astrocytic glutamate uptake, we now tested if A(2A)R blockade could prevent the decrease of astrocytic glutamate uptake caused by Aβ. In cultured astrocytes, Aβ(1-42). (1 μM for 24 hours) triggered an astrogliosis typified by an increased density of GFAP, which was mimicked by the A(2A)R agonist, CGS 26180 (30 nM), and prevented by the A(2A)R antagonist, SCH 58261 (100 nM). Aβ1-42 also decreased D-aspartate uptake by 28 ± 4%, an effect abrogated upon genetic inactivation or pharmacological blockade of A(2A)R. In accordance with the long term control of glutamate transporter expression by A(2A)R, Aβ(1-42). enhanced the expression and density of astrocytic A(2A)R and decreased GLAST and GLT-I expression in astrocytes from wild type, but not from A(2A)R knockout mice. This impact of Aβ(1-42). on glutamate transporters and uptake, dependent on A(2A)R function, was also confirmed in an ex vivo astrocyte preparation (gliosomes) from rats intracerebroventricularly (icv) injected with Aβ(1-42). . These results provide the first demonstration for a direct key role of astrocytic A(2A)R in the ability of Aβ-induced impairment of glutamate uptake, which may underlie glutamatergic synaptic dysfunction and excitotoxicity in AD.

  12. Transducible P11-CNTF rescues the learning and memory impairments induced by amyloid-beta peptide in mice.

    PubMed

    Qu, Heng Yan; Zhang, Ting; Li, Xu Ling; Zhou, Jian Ping; Zhao, Bao Quan; Li, Qian; Sun, Man Ji

    2008-10-10

    Alzheimer's disease is a progressive brain disorder with the loss of memory and other intellectual abilities. Amyloid species and neurofibrillary tangles are the prime suspects in damaging and killing nerve cells. Abnormal accumulation of Amyloid-beta peptide (Abeta) may cause synaptic dysfunction and degeneration of neurons. Drugs that can prevent its formation and accumulation or stimulate its clearance might ultimately be of therapeutic benefit. Ciliary neurotrophic factor (CNTF), a neurotrophic cytokine, promotes the survival of various neurons in brain. However, the blood-brain barrier hinders the systemic delivery of CNTF to brain. Recently the 11-amino acid of protein transduction domain TAT has successfully assisted the delivery of many macromolecules to treat preclinical models of human disease. The present study aimed to evaluate whether P11-CNTF fusion protein (P11-CNTF) is protective against the Abeta25-35-induced dementia in mice. Immunofluorescence experiments showed that P11 effectively carried CNTF to the SH-SY5Y cells in vitro, and to the brains of mice in vivo. The learning and memory impairments of mice induced by Abeta were substantially rescued by supplement with the P11-CNTF. Furthermore, mRNAs of enzymes involved in the Abeta metabolism, e.g. neprilysin (NEP), endothelin-converting enzyme 1 (ECE-1) and insulin degrading enzyme (IDE), increased in the P11-CNTF treated dementia mice, accompanied by the proliferation of nestin- and choline acetyltransferase (ChAT)-positive cells in hippocampus. It implies that the delivery of P11-CNTF may be a novel treatment for Alzheimer's disease. PMID:18644361

  13. Novel Regenerative Peptide TP508 Mitigates Radiation-Induced Gastrointestinal Damage By Activating Stem Cells and Preserving Crypt Integrity

    PubMed Central

    Kantara, Carla; Moya, Stephanie M.; Houchen, Courtney W.; Umar, Shahid; Ullrich, Robert L.; Singh, Pomila; Carney, Darrell H.

    2015-01-01

    In recent years, increasing threats of radiation exposure and nuclear disasters have become a significant concern for the United States and countries worldwide. Exposure to high doses of radiation triggers a number of potentially lethal effects. Among the most severe is the gastrointestinal (GI) toxicity syndrome caused by the destruction of the intestinal barrier, resulting in bacterial translocation, systemic bacteremia, sepsis and death. The lack of effective radioprotective agents capable of mitigating radiation-induced damage has prompted a search for novel countermeasures that can mitigate the effects of radiation post-exposure, accelerate tissue repair in radiation-exposed individuals, and prevent mortality. We report that a single injection of regenerative peptide TP508 (rusalatide acetate, Chrysalin®) 24h after lethal radiation exposure (9Gy, LD100/15) appears to significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity. TP508 treatment post-exposure prevents the disintegration of gastrointestinal crypts, stimulates the expression of adherens junction protein E-cadherin, activates crypt cell proliferation, and decreases apoptosis. TP508 post-exposure treatment also up-regulates the expression of DCLK1 and LGR5 markers of stem cells that have been shown to be responsible for maintaining and regenerating intestinal crypts. Thus, TP508 appears to mitigate the effects of GI toxicity by activating radioresistant stem cells and increasing the stemness potential of crypts to maintain and restore intestinal integrity. These results suggest that TP508 may be an effective emergency nuclear countermeasure that could be delivered within 24h post-exposure to increase survival and delay mortality, giving victims time to reach clinical sites for advanced medical treatment. PMID:26280221

  14. Arthritis-inducing ability of a synthetic adjuvant, N-acetylmuramyl peptides, and bacterial disaccharide peptides related to different oil vehicles and their composition.

    PubMed Central

    Kohashi, O; Tanaka, A; Kotani, S; Shiba, T; Kusumoto, S; Yokogawa, K; Kawata, S; Ozawa, A

    1980-01-01

    A synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), failed to produce polyarthritis with a wide dose range in a water-in-oil emulsion of mineral oil such as liquid paraffin, Drakeol, or heavy mineral oil. MDP, however, produced moderate to severe arthritis with almost 100% incidence in a water-in-oil emulsion made up of Difco incomplete adjuvant, which consists of Bavol F as an oil vehicle and Arlacel A as an emulsifier. N-acetylmuramyl-L-alanyl-L-isoglutamine did not produce arthritis, whereas 4,6-diacetyl-MDP produced the disease. Bacterial peptidoglycans, such as disaccharide peptides which were N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine and N-acetylglucosaminyl-6,o-acetyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine, also produced polyarthritis with low incidence in Difco oil but not in the other mineral oils described above. MDP and bacterial disaccharide peptides were able to produce the disease even in the latter mineral oil only when the concentration of Arlacel A was increased from 15% to 20 to 30% in the oil. We concluded that one of the minimal essential structures responsible for development of this disease is MDP, although the role of the oil vehicle remained uncertain, and there is no direct correlation between granulona formation and arthritogenicity of MDP. PMID:7399708

  15. Two-dimensional self-assembly of amphiphilic peptides; adsorption-induced secondary structural transition on hydrophilic substrate.

    PubMed

    Tanaka, Masayoshi; Abiko, Souhei; Himeiwa, Takahiro; Kinoshita, Takatoshi

    2015-03-15

    Adsorption of sequential amphiphilic peptides on solid substrates triggered the spontaneous construction of nanoscaled architecture. An amphiphilic peptide designed with a cationic amino acid as a hydrophilic residue turned an anionic mica substrate into a water-repellent surface, simply by adsorbing it on the substrate surface. In contrast, an amphiphilic peptide designed with an anionic amino-acid residue formed a precisely controlled fiber array comprising a β-sheet fiber monolayer at the anionic substrate/water interface. This phenomenon was based on the secondary structural transition from random-coil to β-sheet, which occurred specifically when amphiphilic peptide adsorbed on the substrate surface. Such surface-specific nonorder/order transition was implemented by exploiting the strength of adsorption between the peptide and the substrate. A strategic design exploiting weak bonding such as hydrophobic interactions is essential for constructing precisely controlled nano-architectures in two dimensions.

  16. WT1 PEPTIDE VACCINATIONS INDUCE CD4 AND CD8 T CELL IMMUNE RESPONSES IN PATIENTS WITH MESOTHELIOMA AND NON-SMALL CELL LUNG CANCER

    PubMed Central

    Krug, Lee M.; Dao, Tao; Brown, Andrew B.; Maslak, Peter; Travis, William; Bekele, Sara; Korontsvit, Tatyana; Zakhaleva, Victoria; Wolchok, Jedd; Yuan, Jianda; Li, Hao; Tyson, Leslie; Scheinberg, David A.

    2014-01-01

    BACKGROUND The transcription factor, WT1, is highly overexpressed in malignant pleural mesothelioma (MPM) and immunohistochemical stains for WT1 are used routinely to aid in its diagnosis. Using computer prediction analysis we designed analog peptides derived from WT1 sequences by substituting amino acids at key HLA-A0201 binding positions. We tested the safety and immunogenicity of a WT1 vaccine comprised of four class 1 and class 2 peptides in patients with thoracic neoplasms expressing WT1. METHODS Therapy consisted of six subcutaneous vaccinations administered with Montanide adjuvant on weeks 0, 4, 6, 8, 10, and 12, with 6 additional monthly injections for responding patients. Injection sites were pre-stimulated with GM-CSF (70mcg). Immune responses were evaluated by DTH, CD4 T-cell proliferation, CD8 T-cell interferon gamma release, intracellular cytokine staining, WT1 peptide MHC-tetramer staining, and cytotoxicity against WT1 positive tumor cells. RESULTS Nine patients with MPM and 3 with NSCLC were vaccinated, with 8 patients receiving at least 6 vaccinations; in total, 10 patients were evaluable for immune response. Six out of nine patients tested demonstrated CD4 T-cell proliferation to WT1 specific peptides, and five of the six HLA-A0201 patients tested mounted a CD8 T-cell response. Stimulated T cells were capable of cytotoxicity against WT-1 positive cells. Vaccination also induced polyfunctional CD8 T cell responses. CONCLUSIONS This multivalent WT1 peptide analog vaccine induces immune responses in a high proportion of patients with thoracic malignancies with minimal toxicity. A randomized trial testing this vaccine as adjuvant therapy in MPM is planned. PMID:20532500

  17. Evaluation of a Novel Approach for Peptide Sequencing: Laser-induced Acoustic Desorption Combined with P(OCH3)2+ Chemical Ionization and Collision-activated Dissociation in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

    PubMed Central

    Somuramasami, Jayalakshmi; Kenttämaa, Hilkka I.

