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Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library

To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phage-displayed 12-mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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The specific binding to HepG2 cells indicates that HCSP4-fluorescein isothiocyanate (FITC) may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. A, B: HCSP4-FITC binds to HepG2 cells specifically; C, D: the nuclei (C) and membrane (D) of HepG2 cells are displayed using DAPI and Dil staining methods; E, F: the merged photos show that HCSP4-FITC binds to the surfaces of HepG2 cells specifically.

Peptides targeting chemokine receptor CXCR4: structural behavior and biological binding studies

CXCR4 is a G-protein-coupled receptor involved in a number of physiological processes in the hematopoietic and immune systems. CXCL12/CXCR4 axis plays a central role in diseases, such as HIV, cancer, WHIM syndrome, rheumatoid arthritis, pulmonary fibrosis, and lupus and, hence, indicated as putative therapeutic target. Although multiple CXCR4 antagonists have been developed, there is only one marketed drug, plerixafor, indicated for stem cell mobilization in poor mobilizer patients. In this work, we have designed and synthesized two peptides, six and seven residues long, using as template the N-terminal region of CXCL12; analyzed their conformations by CD, NMR, and molecular dynamics simulations; simulated their complexes with CXCR4 by docking methods; and validated these data by in vitro studies. The results showed that the two peptides are rather flexible in aqueous solution lacking ordered secondary structure elements and present a promising affinity for CXCR4. This affinity is not revealed for CXCR7, indicating a specificity for CXCR4. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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In this work, we have designed and synthesized two peptides, six and seven residues long, using as template the N-terminal region of CXCL12; analyzed their conformations by CD, NMR, and molecular dynamics simulations; simulated their complexes with CXCR4 by docking methods; and validated these data by in vitro studies. The results showed that the two peptides are rather flexible in aqueous solution lacking ordered secondary structure elements and present a promising affinity for CXCR4.

Practical, laboratory-scale synthesis of Nin-formyl tryptophan hydrobromide

A range of inorganic and organoelement halides was evaluated as acidic promoters of direct Nin-formylation of tryptophan. In addition to Me3SiBr, the less expensive PBr3 was found to be highly efficient and was selected for further optimization. A convenient and reproducible synthetic procedure for Nin-formyltryptophan hydrobromide developed in this way was scaled to 150 mmol and successfully extended to some derivatives of Trp and closely related indoles as detailed in the present paper. The scope of the method seems to be restricted to indoles substituted at C-3. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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The bromides indicated earlier efficiently promote selective formylation of indole nitrogen in tryptophan, its derivatives and related 3-functionalized indoles. Multigram scale procedure for Trp(For) is detailed.

A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif

A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea-equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled-2 (Dab2) sulfatide-binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane-binding, recombinant peptides that co-purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S-transferase (GST) fusion protein using minimal media containing [15N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea-equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at −80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co-purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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Recombinant Disabled-2 sulfatide-binding motif (SBM) can be efficiently isolated in an intact and active form for NMR studies. The reported approach can be easily applied to the isolation of recombinant peptides for structural studies. The figure displays 1H, 15N-HSQC overlaid spectra of 15N-labeled Disabled-2 SBM in the absence (red) and presence of dodecylphosphocholine micelles (black). Addition of micelles improved resolution of the spectrum of the peptide, suggesting that this region is responsible for Disabled-2 membrane targeting.