    2007-01-01

    A novel mass spectrometric method has been developed for obtaining sequence information on small peptides. The peptides are desorbed as intact neutral molecules into a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR) by means of laser-induced acoustic desorption (LIAD). Reactions of the neutral peptides with the dimethoxyphosphenium ion, P(OCH3)2+, occur predominantly by addition of the peptide to P(OCH3)2+ followed by the loss of two methanol molecules, thus yielding product ions with the composition (peptide + P − 2H)+. Upon sustained off-resonance irradiation for collision-activated dissociation (SORI-CAD), the (peptide + P − 2H)+ ions undergo successive losses of CO and NH = CHR or H2O, CO, and NH = CHR to yield sequence-related fragment ions in addition to the regular an- and bn-type ions. Under the same conditions, SORI-CAD of the analogous protonated peptides predominantly yields the regular an- and bn-type ions. The mechanisms of the reactions of peptides with P(OCH3)2+ and the dissociation of the (peptide + P − 2H)+ ions were examined by using model peptides and molecular orbital calculations. PMID:17157527

  18. Inhibition of ascorbic acid-induced modifications in lens proteins by peptides.

    PubMed

    Argirova, Mariana; Argirov, Ognyan

    2003-03-01

    The effects of three dipeptides L-phenylalanyl-glybine, glycyl-L-phenylalanine,and aspartame (L-aspartyl-L-phenylalanine, methyl ester) as inhibitors of the ascorbic acid-induced modifications in lens proteins were studied. Their efficiency was compared to that of two known inhibitors--aminoguanidine and carnosine. The tested dipeptides diminished protein carbonyl content by 32-58% and most moderated the formation of chromophores, as measured by the absorbency at 325 nm of the glycated proteins. The appearance of non-tryptophan fluorescence (excitation 340 nm/emission 410 nm) was observed for proteins glycated with ascorbic acid. All of the dipeptides examined, as well as aminoguanidine, decreased this glycation-related fluorescence. The potential inhibitors prevented the intensive formation of very high molecular weight aggregates. A competitive mechanism of their inhibitory effect was proposed, based on the reactivity of individual substances toward ascorbic acid. These findings indicate that they have a potential for use as alternatives for aminoguanidine as an anti-glycation agent.

  19. Transmembrane Peptides Stabilize Inverted Cubic Phases in a Biphasic Length-Dependent Manner: Implications for Protein-Induced Membrane Fusion

    PubMed Central

    Siegel, D. P.; Cherezov, V.; Greathouse, D. V.; Koeppe, R. E.; Killian, J. Antoinette; Caffrey, M.

    2006-01-01

    WALP peptides consist of repeating alanine-leucine sequences of different lengths, flanked with tryptophan “anchors” at each end. They form membrane-spanning α-helices in lipid membranes, and mimic protein transmembrane domains. WALP peptides of increasing length, from 19 to 31 amino acids, were incorporated into N-monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) at concentrations up to 0.5 mol % peptide. When pure DOPE-Me is heated slowly, the lamellar liquid crystalline (Lα) phase first forms an inverted cubic (QII) phase, and the inverted hexagonal (HII) phase at higher temperatures. Using time-resolved x-ray diffraction and slow temperature scans (1.5°C/h), WALP peptides were shown to decrease the temperatures of QII and HII phase formation (TQ and TH, respectively) as a function of peptide concentration. The shortest and longest peptides reduced TQ the most, whereas intermediate lengths had weaker effects. These findings are relevant to membrane fusion because the first step in the Lα/QII phase transition is believed to be the formation of fusion pores between pure lipid membranes. These results imply that physiologically relevant concentrations of these peptides could increase the susceptibility of biomembrane lipids to fusion through an effect on lipid phase behavior, and may explain one role of the membrane-spanning domains in the proteins that mediate membrane fusion. PMID:16214859

  20. A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems☆

    PubMed Central

    Massaoka, Mariana H.; Matsuo, Alisson L.; Figueiredo, Carlos R.; Girola, Natalia; Faria, Camyla F.; Azevedo, Ricardo A.; Travassos, Luiz R.

    2014-01-01

    The Wilms tumor protein 1 (WT1) transcription factor has been associated in malignant melanoma with cell survival and metastasis, thus emerging as a candidate for targeted therapy. A lysine–arginine rich peptide, WT1-pTj, derived from the ZF domain of WT1 was evaluated as an antitumor agent against A2058 human melanoma cells and B16F10-Nex2 syngeneic murine melanoma. Peptide WT1-pTj quickly penetrated human melanoma cells and induced senescence, recognized by increased SA-β-galactosidase activity, enhanced transcriptional activity of p53, and induction of the cell cycle inhibitors p21 and p27. Moreover, the peptide bound to p53 and competed with WT1 protein for binding to p53. WT1-pTj treatment led to sustained cell growth suppression, abrogation of clonogenicity and G2/M cell cycle arrest. Notably, in vivo studies showed that WT1-pTj inhibited both the metastases and subcutaneous growth of murine melanoma in syngeneic mice, and prolonged the survival of nude mice challenged with human melanoma cells. The 27-amino acid cell-penetrating WT1-derived peptide, depends on C3 and H16 for effective antimelanoma activity, inhibits proliferation of WT1-expressing human tumor cell lines, and may have an effective role in the treatment of WT1-expressing malignancies. PMID:24490140

  1. Inhibition of dehydration-induced water intake by glucocorticoids is associated with activation of hypothalamic natriuretic peptide receptor-A in rat.

    PubMed

    Liu, Chao; Guan, Jing; Kang, Yunxiao; Xiu, Heming; Chen, Ying; Deng, Bao; Liu, Kunshen

    2010-12-20

    Atrial natriuretic peptide (ANP) provides a potent defense mechanism against volume overload in mammals. Its primary receptor, natriuretic peptide receptor-A (NPR-A), is localized mostly in the kidney, but also is found in hypothalamic areas involved in body fluid volume regulation. Acute glucocorticoid administration produces potent diuresis and natriuresis, possibly by acting in the renal natriuretic peptide system. However, chronic glucocorticoid administration attenuates renal water and sodium excretion. The precise mechanism underlying this paradoxical phenomenon is unclear. We assume that chronic glucocorticoid administration may activate natriuretic peptide system in hypothalamus, and cause volume depletion by inhibiting dehydration-induced water intake. Volume depletion, in turn, compromises renal water excretion. To test this postulation, we determined the effect of dexamethasone on dehydration-induced water intake and assessed the expression of NPR-A in the hypothalamus. The rats were deprived of water for 24 hours to have dehydrated status. Prior to free access to water, the water-deprived rats were pretreated with dexamethasone or vehicle. Urinary volume and water intake were monitored. We found that dexamethasone pretreatment not only produced potent diuresis, but dramatically inhibited the dehydration-induced water intake. Western blotting analysis showed the expression of NPR-A in the hypothalamus was dramatically upregulated by dexamethasone. Consequently, cyclic guanosine monophosphate (the second messenger for the ANP) content in the hypothalamus was remarkably increased. The inhibitory effect of dexamethasone on water intake presented in a time- and dose-dependent manner, which emerged at least after 18-hour dexamethasone pretreatment. This effect was glucocorticoid receptor (GR) mediated and was abolished by GR antagonist RU486. These results indicated a possible physiologic role for glucocorticoids in the hypothalamic control of water intake

  2. Velvet antler peptide prevents pressure overload-induced cardiac fibrosis via transforming growth factor (TGF)-β1 pathway inhibition.

    PubMed

    Zhao, Lihong; Mi, Yang; Guan, Hongya; Xu, Yan; Mei, Yingwu

    2016-07-15

    Velvet antlers (VAs) are commonly used in traditional Chinese medicine and invigorant and contain many functional components for health promotion. The velvet antler peptide sVAP32 is one of active components in VAs; based on structural study, the sVAP32 interacts with TGF-β1 receptors and disrupts the TGF-β1 pathway. We hypothesized that sVAP32 prevents cardiac fibrosis from pressure overload by blocking TGF-β1 signaling. Sprague-Dawley rats underwent transverse aortic constriction (TAC) or a sham operation. After one month, rats received either sVAP32 (15mg/kg/day) or vehicle for an additional one month. TAC surgery induced significant cardiac dysfunction, fibroblast activation and fibrosis; these effects were improved by treatment with sVAP32. In the heart tissue, TAC remarkably increased the expression of TGF-β1 and connective tissue growth factor (CTGF), reactive oxygen species levels, and the phosphorylation levels of Smad2/3 and extracellular signal-regulated kinases 1/2 (ERK1/2). SVAP32 inhibited the increases in reactive oxygen species levels, CTGF expression and the phosphorylation of Smad2/3 and ERK1/2, but not TGF-β1 expression. In cultured cardiac fibroblasts, angiotensin II (Ang II) had similar effects compared to TAC surgery, such as increases in α-SMA-positive cardiac fibroblasts and collagen synthesis. SVAP32 eliminated these effects by disrupting TGF-β1 binding to its receptors and blocking Ang II/TGF-β1 downstream signaling. These results demonstrated that sVAP32 has anti-fibrotic effects by blocking the TGF-β1 pathway in cardiac fibroblasts.

  3. Protein kinase A mediates glucagon-like peptide 1-induced nitric oxide production and muscle microvascular recruitment

    PubMed Central

    Dong, Zhenhua; Chai, Weidong; Wang, Wenhui; Zhao, Lina; Fu, Zhuo; Cao, Wenhong

    2013-01-01

    Glucagon-like peptide-1 (GLP-1) causes vasodilation and increases muscle glucose uptake independent of insulin. Recently, we have shown that GLP-1 recruits muscle microvasculature and increases muscle glucose use via a nitric oxide (NO)-dependent mechanism. Protein kinase A (PKA) is a major signaling intermediate downstream of GLP-1 receptors. To examine whether PKA mediates GLP-1's microvascular action in muscle, GLP-1 was infused to overnight-fasted male rats for 120 min in the presence or absence of H89, a PKA inhibitor. Hindleg muscle microvascular recruitment and glucose use were determined. GLP-1 infusion acutely increased muscle microvascular blood volume within 30 min without altering microvascular blood flow velocity or blood pressure. This effect persisted throughout the 120-min infusion period, leading to a significant increase in muscle microvascular blood flow. These changes were paralleled with an approximately twofold increase in plasma NO levels and hindleg glucose extraction. Systemic infusion of H89 completely blocked GLP-1-mediated muscle microvascular recruitment and increases in NO production and muscle glucose extraction. In cultured endothelial cells, GLP-1 acutely increased PKA activity and stimulated endothelial NO synthase phosphorylation at Ser1177 and NO production. PKA inhibition abolished these effects. In ex vivo studies, perfusion of the distal saphenous artery with GLP-1 induced significant vasorelaxation that was also abolished by pretreatment of the vessels with PKA inhibitor H89. We conclude that GLP-1 recruits muscle microvasculature by expanding microvascular volume and increases glucose extraction in muscle via a PKA/NO-dependent pathway in the vascular endothelium. This may contribute to postprandial glycemic control and complication prevention in diabetes. PMID:23193054

  4. Atypical behavior in the electron capture induced dissociation of biologically relevant transition metal ion complexes of the peptide hormone oxytocin

    NASA Astrophysics Data System (ADS)

    Kleinnijenhuis, Anne J.; Mihalca, Romulus; Heeren, Ron M. A.; Heck, Albert J. R.

    2006-07-01

    Doubly protonated ions of the disulfide bond containing nonapeptide hormone oxytocin and oxytocin complexes with different transition metal ions, that have biological relevance under physiological conditions, were subjected to electron capture dissociation (ECD) to probe their structural features in the gas phase. Although, all the ECD spectra were strikingly different, typical ECD behavior was observed for complexes of the nonapeptide hormone oxytocin with Ni2+, Co2+ and Zn2+, i.e., abundant c/z' and a'/y backbone cleavages and ECD characteristic S-S and S-C bond cleavages were observed. We propose that, although in the oxytocin-transition metal ion complexes the metal ions serve as the main initial capture site, the captured electron is transferred to other sites in the complex to form a hydrogen radical, which drives the subsequent typical ECD fragmentations. The complex of oxytocin with Cu2+ displayed noticeably different ECD behavior. The fragment ions were similar to fragment ions typically observed with low-energy collision induced dissociation (CID). We propose that the electrons captured by the oxytocin-Cu2+ complex might be favorably involved in reducing the Cu2+ metal ion to Cu+. Subsequent energy redistribution would explain the observed low-energy CID-type fragmentations. Electron capture resulted also in quite different specific cleavage sites for the complexes of oxytocin with Ni2+, Co2+ and Zn2+. This is an indication for structural differences in these complexes possibly linked to their significantly different biological effects on oxytocin-receptor binding, and suggests that ECD may be used to study subtle structural differences in transition metal ion-peptide complexes.

  5. Cardiac differentiation potential of human induced pluripotent stem cells in a 3D self-assembling peptide scaffold.

    PubMed

    Puig-Sanvicens, Veronica A C; Semino, Carlos E; Zur Nieden, Nicole I

    2015-01-01

    In the past decade, various strategies for cardiac reparative medicine involving stem cells from multiple sources have been investigated. However, the intra-cardiac implantation of cells with contractile ability may seriously disrupt the cardiac syncytium and de-synchronize cardiac rhythm. For this reason, bioactive cardiac implants, consisting of stem cells embedded in biomaterials that act like band aids, have been exploited to repair the cardiac wall after myocardial infarction. For such bioactive implants to function properly after transplantation, the choice of biomaterial is equally important as the selection of the stem cell source. While adult stem cells have shown promising results, they have various disadvantages including low proliferative potential in vitro, which make their successful usage in human transplants difficult. As a first step towards the development of a bioactive cardiac patch, we investigate here the cardiac differentiation properties of human induced pluripotent stem cells (hiPSCs) when cultured with and without ascorbic acid (AA) and when embedded in RAD16-I, a biomaterial commonly used to develop cardiac implants. In adherent cultures and in the absence of RAD16-I, AA promotes the cardiac differentiation of hiPSCs by enhancing the expression of specific cardiac genes and proteins and by increasing the number of contracting clusters. In turn, embedding in peptide hydrogel based on RAD16-I interferes with the normal cardiac differentiation progression. Embedded hiPSCs up-regulate genes associated with early cardiogenesis by up to 105 times independently of the presence of AA. However, neither connexin 43 nor troponin I proteins, which are related with mature cardiomyocytes, were detected and no contraction was noted in the constructs. Future experiments will need to focus on characterizing the mature cardiac phenotype of these cells when implanted into infarcted myocardia and assess their regenerative potential in vivo. PMID:26707885

  6. Renal Overexpression of Atrial Natriuretic Peptide and Hypoxia Inducible Factor-1α as Adaptive Response to a High Salt Diet

    PubMed Central

    Della Penna, Silvana Lorena; Cao, Gabriel; Carranza, Andrea; Zotta, Elsa; Gorzalczany, Susana; Cerrudo, Carolina Susana; Rukavina Mikusic, Natalia Lucía; Correa, Alicia; Trida, Verónica; Toblli, Jorge Eduardo; Fernández, Belisario Enrique

    2014-01-01

    In the kidney, a high salt intake favors oxidative stress and hypoxia and causes the development of fibrosis. Both atrial natriuretic peptide (ANP) and hypoxia inducible factor (HIF-1α) exert cytoprotective effects. We tested the hypothesis that renal expression of ANP and HIF-1α is involved in a mechanism responding to the oxidative stress produced in the kidneys of rats chronically fed a high sodium diet. Sprague-Dawley rats were fed with a normal salt (0.4% NaCl) (NS) or a high salt (8% NaCl) (HS) diet for 3 weeks, with or without the administration of tempol (T), an inhibitor of oxidative stress, in the drinking water. We measured the mean arterial pressure (MAP), glomerular filtration rate (GFR), and urinary sodium excretion (UVNa). We evaluated the expression of ANP, HIF-1α, and transforming growth factor (TGF-β1) in renal tissues by western blot and immunohistochemistry. The animals fed a high salt diet showed increased MAP and UVNa levels and enhanced renal immunostaining of ANP, HIF-1α, and TGF-β1. The administration of tempol together with the sodium overload increased the natriuresis further and prevented the elevation of blood pressure and the increased expression of ANP, TGF-β1, and HIF-1α compared to their control. These findings suggest that HIF-1α and ANP, synthesized by the kidney, are involved in an adaptive mechanism in response to a sodium overload to prevent or attenuate the deleterious effects of the oxidative stress and the hypoxia on the development of fibrosis. PMID:24689065

  7. Metronomic cyclophosphamide enhances HPV16E7 peptide vaccine induced antigen-specific and cytotoxic T-cell mediated antitumor immune response

    PubMed Central

    Weir, Genevieve M; Hrytsenko, Olga; Stanford, Marianne M; Berinstein, Neil L; Karkada, Mohan; Liwski, Robert S; Mansour, Marc

    2014-01-01

    In clinical trials, metronomic cyclophosphamide (CPA) is increasingly being combined with vaccines to reduce tumor-induced immune suppression. Previous strategies to modulate the immune system during vaccination have involved continuous administration of low dose chemotherapy, studies that have posed unique considerations for clinical trial design. Here, we evaluated metronomic CPA in combination with a peptide vaccine targeting HPV16E7 in an HPV16-induced tumor model, focusing on the cytotoxic T-cell response and timing of low dose metronomic CPA (mCPA) treatment relative to vaccination. Mice bearing C3 tumors were given metronomic CPA on alternating weeks in combination with immunization with a DepoVax vaccine containing HPV16E749–57 peptide antigen every 3 weeks. Only the combination therapy provided significant long-term control of tumor growth. The efficacy of the vaccine was uncompromised if given at the beginning or end of a cycle of metronomic CPA. Metronomic CPA had a pronounced lymphodepletive effect on the vaccine draining lymph node, yet did not reduce the development of antigen-specific CD8+ T cells induced by vaccination. This enrichment correlated with increased cytotoxic activity in the spleen and increased expression of cytotoxic gene signatures in the tumor. Immunity could be passively transferred through CD8+ T cells isolated from tumor-bearing mice treated with the combinatorial treatment regimen. A comprehensive survey of splenocytes indicated that metronomic CPA, in the absence of vaccination, induced transient lymphodepletion marked by a selective expansion of myeloid-derived suppressor cells. These results provide important insights into the multiple mechanisms of metronomic CPA induced immune modulation in the context of a peptide cancer vaccine that may be translated into more effective clinical trial designs. PMID:25960932

  8. Synthesis of the Peptide Ac-Wahx-KTTKS and Evaluation of the Ability to Induce In Vitro Collagen Synthesis.

    PubMed

    Guglielmi, Danila A S; Martinelli, Ariane M; Rissi, Nathália C; Cilli, Eduardo M; Soares, Christiane P; Chiavacci, Leila A

    2016-01-01

    In this work, we report the synthesis of a peptide analogue of the KTTKS, termed Ac- Wahx-KTTKS and evaluate its cytotoxicity and role in biosynthesis of collagen for future application in skin aging. The peptide was obtained with purity higher than 97.5%. In the cytotoxicity assay, we observed non-toxic effects for Ac-WAhx-KTTKS at concentrations below 600 µM for HaCaT and 500 µM for HepG2 cells, respectively. After 24 and 48 h it was possible to observe significant changes in collagen synthesis in the groups treated with various concentrations of the peptide. In conclusion, the Ac-Wahx- KTTKS peptide increased collagen synthesis in fibroblasts by 80% and it is a promising candidate for improving skin aging. PMID:27184584

  9. Protection against morbillivirus-induced encephalitis by immunization with a rationally designed synthetic peptide vaccine containing B- and T-cell epitopes from the fusion protein of measles virus.

    PubMed Central

    Obeid, O E; Partidos, C D; Howard, C R; Steward, M W

    1995-01-01

    Synthetic peptides representing T- and B-cell epitopes from the fusion (F) protein of measles virus (MV) were tested for their ability to induce a protective immune response against intracerebral challenge with neuroadapted strains of MV and canine distemper virus (CDV) in mice. Of the panel of peptides tested, only a chimeric peptide consisting of two copies of a promiscuous T-cell epitope (representing residues 288 to 302 of MV F protein) synthesized at the amino terminus of a B-cell epitope (representing residues 404 to 414 of MV F protein) was able to induce a protective response against challenge with MV and CDV in inbred mice. The protective response induced by this peptide (TTB) was associated with a significant reduction in mortality, histological absence of acute encephalitis, and greatly reduced titers of virus in the brains of TTB-immune mice following challenge compared with the results for nonimmunized controls. A chimeric peptide comprising one copy of the T-cell epitope and one copy of the B-cell epitope (TB) did not induce a protective response. A comparison of the antibody responses induced by the two chimeras suggested that differences in protective efficacy following immunization may be a result of the higher affinity of the antibody induced by the TTB peptide than that of the antibody induced by the TB peptide. In addition, differences in the immunoglobulin G subclass of the antipeptide antibody responses were observed, and these may play a role in the differences in protection observed. These results indicate that appropriately designed synthetic peptides have potential as vaccines for the induction of cross-reactive protection against morbilliviruses. PMID:7531779

  10. Antagonistic Effect of a Salivary Proline-Rich Peptide on the Cytosolic Ca2+ Mobilization Induced by Progesterone in Oral Squamous Cancer Cells.