Spinal ERK2 activation through δ2 – opioid receptors contributes to nociceptive behavior induced by intrathecal injection of Leucine-enkephalin

Publication date: Available online 27 January 2014 Source:Peptides

Author(s): Takaaki Komatsu , Soh Katsuyama , Hirokazu Mizoguchi , Chikai Sakurada , Minoru Tsuzuki , Shinobu Sakurada , Tsukasa Sakurada

Intrathecal (i.t.) injection of leucine-enkephalin (Leu-ENK), co-administered with peptidase inhibitors, phosphoramidon, (an endopeptidase 24.11 inhibitor) and bestatin, (a general aminopeptidase inhibitor), produced behaviors consisting of the biting and/or licking of the hindpaw and the tail along with hindlimb scratching directed toward the flank, which peaked at 10 - 15min after an injection. This characteristic behavior was not observed in mice treated with i.t. Leu-ENK alone. We also investigated the effect of the extracellular signal-regulated kinase (ERK) in spinal processing of nociception induced by i.t. co-administration of Leu-ENK with phospharamidon and bestatin. Western blot analysis of phospho-ERK (pERK) showed a significant increase of pERK2 in the lumbar spinal cord in response to i.t. Leu-ENK co-injected with peptidase inhibitors. The MAP kinase-ERK inhibitor, U0126 dose-dependently attenuated the nociceptive behavior and spinal ERK activation to i.t. Leu-ENK co-injected with peptidase inhibitors. Furthermore, the nociceptive behavior and spinal ERK activation evoked by i.t. Leu-ENK in combination with peptidase inhibitors were inhibited by co-administration of the non-selective δ opioid receptor antagonist, naltrindole, the selective δ 2 -opioid receptor antagonist, naltriben, the non competitive N-methyl-D-aspartate (NMDA) antagonist, MK-801 or the non-selective nitoric oxide synthase inhibitor, L-NAME, the selective nNOS inhibitor, Nω-propyl-L-arginine or the selective iNOS inhibitor, W1400, but not by the selective δ 1 -receptor antagonist, BNTX (7-benzylidenenaltrexone). These results suggest that spontaneous nociceptive behaviors produced by i.t. co-administration of Leu-ENK with peptidase inhibitors may be induced by an activation of the glutamate-NO-ERK pathway through the δ 2 -opioid receptor in the dorsal spinal cord.





Adrenomedullin alleviates pulmonary artery collagen accumulation in rats with pulmonary hypertension induced by high blood flow

Publication date: Available online 27 January 2014 Source:Peptides

Author(s): Lulu Pang , Jianguang Qi , Yang Gao , Hongfang Jin , Junbao Du

Collagen accumulation is one of the important pathologic changes in the development of pulmonary hypertension. Previous research showed that adrenomedullin (ADM) mitigates the development of pulmonary hypertension. The present study explored the role of ADM in the development of pulmonary artery collagen accumulation induced by high pulmonary blood flow, by investigating the effect of ADM [1.5μg/(kg·h)] subcutaneously administered by mini-osmotic pump on pulmonary hemodynamics, pulmonary vascular structure and pulmonary artery collagen accumulation and synthesis in rats with high pulmonary blood flow induced by aortocaval shunting. The results showed that ADM significantly decreased mean pulmonary artery pressure (mPAP) and the ratio of right ventricular mass to left ventricular plus septal mass [RV/(LV+SP)], attenuated the muscularization of small pulmonary vessels and relative medial thickness (RMT) of pulmonary arteries in rats with high pulmonary blood flow. Meanwhile, ADM ameliorated pulmonary artery collagen deposition represented by a decrease in lung tissue hydroxyproline, collagen I and III content and pulmonary artery collagen I and III expression, reduced collagen synthesis represented by a decrease in lung tissue procollagen I and III mRNA expression. The results suggest that ADM plays a protective role in the development of pulmonary hypertension induced by high blood flow, by inhibiting pulmonary procollagen synthesis and alleviating pulmonary artery collagen accumulation





Isolation and characterization of SsmTx-I, a Specific Kv2.1 blocker from the venom of the centipede Scolopendra Subspinipes Mutilans L. Koch