    PubMed

    Palmerini, Carlo Alberto; Mazzoni, Michela; Radicioni, Giorgia; Marzano, Valeria; Granieri, Letizia; Iavarone, Federica; Longhi, Renato; Messana, Irene; Cabras, Tiziana; Sanna, Maria Teresa; Castagnola, Massimo; Vitali, Alberto

    2016-01-01

    A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

  11. Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates a biologically active peptide, prepro-TRH-(160-169), which regulates TRH-induced thyrotropin secretion

    SciTech Connect

    Bulant, M.; Vaudry, H. ); Roussel, J.P.; Astier, H. ); Nicolas, P. )

    1990-06-01

    Rat thyrotropin-releasing hormone (TRH) prohormone contains five copies of the TRH progenitor sequence Gln-His-Pro-Gly linked together by connecting sequences whose biological activity is unknown. Both the predicted connecting peptide prepro-TRH-(160-169) (Ps4) and TRH are predominant storage forms of TRH precursor-related peptides in the hypothalamus. To determine whether Ps4 is co-released with TRH, rat median eminence slices were perfused in vitro. Infusion of depolarizing concentrations of KCl induced stimulation of release of Ps4- and TRH-like immunoreactivity. The possible effect of Ps4 on thyrotropin release was investigated in vitro using quartered anterior pituitaries. Infusion of Ps4 alone had no effect on thyrotropin secretion but potentiated TRH-induced thyrotropin release in a dose-dependent manner. In addition, the occurrence of specific binding sites for {sup 125}I-labeled Tyr-Ps4 in the distal lobe of the pituitary was demonstrated by binding analysis and autoradiographic localization. These findings indicate that these two peptides that arise from a single multifunctional precursor, the TRH prohormone, act in a coordinate manner on the same target cells to promote hormonal secretion. These data suggest that differential processing of the TRH prohormone may have the potential to modulate the biological activity of TRH.

  12. Antibody-Mediated and Cellular Immune Responses Induced in Naive Volunteers by Vaccination with Long Synthetic Peptides Derived from the Plasmodium vivax Circumsporozoite Protein

    PubMed Central

    Arévalo-Herrera, Myriam; Soto, Liliana; Perlaza, Blanca Liliana; Céspedes, Nora; Vera, Omaira; Lenis, Ana Milena; Bonelo, Anilza; Corradin, Giampietro; Herrera, Sócrates

    2011-01-01

    Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial. PMID:21292876

  13. Multifunctional nanoparticles co-delivering Trp2 peptide and CpG adjuvant induce potent cytotoxic T-lymphocyte response against melanoma and its lung metastasis.

    PubMed

    Xu, Zhenghong; Ramishetti, Srinivas; Tseng, Yu-Cheng; Guo, Shutao; Wang, Yuhua; Huang, Leaf

    2013-11-28

    Immunotherapy has shown the potential to become an essential component of the successful treatment of various malignancies. In many cases, such as in melanoma, however, induction of a potent and specific T-cell response against the endogenous antigen or self-antigen still remains a major challenge. To induce a potent MHC I-restricted cytotoxic T-lymphocyte (CTL) response, cytosol delivery of an exogenous antigen into dendritic cells is preferred, if not required. Lipid-calcium-phosphate (LCP) nanoparticles represent a new class of intracellular delivery systems for impermeable drugs. We are interested in exploring the potential of LCP NPs for use as a peptide vaccine delivery system for cancer therapy. To increase the encapsulation of Trp2 peptide into the calcium phosphate precipitate core of LCP, two phosphor-serine residues were added to the N-terminal of the peptide (p-Trp2). CpG ODN was also co-encapsulated with p-Trp2 as an adjuvant. The NPs were further modified with mannose to enhance and prolong the cargo deposit into the lymph nodes (LNs), which ensured persistent antigen loading and stimulation. Compared with free Trp2 peptide/CpG, vaccination with LCP encapsulating p-Trp2 and CpG resulted in superior inhibition of tumor growth in both B16F10 subcutaneous and lung metastasis models. An IFN-γ production assay and in vivo CTL response study revealed that the improved efficacy was a result of a Trp2-specific immune response. Thus, encapsulation of phospho-peptide antigens into LCP may be a promising strategy for enhancing the immunogenicity of poorly immunogenic self-antigens for cancer therapy.

  14. Immunization of mice with a peptide derived from the HTLV-1 TAX1BP1 protein induces cross-reactive antibodies against aquaporin 4.

    PubMed

    Kampylafka, Eleni I; Alexopoulos, Harry; El Hamidieh, Avraam; Dalakas, Marinos C; Andreakos, Evangelos; Tzioufas, Athanasios G

    2015-01-01

    Antibodies against aquaporin-4 (AQP4) are specific and pathogenetic for Neuromyelitis Optica (NMO). In a previous study, three linear intracellular AQP4 B-cell epitopes were uncovered in NMO patients. A particular epitope showed high-sequence similarity with a segment of the human TAX1BP1 protein, which is necessary for the replication of HTLV-1 virus. The aim of the present study was to investigate whether immunization of mice with the TAX1BP1 peptide could produce specific antibodies against AQP4 epitopes or induce symptoms. Eight C57Bl/6 mice were immunized with TAX1BP1pep in Complete Freund's Adjuvant and eight with adjuvant only. Animals received three subcutaneous injections and sera were obtained before each immunization and at sacrifice. All sera were evaluated by ELISA for antibodies against the TAX1BP1peptide, the homologous AQP4 peptide and all linear AQP4 epitopes. Homologous and cross-inhibition assays were performed to ensure binding specificity, and reactivity against conformational AQP4 epitopes was evaluated by a cell-based assay. Sera from immunized animals showed high reactivity against the immunization peptide, and the homologous AQP4 epitope. Inhibition assays confirmed binding specificity. No antibodies were produced against any other epitopes, either linear or conformational. No clinical or brain inflammatory signs were observed in the animals. The induction of antibodies to an AQP4 epitope in mice immunized with the TAX1BP1-derived peptide suggests that a latent HTLV-1 infection could lead to TAX1BP1 antigen presentation and the production of anti-AQP4 antibodies, probably through T cell-mediated mechanisms. Further studies are needed for exploring triggering factors for NMO especially in HTLV-1-endemic regions.

  15. Antimicrobial peptides

    PubMed Central

    2014-01-01

    With increasing antibiotics resistance, there is an urgent need for novel infection therapeutics. Since antimicrobial peptides provide opportunities for this, identification and optimization of such peptides have attracted much interest during recent years. Here, a brief overview of antimicrobial peptides is provided, with focus placed on how selected hydrophobic modifications of antimicrobial peptides can be employed to combat also more demanding pathogens, including multi-resistant strains, without conferring unacceptable toxicity. PMID:24758244

  16. Microinjection of CART (cocaine- and amphetamine-regulated transcript) peptide into the nucleus accumbens inhibits the cocaine-induced upregulation of dopamine receptors and locomotor sensitization.

    PubMed

    Peng, Qinghua; Sun, Xi; Liu, Ziyong; Yang, Jianghua; Oh, Ki-Wan; Hu, Zhenzhen

    2014-09-01

    Repeated exposure to addictive drugs enhances dopamine receptor (DR) signaling and the ultimate phosphorylation of the cyclic adenosine 5'-monophosphate (cAMP)-response element-binding protein (CREB)-regulated cocaine- and amphetamine-regulated transcript (CART) expression in the nucleus accumbens (NAcc). These effects are known to contribute to the expression of behavioral sensitization. CART peptides are neuropeptides that modulate drug reward and reinforcement. The present experiments investigated the effects of CART 55-102 microinjection into the NAcc on (1) the phosphorylation of CREB, (2) cAMP/protein kinase A (PKA) signaling and (3) extracellular signal-regulated kinase (ERK) phosphorylated kinase signaling. Here, we show that repeated microinjections into the NAcc of CART 55-102 peptides (1.0 or 2.5μg, 0.5μl/side) attenuates cocaine-induced enhancements of D1R, D2R and D3R phosphorylation in this sites. Furthermore, the microinjection of CART 55-102 followed by repeated injections of cocaine (15mg/kg) dose-dependently blocked the enhancement of cAMP levels, PKA activity and pERK and pCREB levels on the fifth day of cocaine administration. The cocaine-induced locomotor activity and behavioral sensitization in rats were also inhibited by the 5-day-microinjection of CART peptides. These results suggest that the phosphorylation of CREB by cocaine in the NAcc was blocked by the CART 55-102 peptide via the inhibition of D1R and D2R stimulation, D3R phosphorylation, cAMP/PKA signaling and ERK phosphorylated kinase signaling. These effects may have played a compensatory inhibitory role in the behavioral sensitization of rats that received microinjections of CART 55-102. PMID:24953280

  17. A Celiac Cellular Phenotype, with Altered LPP Sub-Cellular Distribution, Is Inducible in Controls by the Toxic Gliadin Peptide P31-43

    PubMed Central

    Nanayakkara, Merlin; Kosova, Roberta; Lania, Giuliana; Sarno, Marco; Gaito, Alessandra; Galatola, Martina; Greco, Luigi; Cuomo, Marialaura; Troncone, Riccardo; Auricchio, Salvatore

    2013-01-01

    Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a “CD cellular phenotype” in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis. PMID:24278174

  18. Cell-penetrating peptides, targeting the regulation of store-operated channels, slow decay of the progesterone-induced [Ca2+]i signal in human sperm

    PubMed Central

    Morris, Jennifer; Jones, Sarah; Howl, John; Lukanowska, Monika; Lefievre, Linda; Publicover, Stephen

    2015-01-01

    Previous work has provided evidence for involvement of store-operated channels (SOCs) in [Ca2+]i signalling of human sperm, including a contribution to the transient [Ca2+]i elevation that occurs upon activation of CatSper, a sperm-specific cation channel localized to the flagellum, by progesterone. To further investigate the potential involvement of SOCs in the generation of [Ca2+]i signals in human sperm, we have used cell-penetrating peptides containing the important basic sequence KIKKK, part of the STIM–Orai activating region/CRAC activating domain (SOAR/CAD) of the regulatory protein stromal interaction molecule 1. SOAR/CAD plays a key role in controlling the opening of SOCs, which occurs upon mobilization of stored Ca2+. Resting [Ca2+]i temporarily decreased upon application of KIKKK peptide (3–4 min), but scrambled KIKKK peptide had a similar effect, indicating that this action was not sequence-specific. However, in cells pretreated with KIKKK, the transient [Ca2+]i elevation induced by stimulation with progesterone decayed significantly more slowly than in parallel controls and in cells pretreated with scrambled KIKKK peptide. Examination of single-cell responses showed that this effect was due, at least in part, to an increase in the proportion of cells in which the initial transient was maintained for an extended period, lasting up to 10 min in a subpopulation of cells. We hypothesize that SOCs contribute to the progesterone-induced [Ca2+]i transient, and that interference with the regulatory mechanisms of SOC delays their closure, causing a prolongation of the [Ca2+]i transient. PMID:25882543

  19. The Composition of Metals Bound to Class III Metallothionein (Phytochelatin and Its Desglycyl Peptide) Induced by Various Metals in Root Cultures of Rubia tinctorum.

    PubMed Central

    Maitani, T.; Kubota, H.; Sato, K.; Yamada, T.

    1996-01-01

    The induction of phytochelatins (PCs) and their desglycyl peptides (both are referred to as class III metallothionein [CIIIMT]) by exposure to various metals (Ag+, As3+, As5+, Cd2+, Cu2+, Ga3+, Hg2+, In3+, Ni2+, Pb2+, Pd2+, Se4+, and Zn2+) and the metal composition in the CIIIMTs were investigated in root cultures of Rubia tinctorum L. All of these metal species induced PCs to various degrees when analyzed by the postcolumn derivatization high-performance liquid chromatography method. The desglycyl peptides of PCs often were also present. However, only Ag, Cd, and Cu were bound to the CIIIMTs that they induced when analyzed by the high-performance liquid chromatography-inductively coupled plasma-atomic emission spectrometry method. Cu was also bound to the CIIIMTs induced by Ag+, As3+, and Cd2+. After Ag+ exposure, an Fe peak that may be of Fe-CIIIMT was also observed. However, most of the metal species studied were not bound to the CIIIMTs that they induced. PMID:12226248

  20. Measurement of cytoplasmic free Ca2+ concentration in rabbit aorta using the photoprotein, aequorin. Effect of atrial natriuretic peptide on agonist-induced Ca2+ signal generation.

    PubMed Central

    Takuwa, Y; Rasmussen, H

    1987-01-01

    Addition of norepinephrine, angiotensin II, or histamine leads to a transient rise in the cytoplasmic Ca2+ concentration ([Ca2+]i), as measured with aequorin, in rabbit aortic strips. Each induces a [Ca2+]i transient which peaks in 2 min and then falls either back to baseline (angiotensin II) or to a plateau (norepinephrine and histamine). The [Ca2+]i transient is due to the mobilization of Ca2+ from a caffeine-sensitive, intracellular pool. An elevation of [K+] to 35 mM leads to a monotonic sustained rise in [Ca2+]i which depends entirely on extracellular Ca2+, but an increase to 100 mM leads to a [Ca2+]i transient from the mobilization of intracellular Ca2+. Atrial natriuretic peptide does not alter basal [Ca2+]i nor inhibit the [Ca2+]i transient induced by either histamine or angiotensin II, but blocks that induced by norepinephrine, and blocks the plateau phase induced by either histamine or norepinephrine. The peptide inhibits the contractile response to all three agonists and to K+. PMID:2439545

  1. Amyloid-β-induced action potential desynchronization and degradation of hippocampal gamma oscillations is prevented by interference with peptide conformation change and aggregation.

    PubMed

    Kurudenkandy, Firoz Roshan; Zilberter, Misha; Biverstål, Henrik; Presto, Jenny; Honcharenko, Dmytro; Strömberg, Roger; Johansson, Jan; Winblad, Bengt; Fisahn, André

    2014-08-20

    The amyloid-β hypothesis of Alzheimer's Disease (AD) focuses on accumulation of amyloid-β peptide (Aβ) as the main culprit for the myriad physiological changes seen during development and progression of AD including desynchronization of neuronal action potentials, consequent development of aberrant brain rhythms relevant for cognition, and final emergence of cognitive deficits. The aim of this study was to elucidate the cellular and synaptic mechanisms underlying the Aβ-induced degradation of gamma oscillations in AD, to identify aggregation state(s) of Aβ that mediate the peptides neurotoxicity, and to test ways to prevent the neurotoxic Aβ effect. We show that Aβ(1-42) in physiological concentrations acutely degrades mouse hippocampal gamma oscillations in a concentration- and time-dependent manner. The underlying cause is an Aβ-induced desynchronization of action potential generation in pyramidal cells and a shift of the excitatory/inhibitory equilibrium in the hippocampal network. Using purified preparations containing different aggregation states of Aβ, as well as a designed ligand and a BRICHOS chaperone domain, we provide evidence that the severity of Aβ neurotoxicity increases with increasing concentration of fibrillar over monomeric Aβ forms, and that Aβ-induced degradation of gamma oscillations and excitatory/inhibitory equilibrium is prevented by compounds that interfere with Aβ aggregation. Our study provides correlative evidence for a link between Aβ-induced effects on synaptic currents and AD-relevant neuronal network oscillations, identifies the responsible aggregation state of Aβ and proofs that strategies preventing peptide aggregation are able to prevent the deleterious action of Aβ on the excitatory/inhibitory equilibrium and on the gamma rhythm. PMID:25143621

  2. The influence of orientation and number of copies of T and B cell epitopes on the specificity and affinity of antibodies induced by chimeric peptides.

    PubMed

    Partidos, C; Stanley, C; Steward, M

    1992-10-01

    CBA and TO mice were immunized with chimeric peptide immunogens consisting of B cell (residues 404-414) and T cell (residues 288-302) epitopes from the F protein of measles virus. The chimeras were co-linearly synthesized to contain one or two copies of the T cell epitope linked to one or two copies of the B cell epitope via a glycine.glycine spacer. Two orientations were synthesized such that the T cell epitope(s) were located at either the amino or carboxyl terminus of the B cell epitope(s). The levels of antibody induced following immunization with the chimeras were assessed by enzyme-linked immunosorbent assay using microtiter plates coated with either the homologous chimera or the B cell epitope sequence. The affinities of the anti-chimera antibodies for the B cell epitope were assessed by a fluid-phase double-isotope radioimmunoassay. All the chimeras induced good antibody responses in both strains of mice with specificity for the B cell epitope. Chimeras containing two copies of the T cell epitope induced antibodies with higher affinity for the B cell epitope than did chimeras containing one copy of the T cell epitope or two copies of the B cell epitope. Furthermore, the amino terminal location of the T cell epitope in relation to the B cell epitope in the chimera induced higher affinity anti-B cell antibody than did the reverse orientation. These results suggest that orientation of epitopes and amino acid composition of chimeric peptides affect antigen processing and presentation to T cells which govern both the specificity and affinity of antibody produced. Thus, for the production of synthetic peptide immunogens with vaccine potential, attention needs to be given to the number and orientation of the component epitopes required to produce highest affinity antibody.

  3. PID15, a novel 6 kDa secreted peptide, mediates Naja naja venom phospholipase A2 induced apoptosis in isolated human peripheral lymphocytes

    PubMed Central

    2014-01-01

    Background Snake venoms are a complex mixture of active principles mainly peptides and proteins also including amino acids, nucleotides, free lipids, carbohydrates and metallic elements bound to proteins that interfere in several biological systems. In this study, we aimed to understand the mode of action of the apoptosis inducing ability of Naja naja venom phospholipase A2 (NV-PLA2) using isolated human peripheral lymphocytes. Results Human peripheral lymphocytes when incubated with Naja naja venom phospholipase A2 (NV-PLA2) induced up to 68% DNA fragmentation. The dialysed conditioned media obtained by incubating lymphocytes with NV-PLA2 at 15th min induced 44% DNA fragmentation, referred to as cmlp-active. Cmlp-active showed 20.5% increased protein concentration than the corresponding control condition media cmlp-c-15. Test for creatine kinase activity in cmlp-active proved negative and negligible amount of lactate dehydrogenase did not show significant DNA fragmentation. Fractionation of cmlp-active on Sephadex G-25 showed two peaks, major peak induced 38% DNA fragmentation, which was further rechromatographed on Sephadex G-25. The single peak obtained was named PID15 (Phospholipase A 2 Induced DNA fragmentation factor secreted at 15 th min). Q-Tof MS/MS analysis of PID-15 showed it is a 6 kDa peptide. PID15 sequence analysis gave 40 amino acids in the following order, msilpcknvs iwvikdtaas dkevvlgsdr aikflylatg. The homology search for the sequence revealed it to be an Apoptosis Inducing Factor (AIF). Conclusion Results indicate that the secretion of PID15 is dependent on concentration of NV-PLA2 treatment, incubation time and also on temperature and the probable membrane origin of PID15 and not of cytosolic origin with apoptosis inducing ability. PMID:25030355

  4. Antibacterial Activity of Novel Cationic Peptides against Clinical Isolates of Multi-Drug Resistant Staphylococcus pseudintermedius from Infected Dogs

    PubMed Central

    Mohamed, Mohamed F.; Hammac, G. Kenitra; Guptill, Lynn; Seleem, Mohamed N.

    2014-01-01

    Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 “D isoform” demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and

  5. ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release.

    PubMed

    Barthel, T K; Zhang, J; Walker, G C

    2001-10-01

    We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell. PMID:11544208

  6. Correlation between CD8 dependency and determinant density using peptide-induced, Ld-restricted cytotoxic T lymphocytes.