Scolopendra subspinipes mutilans, also known as Chinese red-headed centipede, is a venomous centipede from East Asia and Australasia. Venom from this animal has not been researched as thoroughly as venom from snakes, snails, scorpions, and spiders. In this study, we isolated and characterized SsmTx-I, a novel neurotoxin from the venom of S. subspinipes mutilans. SsmTx-I contains 36 residues with four cysteines forming two disulfide bonds. It had low sequence similarity (<10%) with other identified peptide toxins. By whole-cell recording, SsmTx-I significantly blocked voltage-gated K+ channels in dorsal root ganglion neurons with an IC50 value of 200 nM, but it had no effect on voltage-gated Na+ channels. Among the nine K+ channel subtypes expressed in human embryonic kidney 293 cells, SsmTx-I selectively blocked the Kv2.1 current with an IC50 value of 41.7 nM, but it had little effect on currents mediated by other K+ channel subtypes. Blockage of Kv2.1 by SsmTx-I was not associated with significant alteration of steady-state activation, suggesting that SsmTx-I might act as a simple inhibitor or channel blocker rather than a gating modifier. Our study reported a specific Kv2.1-blocker from centipede venom and provided a basis for future investigations of SsmTx-I, for example on structure–function relationships, mechanism of action, and pharmacological potential. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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SsmTx-I, a novel peptide toxin from the venom of the centipede S subspinipes mutilans, specifically inhibits voltage-gated K+ channel subtype Kv2.1.

Ghrelin administered spinally increases the blood glucose level in mice

Publication date: Available online 25 January 2014 Source:Peptides

Author(s): Yun-Beom Sim , Soo-Hyun Park , Sung-Su Kim , Chea-Ha Kim , Su-Jin Kim , Soo-Min Lim , Jun-Sub Jung , Hong-Won Suh

Ghrelin is known as a regulator of the blood glucose homeostasis and food intake. In the present study, the possible roles of ghrelin located in the spinal cord in the regulation of the blood glucose level were investigated in ICR mice. We found that intrathecal (i.t.) injection with ghrelin (from 1 to 10μg) caused an elevation of the blood glucose level. In addition, i.t. pretreatment with YIL781 (ghrelin receptor antagonist; from 0.1 to 5μg) markedly attenuated ghrelin-induced hyperglycemic effect. The plasma insulin level was increased by ghrelin. The enhanced plasma insulin level by ghrelin was reduced by i.t. pretreatment with YIL781. However, i.t. pretreatment with glucagon-like peptide-1 (GLP-1; 5μg) did not affect the ghrelin-induced hyperglycemia. Furthermore, i.t. administration with ghrelin also elevated the blood glucose level, but in an additive manner, in D-glucose-fed model. Our results suggest that the activation of ghrelin receptors located in the spinal cord plays important roles for the elevation of the blood glucose level.





Irisin as a muscle-derived hormone stimulating thermogenesis–a critical update

Publication date: Available online 26 January 2014 Source:Peptides

Author(s): Tobias Hofmann , Ulf Elbelt , Andreas Stengel

The recently described myokine, irisin is cleaved from fibronectin type III domain containing protein 5 (FNDC5) and has been proposed to be secreted upon exercise to promote the browning of beige fat cells in white adipose tissue that results in enhanced thermogenesis and increased energy expenditure. The initial studies suggested irisin as a treatment option for obesity and associated diseases such as type 2 diabetes mellitus and stimulated further research. However, the results of subsequent studies investigating the regulation of irisin by different types of exercise are partly conflicting and effects were only shown in highly selective patient populations so far. Moreover, other parameters like body weight or fat free mass were shown to influence irisin adding more complexity to the mechanisms regulating this hormone. The present review will describe the discovery of irisin, its potential role in adipose tissue-mediated thermogenesis, its regulation by exercise and lastly, discuss current controversies and highlight gaps of knowledge to be filled by future studies.





Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme

Publication date: Available online 25 January 2014 Source:Peptides

Author(s): Aneta Sowa-Jasiłek , Agnieszka Zdybicka-Barabas , Sylwia Stączek , Jerzy Wydrych , Paweł Mak , Teresa Jakubowicz , Małgorzata Cytryńska

The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella.






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