    PubMed

    Alexander, M A; Damico, C A; Wieties, K M; Hansen, T H; Connolly, J M

    1991-04-01

    We have taken advantage of some unique properties of H-2Ld to investigate the determinant density requirements for cytotoxic T lymphocyte (CTL) priming versus effector function and to correlate the determinant density requirements with CD8 dependency. In a previous study (Lie, W.-R., N. B. Myers, J. Gorka, R. J. Rubocki, J. M. Connolly, and T. H. Hansen. 1990. Nature [Lond.]. 344:439), we demonstrated that culturing normal cells with peptides known to be restricted by H-2Ld led to a two- to fourfold increase in surface Ld expression. In the present study, we demonstrate the generation of Ld-restricted, peptide-specific in vitro primary CTL by culturing spleen cells with murine cytomegalovirus or tum- peptide at concentrations previously shown to result in maximum induction of Ld expression. Target cells can be sensitized for recognition by these CTL with lower dose of peptide than are required for the primary sensitization. This demonstrates differences in the determinant density requirements for priming versus effector function. The in vitro primary CTL generated with peptide can weakly lyse target cells that express the determinant endogenously, and CTL lines and clones capable of strong lysis of endogenous expressors are easily obtained. In both cases, target cells treated with exogenous peptide are lysed better than target cells expressing antigen endogenously. This suggested that there are differences in the determinant density of peptide-fed versus endogenous targets. This interpretation was substantiated when it was observed that the level of lysis of target cells expressing endogenous determinants correlated inversely with the amount of peptide required to sensitize targets for recognition by various tum- -specific CTL clones. Furthermore, simultaneous titration of both the peptide used to treat target cells and the antibody to CD8 revealed that the various CTL clones analyzed displayed widely disparate CD8 dependencies. In each case, the CD8 dependency

  7. Enhanced Peptide Stability Against Protease Digestion Induced by Intrinsic Factor Binding of a Vitamin B12 Conjugate of Exendin-4

    PubMed Central

    Bonaccorso, Ron L.; Chepurny, Oleg G.; Becker-Pauly, Christoph; Holz, George G.; Doyle, Robert P.

    2015-01-01

    Peptide digestion from proteases is a significant limitation in peptide therapeutic development. It has been hypothesized that the dietary pathway of vitamin B12 (B12) may be exploited in this area, but an open question is whether B12-peptide conjugates bound to the B12 gastric uptake protein intrinsic factor (IF) can provide any stability against proteases. Herein, we describe a new conjugate of B12 with the incretin peptide exendin 4 that demonstrates picomolar agonism of the glugacon-like peptide-1 receptor (GLP1-R). Stability studies reveal that Ex-4 is digested by pancreatic proteases trypsin and chymotrypsin and by the kidney endopeptidase meprin β. Prebinding the B12 conjugate to IF, however, resulted in up to a 4-fold greater activity of the B12-Ex-4 conjugate relative to Ex-4, when the IF-B12-Ex-4 complex was exposed to 22 µg/mL of trypsin, 2.3-fold greater activity when exposed to 1.25 µg/mL of chymotrypsin, and there was no decrease in function at up to 5 µg/mL of meprin β. PMID:26260673

  8. Manipulation of unfolding-induced protein aggregation by peptides selected for aggregate-binding ability through phage display library screening.

    PubMed

    Kundu, Bishwajit; Shukla, Anshuman; Guptasarma, Purnananda

    2002-03-01

    A phage-displayed library of peptides (12-mer) was screened for the ability to bind to thermally aggregated bovine carbonic anhydrase (BCA), with a view toward examining whether peptides possessing this ability might bind to partially structured intermediates on the protein's unfolding pathway and, therefore, constitute useful tools for manipulation of the kinetic partitioning of molecules between the unfolded and aggregated states. Two peptides [N-HPSTMGLRTMHP-C and N-TPSAWKTALVKA-C] were identified and tested. While neither showed thermal aggregation autonomously, both peptides individually elicited remarkable increases in the levels of thermal aggregation of BCA. A possible explanation is that both peptides bind to surfaces on molten BCA that are not directly involved in aggregation. Such binding could slow down interconversions between folded and unfolded states and stabilize aggregation-prone intermediate(s) to make them more prone to aggregation, while failing to achieve any steric prevention of aggregation. The approach has the potential of yielding useful aggregation-aiding/inhibiting agents, and may provide clues to whether amorphous aggregates are "immobilized" forms of folding intermediates. PMID:11866450

  9. The involvement of sigma1 receptors in donepezil-induced rescue of hippocampal LTP impaired by beta-amyloid peptide.

    PubMed

    Solntseva, E I; Kapai, N A; Popova, O V; Rogozin, P D; Skrebitsky, V G

    2014-07-01

    Donepezil is a potent acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease (AD). Additional therapeutically relevant target for donepezil is sigma1 receptor (Sig1-R). Beta-amyloid peptide (Aβ) is believed to contribute to the pathogenesis of AD. In our previous work (Kapai et al., 2012), we have shown that donepezil antagonizes the suppressive action of Aβ(1-42) on long-term potentiation (LTP) in rat hippocampal slices. The purpose of the present study was to determine whether Sig1-R is involved into the mechanisms of donepezil action. For this purpose, we have tested whether agonist of Sig1-R PRE-084 mimics, and antagonist of Sig1-R haloperidol abolishes the effect of donepezil. Population spikes (PSs) were recorded from the pyramidal layer of the CA1 region of rat hippocampal slices. Drugs were applied by addition to the perfusate starting 15 min before and ending 5 min after the tetanus. In the control group, the amplitude of PS 30 min post-tetanus reached 153±10%. Aβ (200 nM) markedly suppressed the LTP magnitude or even caused the suppression of baseline PS (82±8%, P<0.001). This suppression of LTP could be markedly prevented when 1 μM donepezil was co-administered with Aβ (136±11%, P<0.05). Further, we co-administered three substances: Aβ, donepezil and 0.5 μM haloperidol and have found that haloperidol antagonized the stimulating effect of donepezil on LTP (92±6%, P<0.05). Agonist of Sig1-R PRE-084 (0.1-10 μM) enhanced control LTP and abolished the inhibitory effect of Aβ on LTP in a concentration-dependent manner. The amplitude of PS 30 min post-tetanus reached 183±7% (P<0.01) for 10 μM PRE-084. The results suggest that activation of Sig1-R is involved into the mechanisms of donepezil-induced rescue of hippocampal LTP impaired by Aβ.

  10. New polycomb group protein enhancer of zeste homolog (EZH) 2-derived peptide with the potential to induce cancer-reactive cytotoxic T lymphocytes in prostate cancer patients with HLA-A3 supertype alleles.

    PubMed

    Minami, Takafumi; Minami, Tomoko; Shimizu, Nobutaka; Yamamoto, Yutaka; De Velasco, Marco A; Nozawa, Masahiro; Yoshimura, Kazuhiro; Harashima, Nanae; Harada, Mamoru; Uemura, Hirotsugu

    2015-05-01

    Analyses on reactivity of anti-cancer cytotoxic T lymphocytes (CTLs) and clinical application of peptide-based anti-cancer vaccine have been mainly focused on patients with HLA-A2 or -A24 alleles. In this study, we identified an enhancer of zeste homolog (EZH) 2-derived peptide applicable for anti-cancer vaccine for prostate cancer patients with HLA-A3 supertype alleles. Five EZH2-derived peptides that were prepared based on the binding motif to the HLA-A3 supertype alleles (HLA-A11, -A31, and -A33) were functionally screened for their potential to induce peptide-specific CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A3 supertype allele(+) prostate cancer patients. As a result, EZH2733-741 peptide was found to efficiently induce peptide-specific CTLs. The EZH2733-741 peptide-stimulated and purified CD8(+) T cells from PBMCs of HLA-A3 supertype allele(+) prostate cancer patients showed higher cytotoxicity against HLA-A3 supertype allele-expressing LNCaP prostate cancer cells than against parental LNCaP cells. This cytotoxicity against HLA-A3 supertype allele-expressing LNCaP cells was partially but significantly inhibited by the addition of EZH2733-741 peptide-pulsed competitive cells. These results indicate that the EZH2733-741 peptide could be a promising candidate for peptide-based immunotherapy for HLA-A3 supertype allele(+) prostate cancer patients.

  11. Light induced drug delivery into cancer cells.

    PubMed

    Shamay, Yosi; Adar, Lily; Ashkenasy, Gonen; David, Ayelet

    2011-02-01

    Cell-penetrating peptides (CPPs) can be used for intracellular delivery of a broad variety of cargoes, including various nanoparticulate pharmaceutical carriers. However, the cationic nature of all CPP sequences, and thus lack of cell specificity, limits their in vivo use for drug delivery applications. Here, we have devised and tested a strategy for site-specific delivery of dyes and drugs into cancer cells by using polymers bearing a light activated caged CPP (cCPP). The positive charge of Lys residues on the minimum sequence of the CPP penetratin ((52)RRMKWKK(58)) was masked with photo-cleavable groups to minimize non-specific adsorption and cellular uptake. Once illuminated by UV light, these protecting groups were cleaved, the positively charged CPP regained its activity and facilitated rapid intracellular delivery of the polymer-dye or polymer-drug conjugates into cancer cells. We have found that a 10-min light illumination time was sufficient to enhance the penetration of the polymer-CPP conjugates bearing the proapoptotic peptide, (D)(KLAKLAK)(2), into 80% of the target cells, and to promote a 'switch' like cytotoxic activity resulting a shift from 100% to 10% in cell viability after 2 h. This report provides an example for tumor targeting by means of light activation of cell-penetrating peptides for intracellular drug delivery. PMID:21074848

  12. Light induced drug delivery into cancer cells.

    PubMed

    Shamay, Yosi; Adar, Lily; Ashkenasy, Gonen; David, Ayelet

    2011-02-01

    Cell-penetrating peptides (CPPs) can be used for intracellular delivery of a broad variety of cargoes, including various nanoparticulate pharmaceutical carriers. However, the cationic nature of all CPP sequences, and thus lack of cell specificity, limits their in vivo use for drug delivery applications. Here, we have devised and tested a strategy for site-specific delivery of dyes and drugs into cancer cells by using polymers bearing a light activated caged CPP (cCPP). The positive charge of Lys residues on the minimum sequence of the CPP penetratin ((52)RRMKWKK(58)) was masked with photo-cleavable groups to minimize non-specific adsorption and cellular uptake. Once illuminated by UV light, these protecting groups were cleaved, the positively charged CPP regained its activity and facilitated rapid intracellular delivery of the polymer-dye or polymer-drug conjugates into cancer cells. We have found that a 10-min light illumination time was sufficient to enhance the penetration of the polymer-CPP conjugates bearing the proapoptotic peptide, (D)(KLAKLAK)(2), into 80% of the target cells, and to promote a 'switch' like cytotoxic activity resulting a shift from 100% to 10% in cell viability after 2 h. This report provides an example for tumor targeting by means of light activation of cell-penetrating peptides for intracellular drug delivery.

  13. Zinc-induced interaction of the metal-binding domain of amyloid-β peptide with DNA.

    PubMed

    Khmeleva, Svetlana A; Mezentsev, Yuri V; Kozin, Sergey A; Tsvetkov, Philipp O; Ivanov, Alexis S; Bodoev, Nikolay V; Makarov, Alexander A; Radko, Sergey P

    2013-01-01

    The interaction of the 16-mer synthetic peptide (Aβ16), which represents the metal-binding domain of the amyloid-β with DNA, was studied employing the surface plasmon resonance technique. It has been shown that Aβ16 binds to the duplex DNA in the presence of zinc ions and thus the metal-binding domain can serve as a zinc-dependent DNA-binding site of the amyloid-β. The interaction of Aβ16 with DNA most probably depends on oligomerization of the peptide and is dominated by interaction with phosphates of the DNA backbone.

  14. Anion Recognition and Induced Self-Assembly of an α,γ-Cyclic Peptide To Form Spherical Clusters.

    PubMed

    Rodríguez-Vázquez, Nuria; Amorín, Manuel; Alfonso, Ignacio; Granja, Juan R

    2016-03-24

    A cyclic octapeptide composed of hydroxy-functionalized γ-amino acids folds in a "V-shaped" conformation that allows the selective recognition of anions such as chloride, nitrate, and carbonate. The process involves the simultaneous self-assembly of six peptide subunits and the recognition of four anions to form a tetrahedral structure, in which the anions are located at the corners of the resulting structure. Each anion is coordinated to three different peptides. The structure was fully characterized by several techniques, including NMR spectroscopy and X-ray diffraction, and the material was able to facilitate the transmembrane transport of chloride ions. PMID:26945782

  15. Generation of the First TCR Transgenic Mouse with CD4(+) T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide.

    PubMed

    Jansen, Manon A A; van Herwijnen, Martijn J C; van Kooten, Peter J S; Hoek, Aad; van der Zee, Ruurd; van Eden, Willem; Broere, Femke

    2016-01-01

    Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such -antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen--specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4(+) T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4(+) T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4(+)CD25(+) Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis. PMID:27014269

  16. Generation of the First TCR Transgenic Mouse with CD4+ T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide

    PubMed Central

    Jansen, Manon A. A.; van Herwijnen, Martijn J. C.; van Kooten, Peter J. S.; Hoek, Aad; van der Zee, Ruurd; van Eden, Willem; Broere, Femke

    2016-01-01

    Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such ­antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen-­specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4+ T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4+ T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4+CD25+ Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis. PMID:27014269

  17. Development of gold nanoparticle based colorimetric method for quantitatively studying the inhibitors of Cu(2+)/Zn(2+) induced β-amyloid peptide assembly.

    PubMed

    Wang, Chengke; Wang, Kun; Wang, Zhenxin

    2015-02-01

    In this paper, a kind of gold nanoparticle (GNP)-based colorimetric assay has been developed for studying the reversible interaction of β-amyloid peptide (Aβ) with Cu(2+) and Zn(2+), and quantitatively analyzing four inhibitors (i.e., EDTA, EGTA, histidine and clioquinol) of Cu(2+)/Zn(2+) induced Aβ assembly. The inhibition efficiencies (e.g., half maximal inhibitory concentration, IC50 value) of these inhibitors could be measured in this work. As far as we know, these IC50 values were reported at the first time. In this assay, the streptavidin conjugated GNPs (SA-GNPs) were employed as indicators to monitor the Cu(2+)/Zn(2+) induced aggregating/disaggregating behaviors of biotin modified β-amyloid 1-16 peptides (Aβ1-16(biotin)). Because of high affinity of streptavidin (SA) with biotin, the aggregating/disaggregating of Aβ1-16(biotin) results in the significant color change of SA-GNPs. Furthermore, we demonstrate that the assay can be used as an effective tool for designing anti-dementia drugs through quantitative analysis of the interactions of four representative inhibitors with Cu(2+)/Zn(2+) induced Aβ assembly. PMID:25597800

  18. Antinociception induced by acute oral administration of sweet substance in young and adult rodents: the role of endogenous opioid peptides chemical mediators and μ(1)-opioid receptors.

    PubMed

    de Freitas, Renato Leonardo; Kübler, João Marcus Lopes; Elias-Filho, Daoud Hibraim; Coimbra, Norberto Cysne

    2012-04-01

    The present work aimed to investigate the effects of acute sucrose treatment on the perception of painful stimuli. Specifically, we sought to determine the involvement of the endogenous opioid peptide-mediated system as well as the role of the μ(1)-opioid receptor in antinociception organisation induced by acute sucrose intake. Nociception was assessed with the tail-flick test in rats (75, 150 and 250 g) of different ages acutely pre-treated with 500 μL of a sucrose solution (25, 50, 150 and 250 g/L) or tap water. Young and Adult rats (250 g) showed antinociception after treatment with 50 g/L (during 5 min) and 150 g/L and 250 g/L (during 20 min) sucrose solutions. Surprisingly, this antinociception was more consistent in mature adult rodents than in pups. To evaluate the role of opioid systems, mature adult rodents were pre-treated with different doses (0.25, 1 or 4 mg/kg) of the non-selective opioid receptor antagonist naloxone, the selective μ(1)-opioid receptor antagonist naloxonazine or vehicle followed by 250 g/L sucrose solution treatment. Sucrose-induced antinociception was reduced by pre-treatment with both naloxone and naloxonazine. The present findings suggest that sweet substance-induced hypo-analgesia is augmented by increasing sucrose concentrations in young and adult rodents. Acute oral sucrose treatment inhibits pain in laboratory animal by mediating endogenous opioid peptide and μ(1)-opioid receptor actions.

  19. Development of gold nanoparticle based colorimetric method for quantitatively studying the inhibitors of Cu(2+)/Zn(2+) induced β-amyloid peptide assembly.

    PubMed

    Wang, Chengke; Wang, Kun; Wang, Zhenxin

    2015-02-01

    In this paper, a kind of gold nanoparticle (GNP)-based colorimetric assay has been developed for studying the reversible interaction of β-amyloid peptide (Aβ) with Cu(2+) and Zn(2+), and quantitatively analyzing four inhibitors (i.e., EDTA, EGTA, histidine and clioquinol) of Cu(2+)/Zn(2+) induced Aβ assembly. The inhibition efficiencies (e.g., half maximal inhibitory concentration, IC50 value) of these inhibitors could be measured in this work. As far as we know, these IC50 values were reported at the first time. In this assay, the streptavidin conjugated GNPs (SA-GNPs) were employed as indicators to monitor the Cu(2+)/Zn(2+) induced aggregating/disaggregating behaviors of biotin modified β-amyloid 1-16 peptides (Aβ1-16(biotin)). Because of high affinity of streptavidin (SA) with biotin, the aggregating/disaggregating of Aβ1-16(biotin) results in the significant color change of SA-GNPs. Furthermore, we demonstrate that the assay can be used as an effective tool for designing anti-dementia drugs through quantitative analysis of the interactions of four representative inhibitors with Cu(2+)/Zn(2+) induced Aβ assembly.

  20. Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys

    PubMed Central

    Desallais, Lucille; Bouchez, Caroline; Mouhsine, Hadley; Moreau, Gabriel; Ratsimandresy, Rojo; Montes, Matthieu; Do, Hervé; Quintin-Colonna, Françoise; Zagury, Jean-François

    2016-01-01

    Interleukin-6 (IL-6) overproduction has been involved in the pathogenesis of several chronic inflammatory diseases and the administration of an anti-IL-6 receptor monoclonal antibody has been proven clinically efficient to treat them. However, the drawbacks of monoclonal antibodies have led our group to develop an innovative anti-IL-6 strategy using a peptide-based active immunization. This approach has previously shown its efficacy in a mouse model of systemic sclerosis. Here the safety, immunogenicity, and efficacy of this strategy was assessed in non human primates. No unscheduled death and clinical signs of toxicity was observed during the study. Furthermore, the cynomolgus monkeys immunized against the IL-6 peptide produced high levels of anti-IL-6 antibodies as well as neutralizing antibodies compared to control groups. They also showed an important decrease of the cumulative inflammatory score following a delayed-type hypersensitivity reaction induced by the Tetanus vaccine compared to control groups (minus 57,9%, P = 0.014). These findings are highly significant because the immunizing IL-6 peptide used in this study is identical in humans and in monkeys and this novel anti-IL-6 strategy could thus represent a promising alternative to monoclonal antibodies. PMID:26782790

  1. Piscidin-1, an antimicrobial peptide from fish (hybrid striped bass morone saxatilis x M. chrysops), induces apoptotic and necrotic activity in HT1080 cells.

    PubMed

    Lin, Heng-Ju; Huang, Tsui-Chin; Muthusamy, Sasikala; Lee, Jheng-Fong; Duann, Yeh-Fang; Lin, Cheng-Hui

    2012-05-01

    Piscidin-1, a 22-residue cationic peptide isolated from mast cells of a hybrid striped bass, has potent antimicrobial activities against both gram-positive and -negative bacteria. To date, there is no report of its antitumor activity on any tumor cell lines. In this study, we examined the antitumor activity of a synthetic piscidin-1 peptide against several human cancer cell lines using an MTS assay and soft-agar colony-formation assay. We found that a low dose of piscidin induces both apoptosis and necrosis in HT1080 cells, as shown by annexin-V/propidium iodide and acridine orange/ethidium bromide staining, and triggers a necrotic cell death pathway in a short period with high-dose treatment. The destruction of cell membranes by piscidin-1 was demonstrated by transmission electron microscopy. Furthermore, piscidin-1 also inhibits the migration of HT1080 cells in a dose-dependent manner. This study provides the first evidence of the anticancer activity of the antimicrobial peptide, piscidin-1, with potential implications for the treatment of cancer. PMID:22559967

  2. NKLP27: a teleost NK-lysin peptide that modulates immune response, induces degradation of bacterial DNA, and inhibits bacterial and viral infection.

    PubMed

    Zhang, Min; Li, Mo-fei; Sun, Li

    2014-01-01

    NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.

  3. Self-assembly-induced far-red/near-infrared fluorescence light-up for detecting and visualizing specific protein-Peptide interactions.

    PubMed

    Wang, Huaimin; Liu, Jie; Han, Aitian; Xiao, Nannan; Xue, Zhaosheng; Wang, Gang; Long, Jiafu; Kong, Deling; Liu, Bin; Yang, Zhimou; Ding, Dan

    2014-02-25

    Understanding specific protein-peptide interactions could offer a deep insight into the development of therapeutics for many human diseases. In this work, we designed and synthesized a far-red/near-infrared (FR/NIR) fluorescence light-up probe (DBT-2EEGWRESAI) by simply integrating two tax-interacting protein-1 (TIP-1)-specific peptide ligands (EEGWRESAI) with one 4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole (DBT) unit. We first demonstrated that DBT is an environment-sensitive fluorophore with FR/NIR fluorescence due to its strong charge transfer character in the excited state. Thanks to the environmental sensitivity of DBT, the probe DBT-2EEGWRESAI is very weakly fluorescent in aqueous solution but lights up its fluorescence when the probe specifically binds to TIP-1 protein or polyprotein (ULD-TIP-1 tetramer). It is found that the DBT-2EEGWRESAI/TIP-1 protein and the DBT-2EEGWRESAI/ULD-TIP-1 tetramer could self-assemble into spherical nanocomplexes and a nanofiber network, respectively, which lead to probe fluorescence turn-on through providing DBT with a hydrophobic microenvironment. By virtue of the self-assembly-induced FR/NIR fluorescence turn-on, DBT-2EEGWRESAI can detect and visualize specific protein/polyprotein-peptide interactions in both solution and live bacteria in a high contrast and selective manner.

  4. Piscidin-1, an antimicrobial peptide from fish (hybrid striped bass morone saxatilis x M. chrysops), induces apoptotic and necrotic activity in HT1080 cells.

    PubMed

    Lin, Heng-Ju; Huang, Tsui-Chin; Muthusamy, Sasikala; Lee, Jheng-Fong; Duann, Yeh-Fang; Lin, Cheng-Hui

    2012-05-01

    Piscidin-1, a 22-residue cationic peptide isolated from mast cells of a hybrid striped bass, has potent antimicrobial activities against both gram-positive and -negative bacteria. To date, there is no report of its antitumor activity on any tumor cell lines. In this study, we examined the antitumor activity of a synthetic piscidin-1 peptide against several human cancer cell lines using an MTS assay and soft-agar colony-formation assay. We found that a low dose of piscidin induces both apoptosis and necrosis in HT1080 cells, as shown by annexin-V/propidium iodide and acridine orange/ethidium bromide staining, and triggers a necrotic cell death pathway in a short period with high-dose treatment. The destruction of cell membranes by piscidin-1 was demonstrated by transmission electron microscopy. Furthermore, piscidin-1 also inhibits the migration of HT1080 cells in a dose-dependent manner. This study provides the first evidence of the anticancer activity of the antimicrobial peptide, piscidin-1, with potential implications for the treatment of cancer.

  5. Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys.

    PubMed

    Desallais, Lucille; Bouchez, Caroline; Mouhsine, Hadley; Moreau, Gabriel; Ratsimandresy, Rojo; Montes, Matthieu; Do, Hervé; Quintin-Colonna, Françoise; Zagury, Jean-François

    2016-01-01

    Interleukin-6 (IL-6) overproduction has been involved in the pathogenesis of several chronic inflammatory diseases and the administration of an anti-IL-6 receptor monoclonal antibody has been proven clinically efficient to treat them. However, the drawbacks of monoclonal antibodies have led our group to develop an innovative anti-IL-6 strategy using a peptide-based active immunization. This approach has previously shown its efficacy in a mouse model of systemic sclerosis. Here the safety, immunogenicity, and efficacy of this strategy was assessed in non human primates. No unscheduled death and clinical signs of toxicity was observed during the study. Furthermore, the cynomolgus monkeys immunized against the IL-6 peptide produced high levels of anti-IL-6 antibodies as well as neutralizing antibodies compared to control groups. They also showed an important decrease of the cumulative inflammatory score following a delayed-type hypersensitivity reaction induced by the Tetanus vaccine compared to control groups (minus 57,9%, P = 0.014). These findings are highly significant because the immunizing IL-6 peptide used in this study is identical in humans and in monkeys and this novel anti-IL-6 strategy could thus represent a promising alternative to monoclonal antibodies. PMID:26782790

  6. Enantioselective Collision-Activated Dissociation of Gas-Phase Tryptophan Induced by Chiral Recognition of Protonated uc(l)-Alanine Peptides

    NASA Astrophysics Data System (ADS)

    Fujihara, Akimasa; Matsuyama, Hiroki; Tajiri, Michiko; Wada, Yoshinao; Hayakawa, Shigeo

    2016-06-01

    Enantioselective dissociation in the gas phase is important for enantiomeric enrichment and chiral transmission processes in molecular clouds regarding the origin of homochirality in biomolecules. Enantioselective collision-activated dissociation (CAD) of tryptophan (Trp) and the chiral recognition ability of uc(l)-alanine peptides (uc(l)-Ala n ; n = 2-4) were examined using a linear ion trap mass spectrometer. CAD spectra of gas-phase heterochiral H+(uc(d)-Trp)(uc(l)-Ala n ) and homochiral H+(uc(l)-Trp)(uc(l)-Ala n ) noncovalent complexes were obtained as a function of the peptide size n. The H2O-elimination product was observed in CAD spectra of both heterochiral and homochiral complexes for n = 2 and 4, and in homochiral H+(uc(l)-Trp)(uc(l)-Ala3), indicating that the proton is attached to the uc(l)-alanine peptide, and H2O loss occurs from H+(uc(l)-Ala n ) in the noncovalent complexes. H2O loss did not occur in heterochiral H+(uc(d)-Trp)(uc(l)-Ala3), where NH3 loss and (H2O + CO) loss were the primary dissociation pathways. In heterochiral H+(uc(d)-Trp)(uc(l)-Ala3), the protonation site is the amino group of uc(d)-Trp, and NH3 loss and (H2O + CO) loss occur from H+(uc(d)-Trp). uc(l)-Ala peptides recognize uc(d)-Trp through protonation of the amino group for peptide size n = 3. NH3 loss and (H2O + CO) loss from H+(uc(d)-Trp) proceeds via enantioselective CAD in gas-phase heterochiral H+(uc(d)-Trp)(uc(l)-Ala3) at room temperature, whereas uc(l)-Trp dissociation was not observed in homochiral H+(uc(l)-Trp)(uc(l)-Ala3). These results suggest that enantioselective dissociation induced by chiral recognition of uc(l)-Ala peptides through protonation could play an important role in enantiomeric enrichment and chiral transmission processes of amino acids.

  7. Energetics and dynamics of the fragmentation reactions of protonated peptides containing methionine sulfoxide or aspartic acid via energy- and time-resolved surface induced dissociation.

    PubMed

    Lioe, Hadi; Laskin, Julia; Reid, Gavin E; O'Hair, Richard A J

    2007-10-25

    The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64 Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility in these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (nonmobile proton conditions) to lysine (partially mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFECs) reveal that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1-2 orders of magnitude lower than nonselective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to nonselective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these processes

  8. Energetics and Dynamics of the Fragmentation Reactions of Protonated Peptides Containing Methionine Sulfoxide or Aspartic Acid via Energy- and Time-Resolved Surface Induced Dissociation

    SciTech Connect

    Lioe, Hadi; Laskin, Julia; Reid, Gavin E.; O'Hair, Richard Aj

    2007-10-25

    The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility on these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (non-mobile proton conditions) to lysine (partially-mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFEC) reveals that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1–2 orders of magnitude lower than non-selective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to non-selective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these

  9. Proinsulin multi-peptide immunotherapy induces antigen-specific regulatory T cells and limits autoimmunity in a humanized model.

    PubMed

    Gibson, V B; Nikolic, T; Pearce, V Q; Demengeot, J; Roep, B O; Peakman, M

    2015-12-01

    Peptide immunotherapy (PIT) is a targeted therapeutic approach, involving administration of disease-associated peptides, with the aim of restoring antigen-specific immunological tolerance without generalized immunosuppression. In type 1 diabetes, proinsulin is a primary antigen targeted by the autoimmune response, and is therefore a strong candidate for exploitation via PIT in this setting. To elucidate the optimal conditions for proinsulin-based PIT and explore mechanisms of action, we developed a preclinical model of proinsulin autoimmunity in a humanized HLA-DRB1*0401 transgenic HLA-DR4 Tg mouse. Once proinsulin-specific tolerance is broken, HLA-DR4 Tg mice develop autoinflammatory responses, including proinsulin-specific T cell proliferation, interferon (IFN)-γ and autoantibody production. These are preventable and quenchable by pre- and post-induction treatment, respectively, using intradermal proinsulin-PIT injections. Intradermal proinsulin-PIT enhances proliferation of regulatory [forkhead box protein 3 (FoxP3(+))CD25(high) ] CD4 T cells, including those capable of proinsulin-specific regulation, suggesting this as its main mode of action. In contrast, peptide delivered intradermally on the surface of vitamin D3-modulated (tolerogenic) dendritic cells, controls autoimmunity in association with proinsulin-specific IL-10 production, but no change in regulatory CD4 T cells. These studies define a humanized, translational model for in vivo optimization of PIT to control autoimmunity in type 1 diabetes and indicate that dominant mechanisms of action differ according to mode of peptide delivery. PMID:26206289

  10. LC-MS/MS Analysis and Comparison of Oxidative Damages on Peptides Induced by Pathogen Reduction Technologies for Platelets

    NASA Astrophysics Data System (ADS)

    Prudent, Michel; Sonego, Giona; Abonnenc, Mélanie; Tissot, Jean-Daniel; Lion, Niels

    2014-04-01

    Pathogen reduction technologies (PRT) are photochemical processes that use a combination of photosensitizers and UV-light to inactivate pathogens in platelet concentrates (PCs), a blood-derived product used to prevent hemorrhage. However, different studies have questioned the impact of PRT on platelet function and transfusion efficacy, and several proteomic analyses revealed possible oxidative damages to proteins. The present work focused on the oxidative damages produced by the two main PRT on peptides. Model peptides containing residues prone to oxidation (tyrosine, histidine, tryptophane, and cysteine) were irradiated with a combination of amotosalen/UVA (Intercept process) or riboflavin/UVB (Mirasol-like process). Modifications were identified and quantified by liquid chromatography coupled to tandem mass spectrometry. Cysteine-containing peptides formed disulfide bridges (R-SS-R, -2 Da; favored following amotosalen/UVA), sulfenic and sulfonic acids (R-SOH, +16 Da, R-SO3H, +48 Da, favored following riboflavin/UVB) upon treatment and the other amino acids exhibited different oxidations revealed by mass shifts from +4 to +34 Da involving different mechanisms; no photoadducts were detected. These amino acids were not equally affected by the PRT and the combination riboflavin/UVB generated more oxidation than amotosalen/UVA. This work identifies the dif